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Zhou Y, Xia XM, Lingle CJ. Disruption of a side portal pathway permits closed-state inactivation by BK β subunit N termini. J Gen Physiol 2025; 157:e202513790. [PMID: 40445147 PMCID: PMC12124225 DOI: 10.1085/jgp.202513790] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2025] [Revised: 04/21/2025] [Accepted: 04/28/2025] [Indexed: 06/02/2025] Open
Abstract
Cytosolic N termini of several BK channel β regulatory subunits mediate rapid inactivation. However, in contrast to Kv channels, inactivation does not occur via a simple, open-channel block mechanism, but involves two steps, an association step in which ion permeation is maintained (O*), then followed by inactivation (I). To produce inactivation, BK β subunit N termini enter the central cavity through a lateral entry pathway ("side portal") separating the transmembrane pore-gate domain and cytosolic gating ring. Comparison of BK conformations reveals an aqueous pathway into the central cavity in the open structure, while in the closed structure, three sequential basic residues (R329K330K331) in the C-linker just following S6 occlude central cavity access. We probed the impact of mutations of the RKK motif (RKK3Q, RKK3E, and RKK3V) on inactivation mediated by the β3a N terminus. All three RKK-mutated constructs differentially reduce depolarization-activated outward current, prolong β3a-mediated tail current upon repolarization, and produce a persistent inward current at potentials down to -240 mV. With depolarization, channels are driven into O*-I inactivated states, and upon repolarization, slow tails and persistent inward currents reflect slow changes in O*-I occupancy. However, evaluation of closed-state occupancy prior to depolarization and at the end of slow tails reveals that some fraction of closed states at negative potentials correspond to resting closed states in voltage-independent equilibrium with N-terminal-occluded closed states. Thus, disruption of the RKK triplet both stabilizes the β3a N terminus in its position of inactivation and permits access of that N terminus to its blocking position in closed states.
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Affiliation(s)
- Yu Zhou
- Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO, USA
| | - Xiao-Ming Xia
- Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO, USA
| | - Christopher J. Lingle
- Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO, USA
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2
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Zhou Y, Xia XM, Lingle CJ. Disruption of a side portal pathway permits closed-state inactivation by BK β subunit N-termini. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.09.642150. [PMID: 40161743 PMCID: PMC11952309 DOI: 10.1101/2025.03.09.642150] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/02/2025]
Abstract
Cytosolic N-termini of several BK channel β regulatory subunits mediate rapid inactivation. However, in contrast to Kv channels, inactivation does not occur via a simple, open channel block mechanism, but involves two steps, an association step in which ion permeation is maintained (O*), then followed by inactivation (I). To produce inactivation, BK β subunit N-termini enter the central cavity through a lateral entry pathway ("side portal") separating the transmembrane pore-gate-domain and cytosolic gating ring. Comparison of BK conformations reveals an aqueous pathway into the central cavity in the open structure, while in the closed structure three sequential basic residues (R 329 K 330 K 331 ) in S6 occlude central cavity access. We probed the impact of mutations of the RKK motif (RKK3Q, RKK3E, and RKK3V) on inactivation mediated by the β3a N-terminus. All three RKK-mutated constructs differentially reduce depolarization-activated outward current, prolong β3a-mediated tail current upon repolarization, and produce a persistent inward current at potentials down to -240 mV. With depolarization channels are driven into O*-I inactivated states and, upon repolarization, slow tails and persistent inward currents reflect slow changes in O*-I occupancy. However, evaluation of closed state occupancy prior to depolarization and at the end of slow tails reveals that some fraction of closed states at negative potentials corresponds to resting closed states in voltage-independent equilibrium with N-terminal-occluded closed-states. Thus, disruption of the RKK triplet both stabilizes the β3a-N-terminus in its position of inactivation and permits access of that N-terminus to its blocking position in closed states. Summary The role of BK S6 residues R329K330K331 and E321/E324 in β subunit-mediated inactivation is probed. WT R329K330K331 hinders inactivation in closed states, while RKK mutations stabilize inactivated states even under conditions where channels are otherwise closed. E321/E324 mutations do not permit closed-state inactivation.
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3
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Tu YC, Lee IC, Chang TW, Lee V, Chao FY, Geltser ER, Tsai MF. Mechanisms of dual modulatory effects of spermine on the mitochondrial calcium uniporter complex. J Biol Chem 2025; 301:108218. [PMID: 39863104 PMCID: PMC11871460 DOI: 10.1016/j.jbc.2025.108218] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2024] [Revised: 01/01/2025] [Accepted: 01/14/2025] [Indexed: 01/27/2025] Open
Abstract
The mitochondrial Ca2+ uniporter is the Ca2+ channel responsible for mitochondrial Ca2+ uptake. It plays crucial physiological roles in regulating oxidative phosphorylation, intracellular Ca2+ signaling, and cell death. The uniporter contains the pore-forming MCU subunit, the auxiliary EMRE protein, and the regulatory MICU1 subunit, which blocks the MCU pore under resting cellular Ca2+ concentrations. It has been known for decades that spermine, a biological polyamine ubiquitously present in animal cells, can enhance mitochondrial Ca2+ uptake, but the underlying mechanisms remain incompletely understood. In this study, we demonstrate that spermine exerts both potentiation and inhibitory effects on the uniporter. At physiological concentrations, spermine binds to membranes and disrupts MCU-MICU1 interactions, thereby opening the uniporter to import more Ca2+. However, at millimolar concentrations, spermine also inhibits the uniporter by targeting the pore-forming region in a MICU1-independent manner. These findings provide molecular insights into how cells can use spermine to control the critical processes of mitochondrial Ca2+ signaling and homeostasis.
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Affiliation(s)
- Yung-Chi Tu
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA; Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia, USA
| | - I-Chi Lee
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA; Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia, USA
| | - Tsai-Wei Chang
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA; School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Vivian Lee
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Fan-Yi Chao
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Eitel R Geltser
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA
| | - Ming-Feng Tsai
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA; Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia, USA.
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4
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Langen JS, Boyle PM, Malan D, Sasse P. Optogenetic quantification of cardiac excitability and electrical coupling in intact hearts to explain cardiac arrhythmia initiation. SCIENCE ADVANCES 2025; 11:eadt4103. [PMID: 40020054 PMCID: PMC11870084 DOI: 10.1126/sciadv.adt4103] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/30/2024] [Accepted: 01/27/2025] [Indexed: 03/03/2025]
Abstract
Increased cardiac excitability and reduced electrical coupling promote cardiac arrhythmia and can be quantified by input resistance (Rm), pacing threshold (Ithr), and cardiac space constant (λ). However, their measurement in the heart was not feasible because the required homogenous current injection cannot be performed with electrical stimulation. We overcame this problem by optogenetic current injection into all illuminated cardiomyocytes of mouse hearts in different action potential phases. Precisely triggered and patterned illumination enabled measuring Rm and λ, which both were smallest at diastole. Pharmacological and depolarization-induced reduction of inwardly rectifying K+ currents (IK1), gap junction block, and cardiac infarction reduced Ithr, showing the importance of high IK1 density and intact cardiomyocyte coupling for preventing arrhythmia initiation. Combining optogenetic current injection and computer simulations was used to classify pro- and anti-arrhythmic mechanisms based on their effects on Rm and Ithr and allowed to quantify IK1 inward rectification in the intact heart, identifying reduced IK1 rectification as anti-arrhythmic concept.
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Affiliation(s)
- Judith S. Langen
- Institute of Physiology I, Medical Faculty, University of Bonn, Bonn, Germany
| | - Patrick M. Boyle
- Department of Bioengineering, University of Washington, Seattle, Washington, USA
- Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA
- Center for Cardiovascular Biology, University of Washington, Seattle, Washington, USA
- eScience Institute, University of Washington, Seattle, Washington, USA
| | - Daniela Malan
- Institute of Physiology I, Medical Faculty, University of Bonn, Bonn, Germany
| | - Philipp Sasse
- Institute of Physiology I, Medical Faculty, University of Bonn, Bonn, Germany
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5
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Schibalski RS, Shulha AS, Tsao BP, Palygin O, Ilatovskaya DV. The role of polyamine metabolism in cellular function and physiology. Am J Physiol Cell Physiol 2024; 327:C341-C356. [PMID: 38881422 PMCID: PMC11427016 DOI: 10.1152/ajpcell.00074.2024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2024] [Revised: 06/07/2024] [Accepted: 06/07/2024] [Indexed: 06/18/2024]
Abstract
Polyamines are molecules with multiple amino groups that are essential for cellular function. The major polyamines are putrescine, spermidine, spermine, and cadaverine. Polyamines are important for posttranscriptional regulation, autophagy, programmed cell death, proliferation, redox homeostasis, and ion channel function. Their levels are tightly controlled. High levels of polyamines are associated with proliferative pathologies such as cancer, whereas low polyamine levels are observed in aging, and elevated polyamine turnover enhances oxidative stress. Polyamine metabolism is implicated in several pathophysiological processes in the nervous, immune, and cardiovascular systems. Currently, manipulating polyamine levels is under investigation as a potential preventive treatment for several pathologies, including aging, ischemia/reperfusion injury, pulmonary hypertension, and cancer. Although polyamines have been implicated in many intracellular mechanisms, our understanding of these processes remains incomplete and is a topic of ongoing investigation. Here, we discuss the regulation and cellular functions of polyamines, their role in physiology and pathology, and emphasize the current gaps in knowledge and potential future research directions.
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Affiliation(s)
- Ryan S Schibalski
- Department of Physiology, Medical College of Georgia, Augusta University, Augusta, Georgia, United States
| | - Anastasia S Shulha
- Department of Physiology, Medical College of Georgia, Augusta University, Augusta, Georgia, United States
| | - Betty P Tsao
- Division of Rheumatology & Immunology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, United States
| | - Oleg Palygin
- Division of Nephrology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, United States
| | - Daria V Ilatovskaya
- Department of Physiology, Medical College of Georgia, Augusta University, Augusta, Georgia, United States
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6
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Mӓnnikkӧ R, Kullmann DM. Structure-function and pharmacologic aspects of ion channels relevant to neurologic channelopathies. HANDBOOK OF CLINICAL NEUROLOGY 2024; 203:1-23. [PMID: 39174242 DOI: 10.1016/b978-0-323-90820-7.00009-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/24/2024]
Abstract
Ion channels are membrane proteins that allow the passage of ions across the membrane. They characteristically contain a pore where the selectivity of certain ion species is determined and gates that open and close the pore are found. The pore is often connected to additional domains or subunits that regulate its function. Channels are grouped into families based on their selectivity for specific ions and the stimuli that control channel opening and closing, such as voltage or ligands. Ion channels are fundamental to the electrical properties of excitable tissues. Dysfunction of channels can lead to abnormal electrical signaling of neurons and muscle cells, accompanied by clinical manifestations, known as channelopathies. Many naturally occurring toxins target ion channels and affect excitable cells where the channels are expressed. Furthermore, ion channels, as membrane proteins and key regulators of a number of physiologic functions, are an important target for drugs in clinical use. In this chapter, we give a general overview of the classification, genetics and structure-function features of the main ion channel families, and address some pharmacologic aspects relevant to neurologic channelopathies.
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Affiliation(s)
- Roope Mӓnnikkӧ
- Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, University College London, London, United Kingdom.
| | - Dimitri M Kullmann
- Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology, University College London, London, United Kingdom.
