|
1
|
Orts DJB, Arcisio-Miranda M. Cell glycosaminoglycans content modulates human voltage-gated proton channel (H V 1) gating. FEBS J 2021; 289:2593-2612. [PMID: 34800064 DOI: 10.1111/febs.16290] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2021] [Revised: 11/01/2021] [Accepted: 11/19/2021] [Indexed: 11/28/2022]
Abstract
Voltage-gated proton channels (HV 1) have been found in many mammalian cells and play a crucial role in the immune system, male fertility, and cancer progression. Glycosaminoglycans play a significant role in various aspects of cell physiology, including the modulation of membrane receptors and ion channel function. We present here evidence that mechanosensitivity of the dimeric HV 1 channel transduce changes on cell membrane fluidity related to the defective biosynthesis of chondroitin sulfate and heparan sulfate in Chinese Hamster Ovary (CHO-745) cells into a leftward shift in the activation voltage dependence. This effect was accompanied by an increase in the H+ current, and an acceleration of the activation kinetics, under symmetrical or asymmetrical pH gradient (ΔpH) conditions. Similar gating alterations were evoked by two naturally occurring HV 1 N-terminal truncated isoforms expressed in wild-type CHO-K1 and CHO-745 cells. On three different monomeric HV 1 constructs, no alterations in the biophysical parameters were observed. Moreover, we have shown that HV 1 gating can be modulated by manipulating CHO-K1 cell membrane fluidity. Our results suggest that the defective biosynthesis of chondroitin sulfate and heparan sulfate on CHO-745 cell increases membrane fluidity and allosterically modulates the coupling between voltage- and ΔpH-sensing through the dimeric HV 1 channel.
Collapse
Affiliation(s)
- Diego J B Orts
- Departamento de Biofísica, Laboratório de Neurobiologia Estrutural e Funcional (LaNEF), Universidade Federal de São Paulo - UNIFESP, Brasil
| | - Manoel Arcisio-Miranda
- Departamento de Biofísica, Laboratório de Neurobiologia Estrutural e Funcional (LaNEF), Universidade Federal de São Paulo - UNIFESP, Brasil
| |
Collapse
|
|
2
|
Alvira-Iraizoz F, Gillard BT, Lin P, Paterson A, Pauža AG, Ali MA, Alabsi AH, Burger PA, Hamadi N, Adem A, Murphy D, Greenwood MP. Multiomic analysis of the Arabian camel (Camelus dromedarius) kidney reveals a role for cholesterol in water conservation. Commun Biol 2021; 4:779. [PMID: 34163009 PMCID: PMC8222267 DOI: 10.1038/s42003-021-02327-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2021] [Accepted: 06/06/2021] [Indexed: 02/05/2023] Open
Abstract
The Arabian camel (Camelus dromedarius) is the most important livestock animal in arid and semi-arid regions and provides basic necessities to millions of people. In the current context of climate change, there is renewed interest in the mechanisms that enable camelids to survive in arid conditions. Recent investigations described genomic signatures revealing evolutionary adaptations to desert environments. We now present a comprehensive catalogue of the transcriptomes and proteomes of the dromedary kidney and describe how gene expression is modulated as a consequence of chronic dehydration and acute rehydration. Our analyses suggested an enrichment of the cholesterol biosynthetic process and an overrepresentation of categories related to ion transport. Thus, we further validated differentially expressed genes with known roles in water conservation which are affected by changes in cholesterol levels. Our datasets suggest that suppression of cholesterol biosynthesis may facilitate water retention in the kidney by indirectly facilitating the AQP2-mediated water reabsorption.
Collapse
Affiliation(s)
- Fernando Alvira-Iraizoz
- Molecular Neuroendocrinology Research Group, Bristol Medical School: Translational Health Sciences, University of Bristol, Bristol, UK.
| | - Benjamin T Gillard
- Molecular Neuroendocrinology Research Group, Bristol Medical School: Translational Health Sciences, University of Bristol, Bristol, UK
| | - Panjiao Lin
- Molecular Neuroendocrinology Research Group, Bristol Medical School: Translational Health Sciences, University of Bristol, Bristol, UK
| | - Alex Paterson
- Molecular Neuroendocrinology Research Group, Bristol Medical School: Translational Health Sciences, University of Bristol, Bristol, UK
| | - Audrys G Pauža
- Molecular Neuroendocrinology Research Group, Bristol Medical School: Translational Health Sciences, University of Bristol, Bristol, UK
| | - Mahmoud A Ali
- Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, AL Ain, United Arab Emirates
| | - Ammar H Alabsi
- College of Medicine, Alfaisal University, Riyadh, Saudi Arabia
| | - Pamela A Burger
- Department of Interdisciplinary Life Sciences, Research Institute of Wildlife Ecology, Vetmeduni Vienna, Vienna, Austria
| | - Naserddine Hamadi
- Department of Life and Environmental Sciences, College of Natural and Health Sciences, Zayed University, Abu Dhabi, United Arab Emirates
| | - Abdu Adem
- Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, AL Ain, United Arab Emirates.
- Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, Khalifa University, Abu Dhabi, United Arab Emirates.
| | - David Murphy
- Molecular Neuroendocrinology Research Group, Bristol Medical School: Translational Health Sciences, University of Bristol, Bristol, UK
| | - Michael P Greenwood
- Molecular Neuroendocrinology Research Group, Bristol Medical School: Translational Health Sciences, University of Bristol, Bristol, UK
| |
Collapse
|
|
3
|
Nakanishi S, Makita M, Denda M. Effects of trans-2-nonenal and olfactory masking odorants on proliferation of human keratinocytes. Biochem Biophys Res Commun 2021; 548:1-6. [PMID: 33631667 DOI: 10.1016/j.bbrc.2021.02.050] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Accepted: 02/12/2021] [Indexed: 11/30/2022]
Abstract
Malodorous compounds induce stress responses, mood changes, an increase of skin conductance, activation of the sympathetic nervous system and other physiological changes, and it has been suggested that sensing malodors could provide warning of danger to health. Furthermore, the human body secretes various malodorous compounds as waste products of metabolism, including trans-2-nonenal ((E)-2-nonenal), the amount of which increases with aging. In the present study, we examined the effects of some endogenous malodorous compounds ((E)-2-nonenal, nonanal, pentanal, hexanal, hexanoic acid, hexylamine and isovaleric acid) on cultured human keratinocytes. (E)-2-Nonenal decreased the viability and promoted apoptosis of cultured keratinocytes. It also reduced the thickness and the number of proliferative cells in a three-dimensional epidermal equivalent model. Co-application of masking odorants (dihydromycenol, benzaldehyde, linalool, phenethyl alcohol, benzyl acetate and anisaldehyde), but not non-masking odorants (1,8-cineol, β-damascone, and o-t-butylcyclohexyl acetate), reduced the effect of (E)-2-nonenal on keratinocyte proliferation, and restored the thickness and number of proliferative cells in a three-dimensional epidermal equivalent model.
Collapse
Affiliation(s)
| | - Mio Makita
- Shiseido Global Innovation Center, Yokohama, Japan
| | | |
Collapse
|
|
4
|
Rovini A, Gurnev PA, Beilina A, Queralt-Martín M, Rosencrans W, Cookson MR, Bezrukov SM, Rostovtseva TK. Molecular mechanism of olesoxime-mediated neuroprotection through targeting α-synuclein interaction with mitochondrial VDAC. Cell Mol Life Sci 2020; 77:3611-3626. [PMID: 31760463 PMCID: PMC7244372 DOI: 10.1007/s00018-019-03386-w] [Citation(s) in RCA: 44] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2019] [Revised: 10/23/2019] [Accepted: 11/13/2019] [Indexed: 01/03/2023]
Abstract
An intrinsically disordered neuronal protein α-synuclein (αSyn) is known to cause mitochondrial dysfunction, contributing to loss of dopaminergic neurons in Parkinson's disease. Through yet poorly defined mechanisms, αSyn crosses mitochondrial outer membrane and targets respiratory complexes leading to bioenergetics defects. Here, using neuronally differentiated human cells overexpressing wild-type αSyn, we show that the major metabolite channel of the outer membrane, the voltage-dependent anion channel (VDAC), is a pathway for αSyn translocation into the mitochondria. Importantly, the neuroprotective cholesterol-like synthetic compound olesoxime inhibits this translocation. By applying complementary electrophysiological and biophysical approaches, we provide mechanistic insights into the interplay between αSyn, VDAC, and olesoxime. Our data suggest that olesoxime interacts with VDAC β-barrel at the lipid-protein interface thus hindering αSyn translocation through the VDAC pore and affecting VDAC voltage gating. We propose that targeting αSyn translocation through VDAC could represent a key mechanism for the development of new neuroprotective strategies.
Collapse
Affiliation(s)
- Amandine Rovini
- Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bldg. 29B, Room 1G09, Bethesda, MD, 20892-0924, USA
- Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC, 29425, USA
| | - Philip A Gurnev
- Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bldg. 29B, Room 1G09, Bethesda, MD, 20892-0924, USA
| | - Alexandra Beilina
- Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute of Aging, National Institutes of Health, Bethesda, MD, 20892, USA
| | - María Queralt-Martín
- Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bldg. 29B, Room 1G09, Bethesda, MD, 20892-0924, USA
| | - William Rosencrans
- Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bldg. 29B, Room 1G09, Bethesda, MD, 20892-0924, USA
- Colgate University, Hamilton, NY, 13346, USA
| | - Mark R Cookson
- Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute of Aging, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Sergey M Bezrukov
- Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bldg. 29B, Room 1G09, Bethesda, MD, 20892-0924, USA
| | - Tatiana K Rostovtseva
- Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bldg. 29B, Room 1G09, Bethesda, MD, 20892-0924, USA.
| |
Collapse
|
|
5
|
Abstract
Cannabidiol (CBD), the non-psychoactive component of Cannabis sativa, acts on a diverse selection of membrane proteins with promising therapeutic potential in epilepsy and chronic pain. One such protein is the voltage-gated sodium channel (Nav). CBD shows a lack of specificity for sodium channels; however, the method of interaction is still unknown. In this review, we will outline the studies that report reproducible results of CBD and other cannabinoids changing membrane channel function, with particular interest on Nav. Nav are implicated in fatal forms of epilepsy and are also associated with chronic pain. This makes Nav potential targets for CBD interaction since it has been reported to reduce pain and seizures. One potential method of interaction that is of interest in this review is whether CBD affects channel function by altering lipid bilayer properties, independent of any possible direct interaction with membrane channels. CBD’s ability to interact with its targets is a novel and important discovery. This discovery will not only prompt further research towards CBD’s characterization, but also promotes the application of cannabinoids as potentially therapeutic compounds for diseases like epilepsy and pain.
Collapse
Affiliation(s)
- Abeline Rose Watkins
- a Department of Biomedical Physiology and Kinesiology , Simon Fraser University , Burnaby , BC , Canada
| |
Collapse
|
|
6
|
Zakany F, Pap P, Papp F, Kovacs T, Nagy P, Peter M, Szente L, Panyi G, Varga Z. Determining the target of membrane sterols on voltage-gated potassium channels. Biochim Biophys Acta Mol Cell Biol Lipids 2018; 1864:312-325. [PMID: 30553843 DOI: 10.1016/j.bbalip.2018.12.006] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2018] [Revised: 11/30/2018] [Accepted: 12/12/2018] [Indexed: 12/18/2022]
Abstract
Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel Kv1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing Kv1.3 and Kv10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-β-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.
Collapse
Affiliation(s)
- Florina Zakany
- Division of Biophysics, Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary
| | - Pal Pap
- Division of Biophysics, Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary; MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary
| | - Ferenc Papp
- Division of Biophysics, Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary; MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary
| | - Tamas Kovacs
- Division of Biophysics, Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary
| | - Peter Nagy
- Division of Biophysics, Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary
| | - Maria Peter
- Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Temesvari Krt. 62, Szeged H-6726, Hungary
| | - Lajos Szente
- CycloLab Cyclodextrin R & D Laboratory Ltd., Illatos u. 7, Budapest H-1097, Hungary
| | - Gyorgy Panyi
- Division of Biophysics, Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary; MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary
| | - Zoltan Varga
- Division of Biophysics, Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary; MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, University of Debrecen, Egyetem ter 1, Debrecen H-4032, Hungary.
| |
Collapse
|
|
7
|
Zhang M, Peyear T, Patmanidis I, Greathouse DV, Marrink SJ, Andersen OS, Ingólfsson HI. Fluorinated Alcohols' Effects on Lipid Bilayer Properties. Biophys J 2018; 115:679-689. [PMID: 30077334 PMCID: PMC6104562 DOI: 10.1016/j.bpj.2018.07.010] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2018] [Revised: 06/23/2018] [Accepted: 07/02/2018] [Indexed: 12/21/2022] Open
Abstract
Fluorinated alcohols (fluoroalcohols) have physicochemical properties that make them excellent solvents of peptides, proteins, and other compounds. Like other alcohols, fluoroalcohols also alter membrane protein function and lipid bilayer properties and stability. Thus, the questions arise: how potent are fluoroalcohols as lipid-bilayer-perturbing compounds, could small residual amounts that remain after adding compounds dissolved in fluoroalcohols alter lipid bilayer properties sufficiently to affect membranes and membrane protein function, and do they behave like other alcohols? To address these questions, we used a gramicidin-based fluorescence assay to determine the bilayer-modifying potency of selected fluoroalcohols: trifluoroethanol (TFE), HFIP, and perfluoro-tert-butanol (PFTB). These fluoroalcohols alter bilayer properties in the low (PFTB) to high (TFE) mM range. Using the same assay, we determined the bilayer partitioning of the alcohols. When referenced to the aqueous concentrations, the fluoroalcohols are more bilayer perturbing than their nonfluorinated counterparts, with the largest fluoroalcohol, PFTB, being the most potent and the smallest, TFE, the least. When referenced to the mole fractions in the membrane, however, the fluoroalcohols have equal or lesser bilayer-perturbing potency than their nonfluorinated counterparts, with TFE being more bilayer perturbing than PFTB. We compared the fluoroalcohols' molecular level bilayer interactions using atomistic molecular dynamics simulations and showed how, at higher concentrations, they can cause bilayer breakdown using absorbance measurements and 31P nuclear magnetic resonance.