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Tu YC, Chao FY, Tsai MF. Mechanisms of dual modulatory effects of spermine on the mitochondrial calcium uniporter complex. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.06.06.543936. [PMID: 37333420 PMCID: PMC10274775 DOI: 10.1101/2023.06.06.543936] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/20/2023]
Abstract
The mitochondrial Ca 2 + uniporter mediates the crucial cellular process of mitochondrial Ca 2 + uptake, which regulates cell bioenergetics, intracellular Ca 2 + signaling, and cell death initiation. The uniporter contains the pore-forming MCU subunit, an EMRE protein that binds to MCU, and the regulatory MICU1 subunit, which can dimerize with MICU1 or MICU2 and under resting cellular [Ca 2 + ] occludes the MCU pore. It has been known for decades that spermine, which is ubiquitously present in animal cells, can enhance mitochondrial Ca 2 + uptake, but the underlying mechanisms remain unclear. Here, we show that spermine exerts dual modulatory effects on the uniporter. In physiological concentrations of spermine, it enhances uniporter activity by breaking the physical interactions between MCU and the MICU1-containing dimers to allow the uniporter to constitutively take up Ca 2 + even in low [Ca 2 + ] conditions. This potentiation effect does not require MICU2 or the EF-hand motifs in MICU1. When [spermine] rises to millimolar levels, it inhibits the uniporter by targeting the pore region in a MICU-independent manner. The MICU1-dependent spermine potentiation mechanism proposed here, along with our previous finding that cardiac mitochondria have very low MICU1, can explain the puzzling observation in the literature that mitochondria in the heart show no response to spermine.
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Affiliation(s)
- Yung-Chi Tu
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045
| | - Fan-Yi Chao
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045
| | - Ming-Feng Tsai
- Department of Physiology and Biophysics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045
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8
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Ríos DS, Malpica-Nieves CJ, Díaz-García A, Eaton MJ, Skatchkov SN. Changes in the Localization of Polyamine Spermidine in the Rat Retina with Age. Biomedicines 2023; 11:1008. [PMID: 37189626 PMCID: PMC10135861 DOI: 10.3390/biomedicines11041008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2023] [Revised: 03/13/2023] [Accepted: 03/15/2023] [Indexed: 05/17/2023] Open
Abstract
Polyamines (PAs) in the nervous system has a key role in regeneration and aging. Therefore, we investigated age-related changes in the expression of PA spermidine (SPD) in the rat retina. Fluorescent immunocytochemistry was used to evaluate the accumulation of SPD in retinae from rats of postnatal days 3, 21, and 120. Glial cells were identified using glutamine synthetase (GS), whereas DAPI, a marker of cell nuclei, was used to differentiate between retinal layers. SPD localization in the retina was strikingly different between neonates and adults. In the neonatal retina (postnatal day 3-P3), SPD is strongly expressed in practically all cell types, including radial glia and neurons. SPD staining showed strong co-localization with the glial marker GS in Müller Cells (MCs) in the outer neuroblast layer. In the weaning period (postnatal day 21-P21), the SPD label was strongly expressed in all MCs, but not in neurons. In early adulthood (postnatal day 120-P120), SPD was localized in MCs only and was co-localized with the glial marker GS. A decline in the expression of PAs in neurons was observed with age while glial cells accumulated SPD after the differentiation stage (P21) and during aging in MC cellular endfoot compartments.
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Affiliation(s)
- David S. Ríos
- College of Science and Health Professions, Universidad Central de Bayamón, Bayamón, PR 00960, USA
| | | | - Amanda Díaz-García
- Department of Physiology, Universidad Central del Caribe, Bayamón, PR 00956, USA
| | - Misty J. Eaton
- Department of Biochemistry, Universidad Central del Caribe, Bayamón, PR 00956, USA
| | - Serguei N. Skatchkov
- Department of Biochemistry, Universidad Central del Caribe, Bayamón, PR 00956, USA
- Department of Physiology, Universidad Central del Caribe, Bayamón, PR 00956, USA
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9
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Li E, Kool W, Woolschot L, van der Heyden MAG. Chronic Propafenone Application Increases Functional K IR2.1 Expression In Vitro. Pharmaceuticals (Basel) 2023; 16:ph16030404. [PMID: 36986503 PMCID: PMC10056987 DOI: 10.3390/ph16030404] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Revised: 02/28/2023] [Accepted: 03/04/2023] [Indexed: 03/30/2023] Open
Abstract
Expression and activity of inwardly rectifying potassium (KIR) channels within the heart are strictly regulated. KIR channels have an important role in shaping cardiac action potentials, having a limited conductance at depolarized potentials but contributing to the final stage of repolarization and resting membrane stability. Impaired KIR2.1 function causes Andersen-Tawil Syndrome (ATS) and is associated with heart failure. Restoring KIR2.1 function by agonists of KIR2.1 (AgoKirs) would be beneficial. The class 1c antiarrhythmic drug propafenone is identified as an AgoKir; however, its long-term effects on KIR2.1 protein expression, subcellular localization, and function are unknown. Propafenone's long-term effect on KIR2.1 expression and its underlying mechanisms in vitro were investigated. KIR2.1-carried currents were measured by single-cell patch-clamp electrophysiology. KIR2.1 protein expression levels were determined by Western blot analysis, whereas conventional immunofluorescence and advanced live-imaging microscopy were used to assess the subcellular localization of KIR2.1 proteins. Acute propafenone treatment at low concentrations supports the ability of propafenone to function as an AgoKir without disturbing KIR2.1 protein handling. Chronic propafenone treatment (at 25-100 times higher concentrations than in the acute treatment) increases KIR2.1 protein expression and KIR2.1 current densities in vitro, which are potentially associated with pre-lysosomal trafficking inhibition.
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Affiliation(s)
- Encan Li
- Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands
| | - Willy Kool
- Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands
| | - Liset Woolschot
- Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands
| | - Marcel A G van der Heyden
- Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands
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Yekefallah M, Rasberry CA, van Aalst EJ, Browning HP, Amani R, Versteeg DB, Wylie BJ. Mutational Insight into Allosteric Regulation of Kir Channel Activity. ACS OMEGA 2022; 7:43621-43634. [PMID: 36506180 PMCID: PMC9730464 DOI: 10.1021/acsomega.2c04456] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Accepted: 11/10/2022] [Indexed: 06/08/2023]
Abstract
Potassium (K+) channels are regulated in part by allosteric communication between the helical bundle crossing, or inner gate, and the selectivity filter, or outer gate. This network is triggered by gating stimuli. In concert, there is an allosteric network which is a conjugated set of interactions which correlate long-range structural rearrangements necessary for channel function. Inward-rectifier K+ (Kir) channels favor inward K+ conductance, are ligand-gated, and help establish resting membrane potentials. KirBac1.1 is a bacterial Kir (KirBac) channel homologous to human Kir (hKir) channels. Additionally, KirBac1.1 is gated by the anionic phospholipid ligand phosphatidylglycerol (PG). In this study, we use site-directed mutagenesis to investigate residues involved in the KirBac1.1 gating mechanism and allosteric network we previously proposed using detailed solid-state NMR (SSNMR) measurements. Using fluorescence-based K+ and sodium (Na+) flux assays, we identified channel mutants with impaired function that do not alter selectivity of the channel. In tandem, we performed coarse grain molecular dynamics simulations, observing changes in PG-KirBac1.1 interactions correlated with mutant channel activity and contacts between the two transmembrane helices and pore helix tied to this behavior. Lipid affinity is closely tied to the proximity of two tryptophan residues on neighboring subunits which lure anionic lipids to a cationic pocket formed by a cluster of arginine residues. Thus, these simulations establish a structural and functional basis for the role of each mutated site in the proposed allosteric network. The experimental and simulated data provide insight into key functional residues involved in gating and lipid allostery of K+ channels. Our findings also have direct implications on the physiology of hKir channels due to conservation of many of the residues identified in this work from KirBac1.1.
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Affiliation(s)
- Maryam Yekefallah
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas79409, United States
| | - Carver A. Rasberry
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas79409, United States
| | - Evan J. van Aalst
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas79409, United States
| | - Holley P. Browning
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas79409, United States
| | - Reza Amani
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas79409, United States
| | - Derek B. Versteeg
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas79409, United States
| | - Benjamin J. Wylie
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas79409, United States
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11
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Rieck J, Skatchkov SN, Derst C, Eaton MJ, Veh RW. Unique Chemistry, Intake, and Metabolism of Polyamines in the Central Nervous System (CNS) and Its Body. Biomolecules 2022; 12:biom12040501. [PMID: 35454090 PMCID: PMC9025450 DOI: 10.3390/biom12040501] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2021] [Revised: 03/16/2022] [Accepted: 03/17/2022] [Indexed: 02/04/2023] Open
Abstract
Polyamines (PAs) are small, versatile molecules with two or more nitrogen-containing positively charged groups and provide widespread biological functions. Most of these aspects are well known and covered by quite a number of excellent surveys. Here, the present review includes novel aspects and questions: (1) It summarizes the role of most natural and some important synthetic PAs. (2) It depicts PA uptake from nutrition and bacterial production in the intestinal system following loss of PAs via defecation. (3) It highlights the discrepancy between the high concentrations of PAs in the gut lumen and their low concentration in the blood plasma and cerebrospinal fluid, while concentrations in cellular cytoplasm are much higher. (4) The present review provides a novel and complete scheme for the biosynthesis of Pas, including glycine, glutamate, proline and others as PA precursors, and provides a hypothesis that the agmatine pathway may rescue putrescine production when ODC knockout seems to be lethal (solving the apparent contradiction in the literature). (5) It summarizes novel data on PA transport in brain glial cells explaining why these cells but not neurons preferentially accumulate PAs. (6) Finally, it provides a novel and complete scheme for PA interconversion, including hypusine, putreanine, and GABA (unique gliotransmitter) as end-products. Altogether, this review can serve as an updated contribution to understanding the PA mystery.
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Affiliation(s)
- Julian Rieck
- Institut für Zell- und Neurobiologie, Centrum 2, Charité—Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany;
| | - Serguei N. Skatchkov
- Department of Physiology, Universidad Central del Caribe, Bayamón, PR 00956, USA
- Department of Biochemistry, Universidad Central del Caribe, Bayamón, PR 00956, USA;
- Correspondence: (S.N.S.); (R.W.V.)
| | - Christian Derst
- Institut für Integrative Neuroanatomie, Centrum 2, Charité—Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany;
| | - Misty J. Eaton
- Department of Biochemistry, Universidad Central del Caribe, Bayamón, PR 00956, USA;
| | - Rüdiger W. Veh
- Institut für Zell- und Neurobiologie, Centrum 2, Charité—Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany;
- Correspondence: (S.N.S.); (R.W.V.)