Collapse
Affiliation(s)
- Mike Zhang
- Department Physiology and Biophysics, Weill Cornell Medicine, New York City, New York; The Bronx High School of Science, New York City, New York
| | - Thasin Peyear
- Department Physiology and Biophysics, Weill Cornell Medicine, New York City, New York
| | - Ilias Patmanidis
- Groningen Biomolecular Science and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands
| | - Denise V Greathouse
- Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas
| | - Siewert J Marrink
- Groningen Biomolecular Science and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands
| | - Olaf S Andersen
- Department Physiology and Biophysics, Weill Cornell Medicine, New York City, New York.
| | - Helgi I Ingólfsson
- Department Physiology and Biophysics, Weill Cornell Medicine, New York City, New York; Groningen Biomolecular Science and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Groningen, the Netherlands; Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California.
| |
Collapse
|
|
8
|
|
|
9
|
Adaptation Independent Modulation of Auditory Hair Cell Mechanotransduction Channel Open Probability Implicates a Role for the Lipid Bilayer. J Neurosci 2016; 36:2945-56. [PMID: 26961949 DOI: 10.1523/jneurosci.3011-15.2016] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection. Hair bundle deflection generates a force by pulling on tip-link proteins connecting adjacent stereocilia. The resting open probability (P(open)) of MET channels determines the linearity and sensitivity to mechanical stimulation. Classically, P(open) is regulated by a calcium-sensitive adaptation mechanism in which lowering extracellular calcium or depolarization increases P(open). Recent data demonstrated that the fast component of adaptation is independent of both calcium and voltage, thus requiring an alternative explanation for the sensitivity of P(open) to calcium and voltage. Using rat auditory hair cells, we characterize a mechanism, separate from fast adaptation, whereby divalent ions interacting with the local lipid environment modulate resting P(open). The specificity of this effect for different divalent ions suggests binding sites that are not an EF-hand or calmodulin model. GsMTx4, a lipid-mediated modifier of cationic stretch-activated channels, eliminated the voltage and divalent sensitivity with minimal effects on adaptation. We hypothesize that the dual mechanisms (lipid modulation and adaptation) extend the dynamic range of the system while maintaining adaptation kinetics at their maximal rates.
Collapse
|
|
10
|
Sachs F. Mechanical transduction by ion channels: A cautionary tale. World J Neurol 2015; 5:74-87. [PMID: 28078202 PMCID: PMC5221657 DOI: 10.5316/wjn.v5.i3.74] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/10/2014] [Revised: 08/23/2014] [Accepted: 07/17/2015] [Indexed: 02/06/2023] Open
Abstract
Mechanical transduction by ion channels occurs in all cells. The physiological functions of these channels have just begun to be elaborated, but if we focus on the upper animal kingdom, these channels serve the common sensory services such as hearing and touch, provide the central nervous system with information on the force and position of muscles and joints, and they provide the autonomic system with information about the filling of hollow organs such as blood vessels. However, all cells of the body have mechanosensitive channels (MSCs), including red cells. Most of these channels are cation selective and are activated by bilayer tension. There are also K+ selective MSCs found commonly in neurons where they may be responsible for both general anesthesia and knockout punches in the boxing ring by hyperpolarizing neurons to reduce excitability. The cationic MSCs are typically inactive under normal mechanical stress, but open under pathologic stress. The channels are normally inactive because they are shielded from stress by the cytoskeleton. The cationic MSCs are specifically blocked by the externally applied peptide GsMtx4 (aka, AT-300). This is the first drug of its class and provides a new approach to many pathologies since it is nontoxic, non-immunogenic, stable in a biological environment and has a long pharmacokinetic lifetime. Pathologies involving excessive stress are common. They produce cardiac arrhythmias, contraction in stretched dystrophic muscle, xerocytotic and sickled red cells, etc. The channels seem to function primarily as “fire alarms”, providing feedback to the cytoskeleton that a region of the bilayer is under excessive tension and needs reinforcing. The eukaryotic forms of MSCs have only been cloned in recent years and few people have experience working with them. “Newbies” need to become aware of the technology, potential artifacts, and the fundamentals of mechanics. The most difficult problem in studying MSCs is that the actual stimulus, the force applied to the channel, is not known. We don’t have direct access to the channels themselves but only to larger regions of the membrane as seen in patches. Cortical forces are shared by the bilayer, the cytoskeleton and the extracellular matrix. How much of an applied stimulus reaches the channel is unknown. Furthermore, many of these channels exist in spatial domains where the forces within a domain are different from forces outside the domain, although we often hope they are proportional. This review is intended to be a guide for new investigators who want to study mechanosensitive ion channels.
Collapse
|
|
11
|
Ashrafuzzaman M. Phenomenology and energetics of diffusion across cell phase states. Saudi J Biol Sci 2015; 22:666-73. [PMID: 26586991 PMCID: PMC4625124 DOI: 10.1016/j.sjbs.2015.05.004] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2015] [Accepted: 05/05/2015] [Indexed: 02/06/2023] Open
Abstract
Cell based transport properties have been mathematically addressed. Cell contained cross boundary diffusion of materials has been explained using valid formalisms and related analytical expressions have been developed. Various distinguishable physical structures and their properties raise different general structure specific diffusion mechanisms and controlled transport related parameters. Some of these parameters play phenomenological roles and some cause regulatory effects. The cell based compartments may be divided into three major physical phase states namely liquid, plasma and solid phase states. Transport of ions, nutrients, small molecules like proteins, etc. across inter phase states and intraphase states follows general transport related formalisms. Creation of some localized permanent and/or temporary structures e.g., ion channels, clustering of constituents, etc. and the transitions between such structures appear as regulators of the transport mechanisms. In this article, I have developed mainly a theoretical analysis of the commonly observed cell transport phenomena. I have attempted to develop formalisms on general cell based diffusion followed by a few numerical computations to address the analytical expression phenomenologically. I have then extended the analysis to adopting with the local structure originated energetics. Independent or correlated molecular transport naturally relies on some general parameters that define the nature of local cell environment as well as on some occasionally raised or transiently active stochastic resonance due to localized interactions. Short and long range interaction energies play crucial roles in this regard. Physical classification of cellular compartments has led us developing analytical expressions on both biologically observed diffusion mechanisms and the diffusions’s occasional stochasticity causing energetics. These analytical expressions help us address the diffusion phenomena generally considering the physical properties of the biostructures across the diffusion pathways. A specific example case of single molecule transport and localized interaction energetics in a specific cell phase has been utilized to address the diffusion quite clearly. This article helps to address the mechanisms of cell based diffusion and nutrient movements and thus helps develop strategic templates to manipulate the diffusion mechanisms. Application of the theoretical knowledge into designing or discovering drugs or small molecule inhibitors targeting cell based structures may open up new avenues in biomedical sciences.
Collapse
|
|
12
|
Speed and sensitivity of phototransduction in Drosophila depend on degree of saturation of membrane phospholipids. J Neurosci 2015; 35:2731-46. [PMID: 25673862 DOI: 10.1523/jneurosci.1150-14.2015] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Drosophila phototransduction is mediated via a G-protein-coupled PLC cascade. Recent evidence, including the demonstration that light evokes rapid contractions of the photoreceptors, suggested that the light-sensitive channels (TRP and TRPL) may be mechanically gated, together with protons released by PLC-mediated PIP2 hydrolysis. If mechanical gating is involved we predicted that the response to light should be influenced by altering the physical properties of the membrane. To achieve this, we used diet to manipulate the degree of saturation of membrane phospholipids. In flies reared on a yeast diet, lacking polyunsaturated fatty acids (PUFAs), mass spectrometry showed that the proportion of polyunsaturated phospholipids was sevenfold reduced (from 38 to ∼5%) but rescued by adding a single species of PUFA (linolenic or linoleic acid) to the diet. Photoreceptors from yeast-reared flies showed a 2- to 3-fold increase in latency and time to peak of the light response, without affecting quantum bump waveform. In the absence of Ca(2+) influx or in trp mutants expressing only TRPL channels, sensitivity to light was reduced up to ∼10-fold by the yeast diet, and essentially abolished in hypomorphic G-protein mutants (Gαq). PLC activity appeared little affected by the yeast diet; however, light-induced contractions measured by atomic force microscopy or the activation of ectopic mechanosensitive gramicidin channels were also slowed ∼2-fold. The results are consistent with mechanosensitive gating and provide a striking example of how dietary fatty acids can profoundly influence sensory performance in a classical G-protein-coupled signaling cascade.
Collapse
|
|
13
|
Patrick JW, Gamez RC, Russell DH. Elucidation of Conformer Preferences for a Hydrophobic Antimicrobial Peptide by Vesicle Capture-Freeze-Drying: A Preparatory Method Coupled to Ion Mobility-Mass Spectrometry. Anal Chem 2014; 87:578-83. [DOI: 10.1021/ac503163g] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Affiliation(s)
- John W. Patrick
- The Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
| | - Roberto C. Gamez
- The Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
| | - David H. Russell
- The Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
| |
Collapse
|
|
14
|
Peptide-induced membrane leakage by lysine derivatives of gramicidin A in liposomes, planar bilayers, and erythrocytes. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2013; 1828:2428-35. [DOI: 10.1016/j.bbamem.2013.06.018] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/25/2013] [Revised: 06/10/2013] [Accepted: 06/13/2013] [Indexed: 11/21/2022]
|
|
15
|
Zhao Y, Vararattanavech A, Li X, Hélixnielsen C, Vissing T, Torres J, Wang R, Fane AG, Tang CY. Effects of proteoliposome composition and draw solution types on separation performance of aquaporin-based proteoliposomes: implications for seawater desalination using aquaporin-based biomimetic membranes. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2013; 47:1496-1503. [PMID: 23311686 DOI: 10.1021/es304306t] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/01/2023]
Abstract
Aquaporins are a large family of water transport proteins in cell membranes. Their high water permeability and solute rejection make them potential building blocks for high-performance biomimetic membranes for desalination. In the current study, proteoliposomes were prepared using AquaporinZ from Escherichia coli cells, and their separation properties were characterized by stopped-flow measurements. The current study systematically investigated the effect of proteoliposome composition (lipid type, protein-to-lipid ratio (PLR), and the addition of cholesterol) on water permeability and NaCl retention. Among the various lipids investigated, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)-based proteoliposomes were found to have excellent osmotic water permeability and NaCl reflection coefficient values. Increasing the PLR of DOPC proteoliposomes up to 1:200 increased their osmotic water permeability. However, further increase in the PLR reduced the osmotic water permeability probably due to the occurrence of defects in the proteoliposomes, whereas the addition of cholesterol improved their osmotic water permeation likely due to defects sealing. The current study also investigated the effect of major dissolved ions in seawater (e.g., Mg(2+) and SO(4)(2-)) on the stability of proteoliposomes, and design criteria for aquaporin-based biomimetic membranes are proposed in the context of desalination.