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12
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Lo J, Forst AL, Warth R, Zdebik AA. EAST/SeSAME Syndrome and Beyond: The Spectrum of Kir4.1- and Kir5.1-Associated Channelopathies. Front Physiol 2022; 13:852674. [PMID: 35370765 PMCID: PMC8965613 DOI: 10.3389/fphys.2022.852674] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Accepted: 02/08/2022] [Indexed: 12/13/2022] Open
Abstract
In 2009, two groups independently linked human mutations in the inwardly rectifying K+ channel Kir4.1 (gene name KCNJ10) to a syndrome affecting the central nervous system (CNS), hearing, and renal tubular salt reabsorption. The autosomal recessive syndrome has been named EAST (epilepsy, ataxia, sensorineural deafness, and renal tubulopathy) or SeSAME syndrome (seizures, sensorineural deafness, ataxia, intellectual disability, and electrolyte imbalance), accordingly. Renal dysfunction in EAST/SeSAME patients results in loss of Na+, K+, and Mg2+ with urine, activation of the renin-angiotensin-aldosterone system, and hypokalemic metabolic alkalosis. Kir4.1 is highly expressed in affected organs: the CNS, inner ear, and kidney. In the kidney, it mostly forms heteromeric channels with Kir5.1 (KCNJ16). Biallelic loss-of-function mutations of Kir5.1 can also have disease significance, but the clinical symptoms differ substantially from those of EAST/SeSAME syndrome: although sensorineural hearing loss and hypokalemia are replicated, there is no alkalosis, but rather acidosis of variable severity; in contrast to EAST/SeSAME syndrome, the CNS is unaffected. This review provides a framework for understanding some of these differences and will guide the reader through the growing literature on Kir4.1 and Kir5.1, discussing the complex disease mechanisms and the variable expression of disease symptoms from a molecular and systems physiology perspective. Knowledge of the pathophysiology of these diseases and their multifaceted clinical spectrum is an important prerequisite for making the correct diagnosis and forms the basis for personalized therapies.
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Affiliation(s)
- Jacky Lo
- Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom
| | - Anna-Lena Forst
- Medical Cell Biology, Institute of Physiology, University of Regensburg, Regensburg, Germany
| | - Richard Warth
- Medical Cell Biology, Institute of Physiology, University of Regensburg, Regensburg, Germany
| | - Anselm A. Zdebik
- Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom
- Centre for Nephrology, University College London, London, United Kingdom
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13
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Lei CL, Fabbri A, Whittaker DG, Clerx M, Windley MJ, Hill AP, Mirams GR, de Boer TP. A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer. Wellcome Open Res 2021; 5:152. [PMID: 34805549 PMCID: PMC8591515 DOI: 10.12688/wellcomeopenres.15968.2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/28/2021] [Indexed: 11/20/2022] Open
Abstract
Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F
− containing internal solution together with a Ca
2+ containing external solution — so that a CaF
2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F
− ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF
2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.
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Affiliation(s)
- Chon Lok Lei
- Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau, China.,Department of Biomedical Sciences, Faculty of Health Sciences, University of Macau, Macau, China.,Department of Computer Science, University of Oxford, Oxford, Oxfordshire, OX1 3QD, UK
| | - Alan Fabbri
- Department of Medical Physiology, Division of Heart and Lungs, University Medical Centre Utrecht, Utrecht, 3584 CX, The Netherlands
| | - Dominic G Whittaker
- Centre for Mathematical Medicine & Biology, School of Mathematical Sciences, University of Nottingham, Nottingham, Nottinghamshire, NG7 2RD, UK
| | - Michael Clerx
- Department of Computer Science, University of Oxford, Oxford, Oxfordshire, OX1 3QD, UK
| | - Monique J Windley
- Molecular Cardiology & Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, 2010, Australia.,St Vincent's Clinical School, UNSW Sydney, Darlinghurst, New South Wales, 2010, Australia
| | - Adam P Hill
- Molecular Cardiology & Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, 2010, Australia.,St Vincent's Clinical School, UNSW Sydney, Darlinghurst, New South Wales, 2010, Australia
| | - Gary R Mirams
- Centre for Mathematical Medicine & Biology, School of Mathematical Sciences, University of Nottingham, Nottingham, Nottinghamshire, NG7 2RD, UK
| | - Teun P de Boer
- Department of Medical Physiology, Division of Heart and Lungs, University Medical Centre Utrecht, Utrecht, 3584 CX, The Netherlands
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14
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Akyuz E, Koklu B, Uner A, Angelopoulou E, Paudel YN. Envisioning the role of inwardly rectifying potassium (Kir) channel in epilepsy. J Neurosci Res 2021; 100:413-443. [PMID: 34713909 DOI: 10.1002/jnr.24985] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2021] [Revised: 09/23/2021] [Accepted: 10/01/2021] [Indexed: 01/29/2023]
Abstract
Epilepsy is a devastating neurological disorder characterized by recurrent seizures attributed to the disruption of the dynamic excitatory and inhibitory balance in the brain. Epilepsy has emerged as a global health concern affecting about 70 million people worldwide. Despite recent advances in pre-clinical and clinical research, its etiopathogenesis remains obscure, and there are still no treatment strategies modifying disease progression. Although the precise molecular mechanisms underlying epileptogenesis have not been clarified yet, the role of ion channels as regulators of cellular excitability has increasingly gained attention. In this regard, emerging evidence highlights the potential implication of inwardly rectifying potassium (Kir) channels in epileptogenesis. Kir channels consist of seven different subfamilies (Kir1-Kir7), and they are highly expressed in both neuronal and glial cells in the central nervous system. These channels control the cell volume and excitability. In this review, we discuss preclinical and clinical evidence on the role of the several subfamilies of Kir channels in epileptogenesis, aiming to shed more light on the pathogenesis of this disorder and pave the way for future novel therapeutic approaches.
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Affiliation(s)
- Enes Akyuz
- Faculty of International Medicine, Department of Biophysics, University of Health Sciences, Istanbul, Turkey
| | - Betul Koklu
- Faculty of Medicine, Namık Kemal University, Tekirdağ, Turkey
| | - Arda Uner
- Faculty of Medicine, Yozgat Bozok University, Yozgat, Turkey
| | - Efthalia Angelopoulou
- Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | - Yam Nath Paudel
- Neuropharmacology Research Strength, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Malaysia
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15
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Control of Biophysical and Pharmacological Properties of Potassium Channels by Ancillary Subunits. Handb Exp Pharmacol 2021; 267:445-480. [PMID: 34247280 DOI: 10.1007/164_2021_512] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Potassium channels facilitate and regulate physiological processes as diverse as electrical signaling, ion, solute and hormone secretion, fluid homeostasis, hearing, pain sensation, muscular contraction, and the heartbeat. Potassium channels are each formed by either a tetramer or dimer of pore-forming α subunits that co-assemble to create a multimer with a K+-selective pore that in most cases is capable of functioning as a discrete unit to pass K+ ions across the cell membrane. The reality in vivo, however, is that the potassium channel α subunit multimers co-assemble with ancillary subunits to serve specific physiological functions. The ancillary subunits impart specific physiological properties that are often required for a particular activity in vivo; in addition, ancillary subunit interaction often alters the pharmacology of the resultant complex. In this chapter the modes of action of ancillary subunits on K+ channel physiology and pharmacology are described and categorized into various mechanistic classes.
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16
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Weaver CD, Denton JS. Next-generation inward rectifier potassium channel modulators: discovery and molecular pharmacology. Am J Physiol Cell Physiol 2021; 320:C1125-C1140. [PMID: 33826405 DOI: 10.1152/ajpcell.00548.2020] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Inward rectifying potassium (Kir) channels play important roles in both excitable and nonexcitable cells of various organ systems and could represent valuable new drug targets for cardiovascular, metabolic, immune, and neurological diseases. In nonexcitable epithelial cells of the kidney tubule, for example, Kir1.1 (KCNJ1) and Kir4.1 (KCNJ10) are linked to sodium reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively, and have been explored as novel-mechanism diuretic targets for managing hypertension and edema. G protein-coupled Kir channels (Kir3) channels expressed in the central nervous system are critical effectors of numerous signal transduction pathways underlying analgesia, addiction, and respiratory-depressive effects of opioids. The historical dearth of pharmacological tool compounds for exploring the therapeutic potential of Kir channels has led to a molecular target-based approach using high-throughput screen (HTS) of small-molecule libraries and medicinal chemistry to develop "next-generation" Kir channel modulators that are both potent and specific for their targets. In this article, we review recent efforts focused specifically on discovery and improvement of target-selective molecular probes. The reader is introduced to fluorescence-based thallium flux assays that have enabled much of this work and then provided with an overview of progress made toward developing modulators of Kir1.1 (VU590, VU591), Kir2.x (ML133), Kir3.X (ML297, GAT1508, GiGA1, VU059331), Kir4.1 (VU0134992), and Kir7.1 (ML418). We discuss what is known about the small molecules' molecular mechanisms of action, in vitro and in vivo pharmacology, and then close with our view of what critical work remains to be done.
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Affiliation(s)
- C David Weaver
- Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.,Department of Chemistry, Vanderbilt University, Nashville, Tennessee.,Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee
| | - Jerod S Denton
- Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.,Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee.,Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee
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17
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Ozturk N, Uslu S, Ozdemir S. Diabetes-induced changes in cardiac voltage-gated ion channels. World J Diabetes 2021; 12:1-18. [PMID: 33520105 PMCID: PMC7807254 DOI: 10.4239/wjd.v12.i1.1] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2020] [Revised: 11/05/2020] [Accepted: 11/13/2020] [Indexed: 02/06/2023] Open
Abstract
Diabetes mellitus affects the heart through various mechanisms such as microvascular defects, metabolic abnormalities, autonomic dysfunction and incompatible immune response. Furthermore, it can also cause functional and structural changes in the myocardium by a disease known as diabetic cardiomyopathy (DCM) in the absence of coronary artery disease. As DCM progresses it causes electrical remodeling of the heart, left ventricular dysfunction and heart failure. Electrophysiological changes in the diabetic heart contribute significantly to the incidence of arrhythmias and sudden cardiac death in diabetes mellitus patients. In recent studies, significant changes in repolarizing K+ currents, Na+ currents and L-type Ca2+ currents along with impaired Ca2+ homeostasis and defective contractile function have been identified in the diabetic heart. In addition, insulin levels and other trophic factors change significantly to maintain the ionic channel expression in diabetic patients. There are many diagnostic tools and management options for DCM, but it is difficult to detect its development and to effectively prevent its progress. In this review, diabetes-associated alterations in voltage-sensitive cardiac ion channels are comprehensively assessed to understand their potential role in the pathophysiology and pathogenesis of DCM.
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Affiliation(s)
- Nihal Ozturk
- Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Serkan Uslu
- Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
| | - Semir Ozdemir
- Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya 07058, Turkey
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18
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Zhang Y, Wang Y, Meng L, Huang Q, Zhu Y, Cui W, Cheng Y, Liu R. Targeted micelles with chemotherapeutics and gene drugs to inhibit the G1/S and G2/M mitotic cycle of prostate cancer. J Nanobiotechnology 2021; 19:17. [PMID: 33422073 PMCID: PMC7796562 DOI: 10.1186/s12951-020-00756-6] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2020] [Accepted: 12/15/2020] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND Chemotherapy and gene therapy are used in clinical practice for the treatment of castration-resistant prostate cancer. However, the poor efficiency of drug delivery and serious systemic side effects remain an obstacle to wider application of these drugs. Herein, we report newly designed PEO-PCL micelles that were self-assembled and modified by spermine ligand, DCL ligand and TAT peptide to carry docetaxel and anti-nucleostemin siRNA. RESULTS The particle size of the micelles was 42 nm, the zeta potential increased from - 12.8 to 15 mV after grafting with spermine, and the optimal N/P ratio was 25:1. Cellular MTT experiments suggested that introduction of the DCL ligand resulted in high toxicity toward PSMA-positive cells and that the TAT peptide enhanced the effect. The expression of nucleostemin was significantly suppressed in vitro and in vivo, and the tumour-inhibition experiment showed that the dual-drug delivery system suppressed CRPC tumour proliferation. CONCLUSIONS This targeted drug delivery system inhibited the G1/S and G2/M mitotic cycle via synergistic interaction of chemotherapeutics and gene drugs.