Collapse
Affiliation(s)
- Yang Zhao
- Singapore Membrane Technology Centre, Nanyang Technological University, Singapore 639798
| | | | | | | | | | | | | | | | | |
Collapse
|
|
16
|
Narang D, Kerr PM, Baserman J, Tam R, Yang W, Searle G, Manning-Fox JE, Paulsen IM, Kozuska JL, MacDonald PE, Light PE, Holt A, Plane F. Triton X-100 inhibits L-type voltage-operated calcium channels. Can J Physiol Pharmacol 2013; 91:316-24. [PMID: 23627843 DOI: 10.1139/cjpp-2012-0257] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Triton X-100 (TX-100) is a nonionic detergent frequently used at millimolar concentrations to disrupt cell membranes and solubilize proteins. At low micromolar concentrations, TX-100 has been reported to inhibit the function of potassium channels. Here, we have used electrophysiological and functional techniques to examine the effects of TX-100 on another class of ion channels, L-type voltage-operated calcium channels (VOCCs). TX-100 (30 nmol·L(-1) to 3 μmol·L(-1)) caused reversible concentration-dependent inhibition of recombinant L-type VOCC (CaV 1.2) currents and of native L-type VOCC currents recorded from rat vascular smooth muscle cells and cardiac myocytes, and murine and human pancreatic β-cells. In functional studies, TX-100 (165 nmol·L(-1) to 3.4 μmol·L(-1)) caused concentration-dependent relaxation of rat isolated mesenteric resistance arteries prestimulated with phenylephrine or KCl. This effect was independent of the endothelium. TX-100 (1.6 μmol·L(-1)) inhibited depolarization-induced exocytosis in both murine and human isolated pancreatic β-cells. These data indicate that at concentrations within the nanomolar to low micromolar range, TX-100 significantly inhibits L-type VOCC activity in a number of cell types, an effect paralleled by inhibition of cell functions dependent upon activation of these channels. This inhibition occurs at concentrations below those used to solubilize proteins and may compromise the use of solutions containing TX-100 in bioassays.
Collapse
Affiliation(s)
- Deepak Narang
- Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2H7, Canada
| | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
17
|
Sorochkina AI, Plotnikov EY, Rokitskaya TI, Kovalchuk SI, Kotova EA, Sychev SV, Zorov DB, Antonenko YN. N-terminally glutamate-substituted analogue of gramicidin A as protonophore and selective mitochondrial uncoupler. PLoS One 2012; 7:e41919. [PMID: 22911866 PMCID: PMC3404012 DOI: 10.1371/journal.pone.0041919] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2012] [Accepted: 06/29/2012] [Indexed: 11/18/2022] Open
Abstract
Limited uncoupling of oxidative phosphorylation could be beneficial for cells by preventing excessive generation of reactive oxygen species. Typical uncouplers are weak organic acids capable of permeating across membranes with a narrow gap between efficacy and toxicity. Aimed at designing a nontoxic uncoupler, the protonatable amino acid residue Glu was substituted for Val at the N-terminus of the pentadecapeptide gramicidin A (gA). The modified peptide [Glu1]gA exhibited high uncoupling activity in isolated mitochondria, in particular, abolishing membrane potential at the inner mitochondrial membrane with the same or even larger efficacy as gA. With mitochondria in cell culture, the depolarizing activity of [Glu1]gA was observed at concentrations by an order of magnitude lower than those of gA. On the contrary, [Glu1]gA was much less potent in forming proton channels in planar lipid bilayers than gA. Remarkably, at uncoupling concentrations, [Glu1]gA did not alter cell morphology and was nontoxic in MTT test, in contrast to gA showing high toxicity. The difference in the behavior of [Glu1]gA and gA in natural and artificial membranes could be ascribed to increased capability of [Glu1]gA to permeate through membranes and/or redistribute between different membranes. Based on the protective role of mild uncoupling, [Glu1]gA and some other proton-conducting gA analogues may be considered as prototypes of prospective therapeutic agents.