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Affiliation(s)
- Yiran Zhang
- Tianjin Institute of Urology & Department of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, People's Republic of China
- Department of Interventional Radiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, No. 600, Yishan Road, Shanghai, 200233, People's Republic of China
- Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin 2nd Road, Shanghai, 200025, People's Republic of China
| | - Yanming Wang
- Tianjin Key Laboratory of Molecular Drug Research, College of Pharmacy, Nankai University College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, People's Republic of China
| | - Li Meng
- Tianjin Key Laboratory of Molecular Drug Research, College of Pharmacy, Nankai University College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, People's Republic of China
| | - Qingqing Huang
- Tianjin Key Laboratory of Molecular Drug Research, College of Pharmacy, Nankai University College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, People's Republic of China
| | - Yueqi Zhu
- Department of Interventional Radiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, No. 600, Yishan Road, Shanghai, 200233, People's Republic of China
| | - Wenguo Cui
- Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197 Ruijin 2nd Road, Shanghai, 200025, People's Republic of China.
| | - Yingsheng Cheng
- Department of Interventional Radiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, No. 600, Yishan Road, Shanghai, 200233, People's Republic of China.
| | - Ranlu Liu
- Tianjin Institute of Urology & Department of Urology, The Second Hospital of Tianjin Medical University, 23 Pingjiang Road, Hexi District, Tianjin, 300211, People's Republic of China.
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19
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Borcik CG, Versteeg DB, Amani R, Yekefallah M, Khan NH, Wylie BJ. The Lipid Activation Mechanism of a Transmembrane Potassium Channel. J Am Chem Soc 2020; 142:14102-14116. [PMID: 32702990 DOI: 10.1021/jacs.0c01991] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Membrane proteins and lipids coevolved to yield unique coregulatory mechanisms. Inward-rectifier K+ (Kir) channels are often activated by anionic lipids endemic to their native membranes and require accessible water along their K+ conductance pathway. To better understand Kir channel activation, we target multiple mutants of the Kir channel KirBac1.1 via solid-state nuclear magnetic resonance (SSNMR) spectroscopy, potassium efflux assays, and Förster resonance energy transfer (FRET) measurements. In the I131C stability mutant (SM), we observe an open-active channel in the presence of anionic lipids with greater activity upon addition of cardiolipin (CL). The introduction of three R to Q mutations (R49/151/153Q (triple Q mutant, TQ)) renders the protein inactive within the same activating lipid environment. Our SSNMR experiments reveal a stark reduction of lipid-protein interactions in the TQ mutant explaining the dramatic loss of channel activity. Water-edited SSNMR experiments further determined the TQ mutant possesses greater overall solvent exposure in comparison to wild-type but with reduced water accessibility along the ion conduction pathway, consistent with the closed state of the channel. These experiments also suggest water is proximal to the selectivity filter of KirBac1.1 in the open-activated state but that it may not directly enter the selectivity filter. Our findings suggest lipid binding initiates a concerted rotation of the cytoplasmic domain subunits, which is stabilized by multiple intersubunit salt bridges. This action buries ionic side chains away from the bulk water, while allowing water greater access to the K+ conduction pathway. This work highlights universal membrane protein motifs, including lipid-protein interactions, domain rearrangement, and water-mediated diffusion mechanisms.
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Affiliation(s)
- Collin G Borcik
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, United States
| | - Derek B Versteeg
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, United States
| | - Reza Amani
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, United States
| | - Maryam Yekefallah
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, United States
| | - Nazmul H Khan
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, United States
| | - Benjamin J Wylie
- Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, United States
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20
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Chen X, Bründl M, Friesacher T, Stary-Weinzinger A. Computational Insights Into Voltage Dependence of Polyamine Block in a Strong Inwardly Rectifying K + Channel. Front Pharmacol 2020; 11:721. [PMID: 32499707 PMCID: PMC7243266 DOI: 10.3389/fphar.2020.00721] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2019] [Accepted: 04/30/2020] [Indexed: 12/30/2022] Open
Abstract
Inwardly rectifying potassium (KIR) channels play important roles in controlling cellular excitability and K+ ion homeostasis. Under physiological conditions, KIR channels allow large K+ influx at potentials negative to the equilibrium potential of K+ but permit little outward current at potentials positive to the equilibrium potential of K+, due to voltage dependent block of outward K+ flux by cytoplasmic polyamines. These polycationic molecules enter the KIR channel pore from the intracellular side. They block K+ ion movement through the channel at depolarized potentials, thereby ensuring, for instance, the long plateau phase of the cardiac action potential. Key questions concerning how deeply these charged molecules migrate into the pore and how the steep voltage dependence arises remain unclear. Recent MD simulations on GIRK2 (=Kir3.2) crystal structures have provided unprecedented details concerning the conduction mechanism of a KIR channel. Here, we use MD simulations with applied field to provide detailed insights into voltage dependent block of putrescine, using the conductive state of the strong inwardly rectifying K+ channel GIRK2 as starting point. Our µs long simulations elucidate details about binding sites of putrescine in the pore and suggest that voltage-dependent rectification arises from a dual mechanism.
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21
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Kuß J, Stallmeyer B, Goldstein M, Rinné S, Pees C, Zumhagen S, Seebohm G, Decher N, Pott L, Kienitz MC, Schulze-Bahr E. Familial Sinus Node Disease Caused by a Gain of GIRK (G-Protein Activated Inwardly Rectifying K + Channel) Channel Function. CIRCULATION-GENOMIC AND PRECISION MEDICINE 2020; 12:e002238. [PMID: 30645171 DOI: 10.1161/circgen.118.002238] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
BACKGROUND Inherited forms of sinus node dysfunction (SND) clinically include bradycardia, sinus arrest, and chronotropic incompetence and may serve as disease models to understand sinus node physiology and impulse generation. Recently, a gain-of-function mutation in the G-protein gene GNB2 led to enhanced activation of the GIRK (G-protein activated inwardly rectifying K+ channel). Thus, human cardiac GIRK channels are important for heart rate regulation and subsequently, genes encoding their subunits Kir3.1 and Kir3.4 ( KCNJ3 and KCNJ5) are potential candidates for inherited SND in human. METHODS We performed a combined approach of targeted sequencing of KCNJ3 and KCNJ5 in 52 patients with idiopathic SND and subsequent whole exome sequencing of additional family members in a genetically affected patient. A putative novel disease-associated gene variant was functionally analyzed by voltage-clamp experiments using various heterologous cell expression systems (Xenopus oocytes, CHO cells, and rat atrial cardiomyocytes). RESULTS In a 3-generation family with SND we identified a novel variant in KCNJ5 which leads to an amino acid substitution (p.Trp101Cys) in the first transmembrane domain of the Kir3.4 subunit of the cardiac GIRK channel. The identified variant cosegregated with the disease in the family and was absent in the Exome Variant Server and Exome Aggregation Consortium databases. Expression of mutant Kir3.4 (±native Kir3.1) in different heterologous cell expression systems resulted in increased GIRK currents ( IK,ACh) and a reduced inward rectification which was not compensated by intracellular spermidine. Moreover, in silico modeling of heterotetrameric mutant GIRK channels indicates a structurally altered binding site for spermine. CONCLUSIONS For the first time, an inherited gain-of-function mutation in the human GIRK3.4 causes familial human SND. The increased activity of GIRK channels is likely to lead to a sustained hyperpolarization of pacemaker cells and thereby reduces heart rate. Modulation of human GIRK channels may pave a way for further treatment of cardiac pacemaking.
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Affiliation(s)
- Johanna Kuß
- Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Germany (J.K., B.S., S.Z., G.S., E.S.-B.)
| | - Birgit Stallmeyer
- Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Germany (J.K., B.S., S.Z., G.S., E.S.-B.)
| | - Matthias Goldstein
- Institute of Physiology and Pathophysiology, Vegetative Physiology, University of Marburg, Germany (M.G., S.R., N.D.)
| | - Susanne Rinné
- Institute of Physiology and Pathophysiology, Vegetative Physiology, University of Marburg, Germany (M.G., S.R., N.D.)
| | - Christiane Pees
- Department of Pediatric Cardiology, University Children's Hospital Vienna, Austria (C.P.)
| | - Sven Zumhagen
- Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Germany (J.K., B.S., S.Z., G.S., E.S.-B.)
| | - Guiscard Seebohm
- Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Germany (J.K., B.S., S.Z., G.S., E.S.-B.)
| | - Niels Decher
- Institute of Physiology and Pathophysiology, Vegetative Physiology, University of Marburg, Germany (M.G., S.R., N.D.)
| | - Lutz Pott
- Department of Cardiovascular Medicine, Institute of Physiology, Ruhr-University Bochum, Germany (L.P., M.-C.K.)
| | - Marie-Cécile Kienitz
- Department of Cardiovascular Medicine, Institute of Physiology, Ruhr-University Bochum, Germany (L.P., M.-C.K.)
| | - Eric Schulze-Bahr
- Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Germany (J.K., B.S., S.Z., G.S., E.S.-B.)
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22
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Upregulation of Potassium Voltage-Gated Channel Subfamily J Member 2 Levels in the Lungs of Patients with Idiopathic Pulmonary Fibrosis. Can Respir J 2020; 2020:3406530. [PMID: 32184906 PMCID: PMC7061125 DOI: 10.1155/2020/3406530] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2019] [Accepted: 01/23/2020] [Indexed: 01/23/2023] Open
Abstract
Background Fibroblast dysfunction is the main pathogenic mechanism underpinning idiopathic pulmonary fibrosis (IPF). Potassium voltage-gated channel subfamily J member 2 (KCNJ2) plays critical roles in the proliferation of myofibroblasts and in the development of cardiac fibrosis. Objectives This study aimed to evaluate the role of KCNJ2 in IPF. Methods KCNJ2 mRNA expression was measured using real-time PCR in fibroblasts from IPF patients and normal controls (NCs). Protein concentrations were measured by ELISA in bronchoalveolar lavage (BAL) fluid obtained from NCs (n = 30), IPF (n = 30), IPF (n = 30), IPF (n = 30), IPF (n = 30), IPF ( Results KCNJ2 mRNA expression was measured using real-time PCR in fibroblasts from IPF patients and normal controls (NCs). Protein concentrations were measured by ELISA in bronchoalveolar lavage (BAL) fluid obtained from NCs (n = 30), IPF (n = 30), IPF (p < 0.001). KCNJ2 protein levels in BAL fluid were significantly higher in IPF (6.587 [1.441–26.01] ng/mL) than in NCs (0.084 [0.00–0.260] ng/mL, p < 0.001). KCNJ2 protein levels in BAL fluid were significantly higher in IPF (6.587 [1.441–26.01] ng/mL) than in NCs (0.084 [0.00–0.260] ng/mL, p < 0.001). KCNJ2 protein levels in BAL fluid were significantly higher in IPF (6.587 [1.441–26.01] ng/mL) than in NCs (0.084 [0.00–0.260] ng/mL, p < 0.001). KCNJ2 protein levels in BAL fluid were significantly higher in IPF (6.587 [1.441–26.01] ng/mL) than in NCs (0.084 [0.00–0.260] ng/mL, p < 0.001). KCNJ2 protein levels in BAL fluid were significantly higher in IPF (6.587 [1.441–26.01] ng/mL) than in NCs (0.084 [0.00–0.260] ng/mL, Conclusion KCNJ2 may participate in the development of IPF, and its protein level may be a candidate diagnostic and therapeutic molecule for IPF.
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23
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Structure-Function Relationship and Physiological Roles of Transient Receptor Potential Canonical (TRPC) 4 and 5 Channels. Cells 2019; 9:cells9010073. [PMID: 31892199 PMCID: PMC7017149 DOI: 10.3390/cells9010073] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2019] [Revised: 12/24/2019] [Accepted: 12/24/2019] [Indexed: 12/11/2022] Open
Abstract
The study of the structure–function relationship of ion channels has been one of the most challenging goals in contemporary physiology. Revelation of the three-dimensional (3D) structure of ion channels has facilitated our understanding of many of the submolecular mechanisms inside ion channels, such as selective permeability, voltage dependency, agonist binding, and inter-subunit multimerization. Identifying the structure–function relationship of the ion channels is clinically important as well since only such knowledge can imbue potential therapeutics with practical possibilities. In a sense, recent advances in the understanding of the structure–relationship of transient receptor potential canonical (TRPC) channels look promising since human TRPC channels are calcium-permeable, non-selective cation channels expressed in many tissues such as the gastrointestinal (GI) tract, kidney, heart, vasculature, and brain. TRPC channels are known to regulate GI contractility and motility, pulmonary hypertension, right ventricular hypertrophy, podocyte injury, seizure, fear, anxiety-like behavior, and many others. In this article, we tried to elaborate recent findings of Cryo-EM (cryogenic-electron microscopy) based structural information of TRPC 4 and 5 channels and domain-specific functions of the channel, such as G-protein mediated activation mechanism, extracellular modification of the channel, homo/hetero-tetramerization, and pharmacological gating mechanisms.
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24
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Shinohara S, Okamoto T, Motose H, Takahashi T. Salt hypersensitivity is associated with excessive xylem development in a thermospermine-deficient mutant of Arabidopsis thaliana. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2019; 100:374-383. [PMID: 31257654 DOI: 10.1111/tpj.14448] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Revised: 06/13/2019] [Accepted: 06/18/2019] [Indexed: 05/21/2023]
Abstract
In Arabidopsis, spermine is produced in most tissues and has been implicated in stress response, while its structural isomer thermospermine is only in xylem precursor cells. Studies on acaulis5 (acl5), a mutant defective in the biosynthesis of thermospermine, have revealed that thermospermine plays a repressive role in xylem development through enhancement of mRNA translation of the SAC51 family. In contrast, the pao5 mutant defective in the degradation of thermospermine has high levels of thermospermine and shows increased salt tolerance, suggesting a role of thermospermine in salt stress response. Here we compared acl5 with a mutant of spermine synthase, spms, in terms of abiotic stress tolerance and found that acl5 was much more sensitive to sodium than the wild-type and spms. A double-mutant of acl5 and sac51-d, which suppresses the excessive xylem phenotype of acl5, recovered normal sensitivity, while a quadruple T-DNA insertion mutant of the SAC51 family, which has an increased thermospermine level but shows excessive xylem development, showed increased salt sensitivity, unlike pao5. Together with the result that the salt tolerance of both wild-type and acl5 seedlings was improved by long-term treatment with thermospermine, we suggest a correlation of the salt tolerance with reduced xylem development rather than with the thermospermine level. We further found that the mutants containing high thermospermine levels showed increased tolerance to drought and heat stress, suggesting another role of thermospermine that may be common with that of spermine and secondary to that in restricting excess xylem development associated with salt hypersensitivity.
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Affiliation(s)
- Shiori Shinohara
- Division of Earth, Life, and Molecular Sciences, Graduate School of Natural Science and Technology, Okayama University, 700-8530, Okayama, Japan
| | - Takashi Okamoto
- Division of Earth, Life, and Molecular Sciences, Graduate School of Natural Science and Technology, Okayama University, 700-8530, Okayama, Japan
| | - Hiroyasu Motose
- Division of Earth, Life, and Molecular Sciences, Graduate School of Natural Science and Technology, Okayama University, 700-8530, Okayama, Japan
| | - Taku Takahashi
- Division of Earth, Life, and Molecular Sciences, Graduate School of Natural Science and Technology, Okayama University, 700-8530, Okayama, Japan
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25
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Selakovic V, Arsenijevic L, Jovanovic M, Sivcev S, Jovanovic N, Leontijevic M, Stojanovic M, Radenkovic M, Andjus P, Radenovic L. Functional and pharmacological analysis of agmatine administration in different cerebral ischemia animal models. Brain Res Bull 2019; 146:201-212. [PMID: 30641119 DOI: 10.1016/j.brainresbull.2019.01.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2018] [Revised: 12/18/2018] [Accepted: 01/03/2019] [Indexed: 11/17/2022]
Abstract
Agmatine (AgM, 100 mg/kg i.p.) effect was tested in parallel at two animal models of cerebral ischemia - rat MCAO model (60'/24 h, 60'/48 h, 90'/24 h, 90'/48 h) and gerbil global ischemia (10') model, administrated 5 min after reperfusion. Aim was to evaluate AgM effect on functional outcome 24 and 48 h after MCAO on neurological and sensor-motor function, and coordination in rats. AgM administration significantly reduced infarct volume, improved neurological score and improved post-ischemic oxidative status. Results of behavioral tests (cylinder test, beam walking test, and adhesive removal test) have shown very effective functional recovery after AgM administration. Efficiency of AgM administration in gerbils was observed in forebrain cortex, striatum, hippocampus, and cerebellum at the level of each examined oxidative stress parameter (nitric oxide level, superoxide production, superoxide dismutase activity, and index of lipid peroxidation) measured in four different time points starting at 3 h up to 48 h after reperfusion. The highest levels were obtained 6 h after the insult. The most sensitive oxidative stress parameter to AgM was nitric oxide. Additionally, we performed pharmacological analysis of AgM on rat isolated common carotid arteries. The findings imply that mixed population of potassium channels located on the smooth muscle cells was involved in common carotid artery response to AgM, with predominance of inward rectifying K+ channels. In our comparative experimental approach, judged by behavioral, biochemical, as well as pharmacological data, the AgM administration showed an effective reduction of ischemic neurological damage and oxidative stress, hence indicating a direction towards improving post-stroke recovery.
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Affiliation(s)
- V Selakovic
- Institute of Medical Research, Medical Faculty Military Medical Academy, University of Defense, Serbia
| | | | - M Jovanovic
- Faculty of Biology, University of Belgrade, Serbia
| | - S Sivcev
- Faculty of Biology, University of Belgrade, Serbia
| | - N Jovanovic
- Faculty of Biology, University of Belgrade, Serbia
| | | | - M Stojanovic
- Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, Serbia
| | - M Radenkovic
- Department of Pharmacology, Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Belgrade, Serbia
| | - P Andjus
- Faculty of Biology, University of Belgrade, Serbia
| | - L Radenovic
- Faculty of Biology, University of Belgrade, Serbia.
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26
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Abstract
Potassium channels that exhibit the property of inward rectification (Kir channels) are present in most cells. Cloning of the first Kir channel genes 25 years ago led to recognition that inward rectification is a consequence of voltage-dependent block by cytoplasmic polyamines, which are also ubiquitously present in animal cells. Upon cellular depolarization, these polycationic metabolites enter the Kir channel pore from the intracellular side, blocking the movement of K+ ions through the channel. As a consequence, high K+ conductance at rest can provide very stable negative resting potentials, but polyamine-mediated blockade at depolarized potentials ensures, for instance, the long plateau phase of the cardiac action potential, an essential feature for a stable cardiac rhythm. Despite much investigation of the polyamine block, where exactly polyamines get to within the Kir channel pore and how the steep voltage dependence arises remain unclear. This Minireview will summarize current understanding of the relevance and molecular mechanisms of polyamine block and offer some ideas to try to help resolve the fundamental issue of the voltage dependence of polyamine block.
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Affiliation(s)
- Colin G Nichols
- From the Department of Cell Biology and Physiology, Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, Saint Louis, Missouri 63110
| | - Sun-Joo Lee
- From the Department of Cell Biology and Physiology, Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, Saint Louis, Missouri 63110
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27
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Binda A, Rivolta I, Villa C, Chisci E, Beghi M, Cornaggia CM, Giovannoni R, Combi R. A Novel KCNJ2 Mutation Identified in an Autistic Proband Affects the Single Channel Properties of Kir2.1. Front Cell Neurosci 2018; 12:76. [PMID: 29615871 PMCID: PMC5869910 DOI: 10.3389/fncel.2018.00076] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2017] [Accepted: 03/06/2018] [Indexed: 11/29/2022] Open
Abstract
Inwardly rectifying potassium channels (Kir) have been historically associated to several cardiovascular disorders. In particular, loss-of-function mutations in the Kir2.1 channel have been reported in cases affected by Andersen-Tawil syndrome while gain-of-function mutations in the same channel cause the short QT3 syndrome. Recently, a missense mutation in Kir2.1, as well as mutations in the Kir4.1, were reported to be involved in autism spectrum disorders (ASDs) suggesting a role of potassium channels in these diseases and introducing the idea of the existence of K+ channel ASDs. Here, we report the identification in an Italian affected family of a novel missense mutation (p.Phe58Ser) in the KCNJ2 gene detected in heterozygosity in a proband affected by autism and borderline for short QT syndrome type 3. The mutation is located in the N-terminal region of the gene coding for the Kir2.1 channel and in particular in a very conserved domain. In vitro assays demonstrated that this mutation results in an increase of the channel conductance and in its open probability. This gain-of-function of the protein is consistent with the autistic phenotype, which is normally associated to an altered neuronal excitability.
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Affiliation(s)
- Anna Binda
- School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
| | - Ilaria Rivolta
- School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
| | - Chiara Villa
- School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
| | - Elisa Chisci
- School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
| | | | - Cesare M Cornaggia
- School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
| | - Roberto Giovannoni
- School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
| | - Romina Combi
- School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy
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28
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Jackson WF. Boosting the signal: Endothelial inward rectifier K + channels. Microcirculation 2018; 24. [PMID: 27652592 DOI: 10.1111/micc.12319] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2016] [Accepted: 09/12/2016] [Indexed: 12/19/2022]
Abstract
Endothelial cells express a diverse array of ion channels including members of the strong inward rectifier family composed of KIR 2 subunits. These two-membrane spanning domain channels are modulated by their lipid environment, and exist in macromolecular signaling complexes with receptors, protein kinases and other ion channels. Inward rectifier K+ channel (KIR ) currents display a region of negative slope conductance at membrane potentials positive to the K+ equilibrium potential that allows outward current through the channels to be activated by membrane hyperpolarization, permitting KIR to amplify hyperpolarization induced by other K+ channels and ion transporters. Increases in extracellular K+ concentration activate KIR allowing them to sense extracellular K+ concentration and transduce this change into membrane hyperpolarization. These properties position KIR to participate in the mechanism of action of hyperpolarizing vasodilators and contribute to cell-cell conduction of hyperpolarization along the wall of microvessels. The expression of KIR in capillaries in electrically active tissues may allow KIR to sense extracellular K+ , contributing to functional hyperemia. Understanding the regulation of expression and function of microvascular endothelial KIR will improve our understanding of the control of blood flow in the microcirculation in health and disease and may provide new targets for the development of therapeutics in the future.