Collapse
Affiliation(s)
- Alexandra I. Sorochkina
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
| | - Egor Y. Plotnikov
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
| | - Tatyana I. Rokitskaya
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
| | - Sergei I. Kovalchuk
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
| | - Elena A. Kotova
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
| | - Sergei V. Sychev
- Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
| | - Dmitry B. Zorov
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
| | - Yuri N. Antonenko
- Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
- * E-mail:
| |
Collapse
|
|
18
|
Ingólfsson HI, Andersen OS. Alcohol's effects on lipid bilayer properties. Biophys J 2011; 101:847-55. [PMID: 21843475 DOI: 10.1016/j.bpj.2011.07.013] [Citation(s) in RCA: 119] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2011] [Revised: 07/13/2011] [Accepted: 07/15/2011] [Indexed: 11/26/2022] Open
Abstract
Alcohols are known modulators of lipid bilayer properties. Their biological effects have long been attributed to their bilayer-modifying effects, but alcohols can also alter protein function through direct protein interactions. This raises the question: Do alcohol's biological actions result predominantly from direct protein-alcohol interactions or from general changes in the membrane properties? The efficacy of alcohols of various chain lengths tends to exhibit a so-called cutoff effect (i.e., increasing potency with increased chain length, which that eventually levels off). The cutoff varies depending on the assay, and numerous mechanisms have been proposed such as: limited size of the alcohol-protein interaction site, limited alcohol solubility, and a chain-length-dependent lipid bilayer-alcohol interaction. To address these issues, we determined the bilayer-modifying potency of 27 aliphatic alcohols using a gramicidin-based fluorescence assay. All of the alcohols tested (with chain lengths of 1-16 carbons) alter the bilayer properties, as sensed by a bilayer-spanning channel. The bilayer-modifying potency of the short-chain alcohols scales linearly with their bilayer partitioning; the potency tapers off at higher chain lengths, and eventually changes sign for the longest-chain alcohols, demonstrating an alcohol cutoff effect in a system that has no alcohol-binding pocket.
Collapse
Affiliation(s)
- Helgi I Ingólfsson
- Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York, USA.
| | | |
Collapse
|
|
19
|
Mereuta L, Asandei A, Luchian T. Meet me on the other side: trans-bilayer modulation of a model voltage-gated ion channel activity by membrane electrostatics asymmetry. PLoS One 2011; 6:e25276. [PMID: 21980414 PMCID: PMC3181326 DOI: 10.1371/journal.pone.0025276] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2011] [Accepted: 08/30/2011] [Indexed: 12/03/2022] Open
Abstract
While it is accepted that biomembrane asymmetry is generated by proteins and phospholipids distribution, little is known about how electric changes manifested in a monolayer influence functional properties of proteins localized on the opposite leaflet. Herein we used single-molecule electrophysiology and investigated how asymmetric changes in the electrostatics of an artificial lipid membrane monolayer, generated oppositely from where alamethicin - a model voltage-gated ion channel - was added, altered peptide activity. We found that phlorizin, a membrane dipole potential lowering amphiphile, augmented alamethicin activity and transport features, whereas the opposite occurred with RH-421, which enhances the monolayer dipole potential. Further, the monolayer surface potential was decreased via adsorption of sodium dodecyl sulfate, and demonstrated that vectorial modification of it also affected the alamethicin activity in a predictive manner. A new paradigm is suggested according to which asymmetric changes in the monolayer dipole and surface potential extend their effects spatially by altering the intramembrane potential, whose gradient is sensed by distantly located peptides.
Collapse
Affiliation(s)
- Loredana Mereuta
- Department of Physics, Laboratory of Molecular Biophysics and Medical Physics, Alexandru I. Cuza University, Iasi, Romania
| | - Alina Asandei
- Department of Physics, Laboratory of Molecular Biophysics and Medical Physics, Alexandru I. Cuza University, Iasi, Romania
| | - Tudor Luchian
- Department of Physics, Laboratory of Molecular Biophysics and Medical Physics, Alexandru I. Cuza University, Iasi, Romania
- * E-mail:
| |
Collapse
|
|
20
|
Ingólfsson HI, Koeppe RE, Andersen OS. Effects of green tea catechins on gramicidin channel function and inferred changes in bilayer properties. FEBS Lett 2011; 585:3101-5. [PMID: 21896274 DOI: 10.1016/j.febslet.2011.08.040] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2011] [Revised: 08/24/2011] [Accepted: 08/25/2011] [Indexed: 12/21/2022]
Abstract
Green tea's health benefits have been attributed to its major polyphenols, the catechins: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), and epicatechin (EC). Catechins (especially EGCG) modulate a wide range of biologically important molecules, including many membrane proteins. Yet, little is known about their mechanism(s) of action. We tested the catechins' bilayer-modifying potency using gramicidin A (gA) channels as molecular force probes. All the catechins alter gA channel function and modify bilayer properties, with a 500-fold range in potency (EGCG>ECG≫EGC>EC). Additionally, the gallate group causes current block, as evident by brief downward current transitions (flickers).