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Affiliation(s)
- William F Jackson
- Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA
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29
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Aréchiga-Figueroa IA, Marmolejo-Murillo LG, Cui M, Delgado-Ramírez M, van der Heyden MAG, Sánchez-Chapula JA, Rodríguez-Menchaca AA. High-potency block of Kir4.1 channels by pentamidine: Molecular basis. Eur J Pharmacol 2017; 815:56-63. [PMID: 28993158 DOI: 10.1016/j.ejphar.2017.10.009] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2017] [Revised: 10/03/2017] [Accepted: 10/05/2017] [Indexed: 02/06/2023]
Abstract
Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and contribute to a wide range of physiological processes. Kir4.1 channel expression is found in the brain, inner ear, eye, and kidney. Loss-of-function mutations in the pore-forming Kir4.1 subunit cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (SeSAME/EST syndrome). Despite its importance in physiological and pathological conditions, pharmacological research of Kir4.1 is limited. Here, we characterized the effect of pentamidine on Kir4.1 channels using electrophysiology, mutagenesis and computational methods. Pentamidine potently inhibited Kir4.1 channels when applied to the cytoplasmic side under inside-out patch clamp configuration (IC50 = 97nM). The block was voltage dependent. Molecular modeling predicted the binding of pentamidine to the transmembrane pore region of Kir4.1 at aminoacids T127, T128 and E158. Mutation of each of these residues reduced the potency of pentamidine to block Kir4.1 channels. A pentamidine analog (PA-6) inhibited Kir4.1 with similar potency (IC50 = 132nM). Overall, this study shows that pentamidine blocks Kir4.1 channels interacting with threonine and glutamate residues in the transmembrane pore region. These results can be useful to design novel compounds with major potency and specificity over Kir4.1 channels.
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Affiliation(s)
- Iván A Aréchiga-Figueroa
- CONACYT, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP, Mexico
| | | | - Meng Cui
- Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA
| | - Mayra Delgado-Ramírez
- Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, SLP, Mexico
| | - Marcel A G van der Heyden
- Department of Medical Physiology, Division Heart & Lungs, University Medical Center Utrecht, Utrecht, The Netherlands
| | - José A Sánchez-Chapula
- Centro Universitario de Investigaciones Biomédicas, Universidad de Colima, Colima, Col, Mexico
| | - Aldo A Rodríguez-Menchaca
- Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, SLP, Mexico.
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30
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Jeevaratnam K, Chadda KR, Huang CLH, Camm AJ. Cardiac Potassium Channels: Physiological Insights for Targeted Therapy. J Cardiovasc Pharmacol Ther 2017; 23:119-129. [PMID: 28946759 PMCID: PMC5808825 DOI: 10.1177/1074248417729880] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The development of novel drugs specifically directed at the ion channels underlying particular features of cardiac action potential (AP) initiation, recovery, and refractoriness would contribute to an optimized approach to antiarrhythmic therapy that minimizes potential cardiac and extracardiac toxicity. Of these, K+ channels contribute numerous and diverse currents with specific actions on different phases in the time course of AP repolarization. These features and their site-specific distribution make particular K+ channel types attractive therapeutic targets for the development of pharmacological agents attempting antiarrhythmic therapy in conditions such as atrial fibrillation. However, progress in the development of such temporally and spatially selective antiarrhythmic drugs against particular ion channels has been relatively limited, particularly in view of our incomplete understanding of the complex physiological roles and interactions of the various ionic currents. This review summarizes the physiological properties of the main cardiac potassium channels and the way in which they modulate cardiac electrical activity and then critiques a number of available potential antiarrhythmic drugs directed at them.
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Affiliation(s)
- Kamalan Jeevaratnam
- 1 Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.,2 School of Medicine, Perdana University-Royal College of Surgeons Ireland, Serdang, Selangor Darul Ehsan, Malaysia
| | - Karan R Chadda
- 1 Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom.,3 Physiological Laboratory, University of Cambridge, Cambridge, United Kingdom
| | - Christopher L-H Huang
- 3 Physiological Laboratory, University of Cambridge, Cambridge, United Kingdom.,4 Division of Cardiovascular Biology, Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
| | - A John Camm
- 5 Cardiac Clinical Academic Group, St George's Hospital Medical School, University of London, Cranmer Terrace, London, United Kingdom
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31
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Hydrocinnamic Acid Inhibits the Currents of WT and SQT3 Syndrome-Related Mutants of Kir2.1 Channel. J Membr Biol 2017; 250:425-432. [PMID: 28660286 DOI: 10.1007/s00232-017-9964-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2016] [Accepted: 06/01/2017] [Indexed: 01/27/2023]
Abstract
Gain of function in mutations, D172N and E299V, of Kir2.1 will induce type III short QT syndrome. In our previous work, we had identified that a mixture of traditional Chinese medicine, styrax, is a blocker of Kir2.1. Here, we determined a monomer, hydrocinnamic acid (HA), as the effective component from 18 compounds of styrax. Our data show that HA can inhibit the currents of Kir2.1 channel in both excised inside-out and whole-cell patch with the IC50 of 5.21 ± 1.02 and 10.08 ± 0.46 mM, respectively. The time course of HA blockage and washout are 2.3 ± 0.6 and 10.5 ± 2.6 s in the excised inside-out patch. Moreover, HA can also abolish the currents of D172N and E299V with the IC50 of 6.66 ± 0.57 and 5.81 ± 1.10 mM for D172N and E299V, respectively. Molecular docking results determine that HA binds with Kir2.1 at K182, K185, and K188, which are phosphatidylinositol 4,5-bisphosphate (PIP2) binding residues. Our results indicate that HA competes with PIP2 to bind with Kir2.1 and inhibits the currents.
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32
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Hu B, Cilz NI, Lei S. Somatostatin depresses the excitability of subicular bursting cells: Roles of inward rectifier K + channels, KCNQ channels and Epac. Hippocampus 2017; 27:971-984. [PMID: 28558129 DOI: 10.1002/hipo.22744] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2017] [Revised: 05/08/2017] [Accepted: 05/18/2017] [Indexed: 12/17/2022]
Abstract
The hippocampus is a crucial component for cognitive and emotional processing. The subiculum provides much of the output for this structure but the modulation and function of this region is surprisingly under-studied. The neuromodulator somatostatin (SST) interacts with five subtypes of SST receptors (sst1 to sst5 ) and each of these SST receptor subtypes is coupled to Gi proteins resulting in inhibition of adenylyl cyclase (AC) and decreased level of intracellular cAMP. SST modulates many physiological functions including cognition, emotion, autonomic responses and locomotion. Whereas SST has been shown to depress neuronal excitability in the subiculum, the underlying cellular and molecular mechanisms have not yet been determined. Here, we show that SST hyperpolarized two classes of subicular neurons with a calculated EC50 of 0.1 μM. Application of SST (1 μM) induced outward holding currents by primarily activating K+ channels including the G-protein-activated inwardly-rectifying potassium channels (GIRK) and KCNQ (M) channels, although inhibition of cation channels in some cells may also be implicated. SST-elicited hyperpolarization was mediated by activation of sst2 receptors and required the function of G proteins. The SST-induced hyperpolarization resulted from decreased activity of AC and reduced levels of cAMP but did not require the activity of either PKA or PKC. Inhibition of Epac2, a guanine nucleotide exchange factor, partially blocked SST-mediated hyperpolarization of subicular neurons. Furthermore, application of SST resulted in a robust depression of subicular action potential firing and the SST-induced hyperpolarization was responsible for its inhibitory action on LTP at the CA1-subicilum synapses. Our results provide a novel cellular and molecular mechanism that may explain the roles of SST in modulation of subicular function and be relevant to SST-related physiological functions.
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Affiliation(s)
- Binqi Hu
- Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58203
| | - Nicholas I Cilz
- Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58203
| | - Saobo Lei
- Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota 58203
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33
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Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy. J Mol Biol 2017; 429:2192-2210. [PMID: 28535993 DOI: 10.1016/j.jmb.2017.05.015] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2017] [Revised: 04/21/2017] [Accepted: 05/16/2017] [Indexed: 12/25/2022]
Abstract
The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH while W41 ensures proton conduction only from the N terminus to the C terminus and prevents reverse current under low internal pH. Here, we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low internal pH. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type (WT) channel but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted toward the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C terminus. At low pH, the mutant channel contains more cationic tetrads than the WT channel, consistent with faster reverse protonation from the C terminus. 15N NMR spectra allow the extraction of four H37 pKas and show that the pKas are more clustered in the mutant channel compared to WT M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in WT M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in WT M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants.
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34
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Scherer D, Schworm B, Seyler C, Xynogalos P, Scholz EP, Thomas D, Katus HA, Zitron E. Inhibition of inwardly rectifying Kir2.x channels by the novel anti-cancer agent gambogic acid depends on both pore block and PIP 2 interference. Naunyn Schmiedebergs Arch Pharmacol 2017; 390:701-710. [PMID: 28365825 DOI: 10.1007/s00210-017-1372-5] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2017] [Accepted: 03/23/2017] [Indexed: 12/21/2022]
Abstract
The caged xanthone gambogic acid (GA) is a novel anti-cancer agent which exhibits anti-proliferative, anti-inflammatory and cytotoxic effects in many types of cancer tissues. In a recent phase IIa study, GA exhibits a favourable safety profile. However, limited data are available concerning its interaction with cardiac ion channels. Heteromeric assembly of Kir2.x channels underlies the cardiac inwardly rectifying IK1 current which is responsible for the stabilization of the diastolic resting membrane potential. Inhibition of the cardiac IK1 current may lead to ventricular arrhythmia due to delayed afterdepolarizations. Compared to Kv2.1, hERG and Kir1.1, a slow, delayed inhibition of Kir2.1 channels by GA in a mammalian cell line was reported before but no data exist in literature concerning action of GA on homomeric Kir2.2 and Kir2.3 and heteromeric Kir2.x channels. Therefore, the aim of this study was to provide comparative data on the effect of GA on homomeric and heteromeric Kir2.x channels. Homomeric and heteromeric Kir2.x channels were heterologously expressed in Xenopus oocytes, and the two-microelectrode voltage-clamp technique was used to record Kir2.x currents. To investigate the mechanism of the channel inhibition by GA, alanine-mutated Kir2.x channels with modifications in the channels pore region or at phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sites were employed. GA caused a slow inhibition of homomeric and heteromeric Kir2.x channels at low micromolar concentrations (with IC50 Kir2.1/2.2 < Kir2.2 < Kir2.2/2.3 < Kir2.3 < Kir2.1 < Kir2.1/2.3). The effect did not reach saturation within 60 min and was not reversible upon washout for 30 min. The inhibition showed no strong voltage dependence. We provide evidence for a combination of direct channel pore blockade and a PIP2-dependent mechanism as a molecular basis for the observed effect. We conclude that Kir2.x channel inhibition by GA may be relevant in patients with pre-existing cardiac disorders such as chronic heart failure or certain rhythm disorders and recommend a close cardiac monitoring for those patients when treated with GA.
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Affiliation(s)
- Daniel Scherer
- Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany.
| | - Benedikt Schworm
- Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany
| | - Claudia Seyler
- Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, Heidelberg, Germany
| | - Panagiotis Xynogalos
- Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany
| | - Eberhard P Scholz
- Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany
| | - Dierk Thomas
- Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, Heidelberg, Germany
| | - Hugo A Katus
- Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, Heidelberg, Germany
| | - Edgar Zitron
- Department of Cardiology, Medical University Hospital Heidelberg, Im Neuenheimer Feld 410, D-69120, Heidelberg, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, Heidelberg, Germany
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35
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Tykocki NR, Boerman EM, Jackson WF. Smooth Muscle Ion Channels and Regulation of Vascular Tone in Resistance Arteries and Arterioles. Compr Physiol 2017; 7:485-581. [PMID: 28333380 DOI: 10.1002/cphy.c160011] [Citation(s) in RCA: 241] [Impact Index Per Article: 30.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Vascular tone of resistance arteries and arterioles determines peripheral vascular resistance, contributing to the regulation of blood pressure and blood flow to, and within the body's tissues and organs. Ion channels in the plasma membrane and endoplasmic reticulum of vascular smooth muscle cells (SMCs) in these blood vessels importantly contribute to the regulation of intracellular Ca2+ concentration, the primary determinant of SMC contractile activity and vascular tone. Ion channels provide the main source of activator Ca2+ that determines vascular tone, and strongly contribute to setting and regulating membrane potential, which, in turn, regulates the open-state-probability of voltage gated Ca2+ channels (VGCCs), the primary source of Ca2+ in resistance artery and arteriolar SMCs. Ion channel function is also modulated by vasoconstrictors and vasodilators, contributing to all aspects of the regulation of vascular tone. This review will focus on the physiology of VGCCs, voltage-gated K+ (KV) channels, large-conductance Ca2+-activated K+ (BKCa) channels, strong-inward-rectifier K+ (KIR) channels, ATP-sensitive K+ (KATP) channels, ryanodine receptors (RyRs), inositol 1,4,5-trisphosphate receptors (IP3Rs), and a variety of transient receptor potential (TRP) channels that contribute to pressure-induced myogenic tone in resistance arteries and arterioles, the modulation of the function of these ion channels by vasoconstrictors and vasodilators, their role in the functional regulation of tissue blood flow and their dysfunction in diseases such as hypertension, obesity, and diabetes. © 2017 American Physiological Society. Compr Physiol 7:485-581, 2017.
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Affiliation(s)
- Nathan R Tykocki
- Department of Pharmacology, University of Vermont, Burlington, Vermont, USA
| | - Erika M Boerman
- Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri, USA
| | - William F Jackson
- Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan, USA
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36
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Chloroquine blocks the Kir4.1 channels by an open-pore blocking mechanism. Eur J Pharmacol 2017; 800:40-47. [PMID: 28216048 DOI: 10.1016/j.ejphar.2017.02.024] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2016] [Revised: 02/03/2017] [Accepted: 02/15/2017] [Indexed: 11/23/2022]
Abstract
Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch-clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK-293 cells. CQ produced a fast-onset voltage-dependent pore-blocking effect on these channels. In inside-out patches, CQ showed notable higher potency (IC50 ≈0.5μM at +50mV) and faster onset of block when compared to whole-cell configuration (IC50 ≈7μM at +60mV). Also, CQ showed a voltage-dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore-plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ-Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.
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Ren S, Pang C, Li J, Huang Y, Zhang S, Zhan Y, An H. Styrax blocks inward and outward current of Kir2.1 channel. Channels (Austin) 2017; 11:46-54. [PMID: 27540685 DOI: 10.1080/19336950.2016.1207022] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
Kir2.1 plays key roles in setting rest membrane potential and modulation of cell excitability. Mutations of Kir2.1, such as D172N or E299V, inducing gain-of-function, can cause type3 short QT syndrome (SQT3) due to the enlarged outward currents. So far, there is no clinical drug target to block the currents of Kir2.1. Here, we identified a novel blocker of Kir2.1, styrax, which is a kind of natural compound selected from traditional Chinese medicine. Our data show that styrax can abolish the inward and outward currents of Kir2.1. The IC50 of styrax for WT, D172N and E299V are 0.0113 ± 0.00075, 0.0204 ± 0.0048 and 0.0122 ± 0.0012 (in volume), respectively. The results indicate that styrax can serve as a novel blocker for Kir2.1.
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Affiliation(s)
- Shuxi Ren
- a Key Laboratory of Molecular Biophysics, Hebei Province, Institute of Biophysics, School of Sciences, Hebei University of Technology , Tianjin , China
| | - Chunli Pang
- a Key Laboratory of Molecular Biophysics, Hebei Province, Institute of Biophysics, School of Sciences, Hebei University of Technology , Tianjin , China
| | - Junwei Li
- a Key Laboratory of Molecular Biophysics, Hebei Province, Institute of Biophysics, School of Sciences, Hebei University of Technology , Tianjin , China
| | - Yayue Huang
- a Key Laboratory of Molecular Biophysics, Hebei Province, Institute of Biophysics, School of Sciences, Hebei University of Technology , Tianjin , China
| | - Suhua Zhang
- a Key Laboratory of Molecular Biophysics, Hebei Province, Institute of Biophysics, School of Sciences, Hebei University of Technology , Tianjin , China
| | - Yong Zhan
- a Key Laboratory of Molecular Biophysics, Hebei Province, Institute of Biophysics, School of Sciences, Hebei University of Technology , Tianjin , China
| | - Hailong An
- a Key Laboratory of Molecular Biophysics, Hebei Province, Institute of Biophysics, School of Sciences, Hebei University of Technology , Tianjin , China
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Raltschev C, Hetsch F, Winkelmann A, Meier JC, Semtner M. Electrophysiological Signature of Homomeric and Heteromeric Glycine Receptor Channels. J Biol Chem 2016; 291:18030-40. [PMID: 27382060 DOI: 10.1074/jbc.m116.735084] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2016] [Indexed: 11/06/2022] Open
Abstract
Glycine receptors are chloride-permeable, ligand-gated ion channels and contribute to the inhibition of neuronal firing in the central nervous system or to facilitation of neurotransmitter release if expressed at presynaptic sites. Recent structure-function studies have provided detailed insights into the mechanisms of channel gating, desensitization, and ion permeation. However, most of the work has focused only on comparing a few isoforms, and among studies, different cellular expression systems were used. Here, we performed a series of experiments using recombinantly expressed homomeric and heteromeric glycine receptor channels, including their splice variants, in the same cellular expression system to investigate and compare their electrophysiological properties. Our data show that the current-voltage relationships of homomeric channels formed by the α2 or α3 subunits change upon receptor desensitization from a linear to an inwardly rectifying shape, in contrast to their heteromeric counterparts. The results demonstrate that inward rectification depends on a single amino acid (Ala(254)) at the inner pore mouth of the channels and is closely linked to chloride permeation. We also show that the current-voltage relationships of glycine-evoked currents in primary hippocampal neurons are inwardly rectifying upon desensitization. Thus, the alanine residue Ala(254) determines voltage-dependent rectification upon receptor desensitization and reveals a physio-molecular signature of homomeric glycine receptor channels, which provides unprecedented opportunities for the identification of these channels at the single cell level.
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Affiliation(s)
- Constanze Raltschev
- From the Department of Biomedicine, Cellular Neurophysiology, University of Basel, Pestalozzistrasse 20, 4056 Basel, Switzerland
| | - Florian Hetsch
- the Division of Cell Physiology, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany, and
| | - Aline Winkelmann
- the Division of Cell Physiology, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany, and
| | - Jochen C Meier
- the Division of Cell Physiology, Technische Universität Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany, and
| | - Marcus Semtner
- Cellular Neurosciences, Max-Delbrück-Centrum für Molekulare Medizin (MDC), Robert-Rössle-Strasse 10, 13092 Berlin, Germany
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Skatchkov SN, Antonov SM, Eaton MJ. Glia and glial polyamines. Role in brain function in health and disease. BIOCHEMISTRY MOSCOW SUPPLEMENT SERIES A-MEMBRANE AND CELL BIOLOGY 2016. [DOI: 10.1134/s1990747816010116] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
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De Angeli A, Thomine S, Frachisse JM. Anion Channel Blockage by ATP as a Means for Membranes to Perceive the Energy Status of the Cell. MOLECULAR PLANT 2016; 9:320-322. [PMID: 26785050 DOI: 10.1016/j.molp.2016.01.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/03/2015] [Revised: 12/25/2015] [Accepted: 01/08/2016] [Indexed: 05/08/2023]
Affiliation(s)
- Alexis De Angeli
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France
| | - Sébastien Thomine
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France
| | - Jean-Marie Frachisse
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette cedex, France.
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Downregulation of Spermine Augments Dendritic Persistent Sodium Currents and Synaptic Integration after Status Epilepticus. J Neurosci 2016; 35:15240-53. [PMID: 26586813 DOI: 10.1523/jneurosci.0493-15.2015] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
UNLABELLED Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na(+) current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendritic INaP is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic INaP causes augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy. SIGNIFICANCE STATEMENT In this paper, we describe a novel mechanism that causes increased dendritic persistent Na(+) current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na(+) currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.
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Fechner S, Alvarez L, Bönigk W, Müller A, Berger TK, Pascal R, Trötschel C, Poetsch A, Stölting G, Siegfried KR, Kremmer E, Seifert R, Kaupp UB. A K(+)-selective CNG channel orchestrates Ca(2+) signalling in zebrafish sperm. eLife 2015; 4:e07624. [PMID: 26650356 PMCID: PMC4749565 DOI: 10.7554/elife.07624] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2015] [Accepted: 12/09/2015] [Indexed: 01/03/2023] Open
Abstract
Calcium in the flagellum controls sperm navigation. In sperm of marine invertebrates and mammals, Ca(2+) signalling has been intensely studied, whereas for fish little is known. In sea urchin sperm, a cyclic nucleotide-gated K(+) channel (CNGK) mediates a cGMP-induced hyperpolarization that evokes Ca(2+) influx. Here, we identify in sperm of the freshwater fish Danio rerio a novel CNGK family member featuring non-canonical properties. It is located in the sperm head rather than the flagellum and is controlled by intracellular pH, but not cyclic nucleotides. Alkalization hyperpolarizes sperm and produces Ca(2+) entry. Ca(2+) induces spinning-like swimming, different from swimming of sperm from other species. The "spinning" mode probably guides sperm into the micropyle, a narrow entrance on the surface of fish eggs. A picture is emerging of sperm channel orthologues that employ different activation mechanisms and serve different functions. The channel inventories probably reflect adaptations to species-specific challenges during fertilization.
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Affiliation(s)
- Sylvia Fechner
- Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
| | - Luis Alvarez
- Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
| | - Wolfgang Bönigk
- Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
| | - Astrid Müller
- Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
| | - Thomas K Berger
- Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
| | - Rene Pascal
- Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
| | | | - Ansgar Poetsch
- Lehrstuhl Biochemie der Pflanzen, Ruhr-Universität Bochum, Bochum, Germany
| | - Gabriel Stölting
- Institute of Complex Systems 4, Forschungszentrum Jülich, Jülich, Germany
| | - Kellee R Siegfried
- Biology Department, University of Massachusetts Boston, Boston, United States
| | - Elisabeth Kremmer
- Institut für Molekulare Immunologie, Helmholtz-Zentrum München, München, Germany
| | - Reinhard Seifert
- Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
| | - U Benjamin Kaupp
- Abteilung Molekulare Neurosensorik, Center of Advanced European Studies and Research, Bonn, Germany
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Kim J, Moon SH, Shin YC, Jeon JH, Park KJ, Lee KP, So I. Intracellular spermine blocks TRPC4 channel via electrostatic interaction with C-terminal negative amino acids. Pflugers Arch 2015; 468:551-61. [PMID: 26631167 DOI: 10.1007/s00424-015-1753-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2015] [Revised: 10/27/2015] [Accepted: 10/29/2015] [Indexed: 01/09/2023]
Abstract
Transient receptor potential canonical (TRPC) 4 channels are calcium-permeable, nonselective cation channels and are widely expressed in mammalian tissue, especially in the GI tract and brain. TRPC4 channels are known to be involved in neurogenic contraction of ileal smooth muscle cells via generating cationic current after muscarinic stimulation (muscarinic cationic current (mIcat)). Polyamines exist in numerous tissues and are believed to be involved in cell proliferation, differentiation, scar formation, wound healing, and carcinogenesis. Besides, physiological polyamines are essential to maintain inward rectification of cardiac potassium channels (Kir2.1). At membrane potentials more positive than equilibrium potential, intracellular polyamines plug the cytosolic surface of the Kir2.1 so that potassium ions cannot pass through the pore. Recently, it was reported that polyamines inhibit not only cardiac potassium channels but also nonselective cation channels that mediate the generation of mIcat. Here, we report that TRPC4, a definite mIcat mediator, is inhibited by intracellular spermine with great extent. The inhibition was specific to TRPC4 and TRPC5 channels but was not effective to TRPC1/4, TRPC1/5, and TRPC3 channels. For this inhibition to occur, we found that glutamates at 728th and 729th position of TRPC4 channels are essential whereby we conclude that spermine blocks the TRPC4 channel with electrostatic interaction between negative amino acids at the C-terminus of the channel.
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Affiliation(s)
- Jinsung Kim
- College of Medicine, Catholic University of Korea, Seoul, 137-701, Republic of Korea.,Department of Physiology, College of Medicine, Seoul National University, Seoul, 110-799, Republic of Korea
| | - Sang Hui Moon
- Department of Surgery, College of Medicine, Seoul National University, Seoul, 110-799, Republic of Korea
| | - Young-Cheul Shin
- Department of Physiology, College of Medicine, Seoul National University, Seoul, 110-799, Republic of Korea
| | - Ju-Hong Jeon
- Department of Physiology, College of Medicine, Seoul National University, Seoul, 110-799, Republic of Korea
| | - Kyu Joo Park
- Department of Surgery, College of Medicine, Seoul National University, Seoul, 110-799, Republic of Korea
| | - Kyu Pil Lee
- Department of Physiology, College of Veterinary Medicine, Chungnam National University, Daejeon, 305-764, Republic of Korea.
| | - Insuk So
- Department of Physiology, College of Medicine, Seoul National University, Seoul, 110-799, Republic of Korea.
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Hoffmann B, Klöcker N, Benndorf K, Biskup C. Visualization of the dynamics of PSD-95 and Kir2.1 interaction by fluorescence lifetime-based resonance energy transfer imaging. ACTA ACUST UNITED AC 2015. [DOI: 10.1016/j.medpho.2014.11.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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45
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Okada M, Corzo G, Romero-Perez GA, Coronas F, Matsuda H, Possani LD. A pore forming peptide from spider Lachesana sp. venom induced neuronal depolarization and pain. Biochim Biophys Acta Gen Subj 2015; 1850:657-66. [DOI: 10.1016/j.bbagen.2014.11.022] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2014] [Revised: 11/12/2014] [Accepted: 11/28/2014] [Indexed: 10/24/2022]
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Abstract
This review focuses on the roles of glia and polyamines (PAs) in brain function and dysfunction, highlighting how PAs are one of the principal differences between glia and neurons. The novel role of PAs, such as putrescine, spermidine, and spermine and their precursors and derivatives, is discussed. However, PAs have not yet been a focus of much glial research. They affect many neuronal and glial receptors, channels, and transporters. They are therefore key elements in the development of many diseases and syndromes, thus forming the rationale for PA-focused and glia-focused therapy for these conditions.
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Affiliation(s)
- Serguei N Skatchkov
- Department of Biochemistry, School of Medicine, Universidad, Central del Caribe, PO Box 60-327, Bayamón, PR 00960-6032, USA; Department of Physiology, School of Medicine, Universidad, Central del Caribe, PO Box 60-327, Bayamón, PR 00960-6032, USA.
| | - Michel A Woodbury-Fariña
- Department of Psychiatry, University of Puerto Rico School of Medicine, 307 Calle Eleonor Roosevelt, San Juan, PR 00918-2720, USA
| | - Misty Eaton
- Department of Biochemistry, School of Medicine, Universidad, Central del Caribe, PO Box 60-327, Bayamón, PR 00960-6032, USA
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Cervera J, Alcaraz A, Mafe S. Membrane potential bistability in nonexcitable cells as described by inward and outward voltage-gated ion channels. J Phys Chem B 2014; 118:12444-50. [PMID: 25286866 DOI: 10.1021/jp508304h] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The membrane potential of nonexcitable cells, defined as the electrical potential difference between the cell cytoplasm and the extracellular environment when the current is zero, is controlled by the individual electrical conductance of different ion channels. In particular, inward- and outward-rectifying voltage-gated channels are crucial for cell hyperpolarization/depolarization processes, being amenable to direct physical study. High (in absolute value) negative membrane potentials are characteristic of terminally differentiated cells, while low membrane potentials are found in relatively depolarized, more plastic cells (e.g., stem, embryonic, and cancer cells). We study theoretically the hyperpolarized and depolarized values of the membrane potential, as well as the possibility to obtain a bistability behavior, using simplified models for the ion channels that regulate this potential. The bistability regions, which are defined in the multidimensional state space determining the cell state, can be relevant for the understanding of the different model cell states and the transitions between them, which are triggered by changes in the external environment.
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Affiliation(s)
- Javier Cervera
- Departament de Termodinàmica, Universitat de València , E-46100 Burjassot, Spain
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48
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Baronas VA, Kurata HT. Inward rectifiers and their regulation by endogenous polyamines. Front Physiol 2014; 5:325. [PMID: 25221519 PMCID: PMC4145359 DOI: 10.3389/fphys.2014.00325] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2014] [Accepted: 08/06/2014] [Indexed: 12/02/2022] Open
Abstract
Inwardly-rectifying potassium (Kir) channels contribute to maintenance of the resting membrane potential and regulation of electrical excitation in many cell types. Strongly rectifying Kir channels exhibit a very steep voltage dependence resulting in silencing of their activity at depolarized membrane voltages. The mechanism underlying this steep voltage dependence is blockade by endogenous polyamines. These small multifunctional, polyvalent metabolites enter the long Kir channel pore from the intracellular side, displacing multiple occupant ions as they migrate to a stable binding site in the transmembrane region of the channel. Numerous structure-function studies have revealed structural elements of Kir channels that determine their susceptibility to polyamine block, and enable the steep voltage dependence of this process. In addition, various channelopathies have been described that result from alteration of the polyamine sensitivity or activity of strongly rectifying channels. The primary focus of this article is to summarize current knowledge of the molecular mechanisms of polyamine block, and provide some perspective on lingering uncertainties related to this physiologically important mechanism of ion channel blockade. We also briefly review some of the important and well understood physiological roles of polyamine sensitive, strongly rectifying Kir channels, primarily of the Kir2 family.
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Affiliation(s)
- Victoria A Baronas
- Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia Vancouver, BC, Canada
| | - Harley T Kurata
- Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia Vancouver, BC, Canada
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Schmitt N, Grunnet M, Olesen SP. Cardiac potassium channel subtypes: new roles in repolarization and arrhythmia. Physiol Rev 2014; 94:609-53. [PMID: 24692356 DOI: 10.1152/physrev.00022.2013] [Citation(s) in RCA: 174] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
About 10 distinct potassium channels in the heart are involved in shaping the action potential. Some of the K+ channels are primarily responsible for early repolarization, whereas others drive late repolarization and still others are open throughout the cardiac cycle. Three main K+ channels drive the late repolarization of the ventricle with some redundancy, and in atria this repolarization reserve is supplemented by the fairly atrial-specific KV1.5, Kir3, KCa, and K2P channels. The role of the latter two subtypes in atria is currently being clarified, and several findings indicate that they could constitute targets for new pharmacological treatment of atrial fibrillation. The interplay between the different K+ channel subtypes in both atria and ventricle is dynamic, and a significant up- and downregulation occurs in disease states such as atrial fibrillation or heart failure. The underlying posttranscriptional and posttranslational remodeling of the individual K+ channels changes their activity and significance relative to each other, and they must be viewed together to understand their role in keeping a stable heart rhythm, also under menacing conditions like attacks of reentry arrhythmia.
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Shen N, Zheng J, Liu H, Xu K, Chen Q, Chen Y, Shen Y, Jiang L, Chen Y. Barium chloride impaired Kir2.1 inward rectification in its stably transfected HEK 293 cell lines. Eur J Pharmacol 2014; 730:164-70. [PMID: 24631257 DOI: 10.1016/j.ejphar.2014.02.025] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2014] [Revised: 02/16/2014] [Accepted: 02/20/2014] [Indexed: 11/16/2022]
Abstract
Kir2.1 channel is a typical inward rectified channel with little outward currents when the membrane depolarized. Barium blocks the inward Kir2.1 currents in a voltage-dependent manner. However, in this study we found that barium would impair the rectification and open Kir2.1 outward currents at a depolarized voltage, causing increment of outward current amplitudes by 43±7% (n=5, P<0.01) after 200s barium application. In the meanwhile, a higher barium concentration did block the outward currents by 17.5±4.3% (n=4, P<0.01) and temporarily twisted current upward tendency. The increment was likely barium specific since both calcium and Kir2.1 specific blocker, Chloroethylclonidine (CEC), did not enhance the current amplitudes. The rectification of Kir2.1 was not recovered by washing barium off, which suggested a non-competitive mechanism. Since the currents occurred at phase 1, 2 of cardiac action potential, it would likely shorten the action potential plateau and it would decrease QT duration in electrocardiography (ECG).
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Affiliation(s)
- Naiji Shen
- Cardiovascular Division, Zhejiang Province People׳s Hospital, 158 Shangtang Road, Hangzhou 310014, PR China
| | - Jifeng Zheng
- Cardiovascular Division, Jiaxing No. 2 Hospital, 1882 Central Circle Road South, Jiaxing 314001, PR China
| | - Hualiang Liu
- Chinese Herb Medicine Division, The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, 88 North Circle Road, Lin'an 311300, PR China
| | - Kai Xu
- Safety Pharmacology Division, Olivepharmasolutions Ltd., 333 Changhong Road, Wukang 313200, PR China
| | - Qingmao Chen
- Safety Pharmacology Division, Olivepharmasolutions Ltd., 333 Changhong Road, Wukang 313200, PR China
| | - Yingying Chen
- Pathophysiolog department, Medical School, Zhejiang University, Hangzhou 310058, PR China
| | - Yueliang Shen
- Pathophysiolog department, Medical School, Zhejiang University, Hangzhou 310058, PR China
| | - Liqing Jiang
- Cardiovascular Division, Jiaxing No. 2 Hospital, 1882 Central Circle Road South, Jiaxing 314001, PR China
| | - Yuan Chen
- Chinese Herb Medicine Division, The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, 88 North Circle Road, Lin'an 311300, PR China.
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