Collapse
Affiliation(s)
- Helgi I Ingólfsson
- Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY 10065, United States.
| | | | | |
Collapse
|
|
21
|
Ander BP, Dupasquier CM, Prociuk MA, Pierce GN. Polyunsaturated fatty acids and their effects on cardiovascular disease. Exp Clin Cardiol 2011; 19:6858-63. [PMID: 19649216 DOI: 10.2174/138161281939131127111018] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2013] [Accepted: 04/09/2013] [Indexed: 11/22/2022]
Abstract
Dietary polyunsaturated fatty acids (PUFAs) affect a wide variety of physiological processes. Much attention has been given to the n-3 PUFAs and their role in the prevention and treatment of cardiovascular disease, stemming from evidence obtained through a number of epidemiological studies and clinical trials. Investigators are now focused on elucidating the pathways and mechanisms for the biological action of n-3 PUFAs. Dietary intervention is recognized as a key measure in patient therapy and in the maintenance of human health in general. This review provides a summary of several important clinical trials, and while the exact modes of action of n-3 PUFA are not known, current viewpoints regarding the mechanisms of these fatty acids on atherosclerosis, circulating lipid profile, cell membranes, cell proliferation, platelet aggregation and cardiac arrhythmias are discussed.
Collapse
Affiliation(s)
- Bradley P Ander
- National Centre for Agri-Food Research in Medicine and the Division of Stroke and Vascular Disease, St Boniface Hospital Research Centre, and the Department of Physiology, Faculties of Medicine and Pharmacy, University of Manitoba, Winnipeg, Manitoba
| | | | | | | |
Collapse
|
|
22
|
Kishino Y, Kato H, Kurahashi T, Takeuchi H. Chemical structures of odorants that suppress ion channels in the olfactory receptor cell. J Physiol Sci 2011; 61:231-45. [PMID: 21431980 PMCID: PMC10717247 DOI: 10.1007/s12576-011-0142-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2011] [Accepted: 03/08/2011] [Indexed: 01/30/2023]
Abstract
It has been proposed that odorant suppression of the cyclic nucleotide-gated (CNG) channel is responsible for olfactory masking. In this study, the effect of odorant chain length and functional group on this suppression was investigated. Because similar suppression has been observed for voltage-gated channels also, we used voltage-gated Na channels in the olfactory receptor cell as a tool for substance screening. These features were then re-examined using CNG channels. Interestingly, both CNG and Na channels were suppressed in a similar manner-carboxylic acids had little effect and suppression became stronger when the chain length of the alcohol or ester was increased. Degree of suppression was correlated with the distribution coefficients (Log D), irrespective of the molecules used. Results obtained here may provide information for the development of novel masking agents based on molecular architecture.
Collapse
Affiliation(s)
- Yukako Kishino
- Graduate School of Frontier Bioscience, Osaka University, Osaka, 560-8531 Japan
| | - Hiroyuki Kato
- Departments of Chemistry and Materials Science, Graduate School of Science, Osaka City University, Osaka, 558-8585 Japan
| | - Takashi Kurahashi
- Graduate School of Frontier Bioscience, Osaka University, Osaka, 560-8531 Japan
| | - Hiroko Takeuchi
- Graduate School of Frontier Bioscience, Osaka University, Osaka, 560-8531 Japan
| |
Collapse
|
|
23
|
Le Guennec JY, Jude S, Besson P, Martel E, Champeroux P. Cardioprotection by omega-3 fatty acids: involvement of PKCs? Prostaglandins Leukot Essent Fatty Acids 2010; 82:173-7. [PMID: 20189372 DOI: 10.1016/j.plefa.2010.02.024] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/15/2009] [Indexed: 11/15/2022]
Abstract
It has been known since the 1970s that an increased consumption of n-3 long chain polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid has cardioprotective effects. Epidemiological studies have reported that this effect is due to the prevention of the arrhythmias responsible for sudden cardiac death. Mechanistically, different hypotheses have been put forward to give an explanation. Among them, there are a direct effect of the polyunsaturated fatty acids on ion channels and/or a modification of the regulation of ion channels by protein kinase C's.
Collapse
Affiliation(s)
- J-Y Le Guennec
- Inserm U637, Université Montpellier-2, Montpellier, France.
| | | | | | | | | |
Collapse
|
|
24
|
Gillet L, Roger S, Bougnoux P, Le Guennec JY, Besson P. Beneficial effects of omega-3 long-chain fatty acids in breast cancer and cardiovascular diseases: voltage-gated sodium channels as a common feature? Biochimie 2010; 93:4-6. [PMID: 20167245 DOI: 10.1016/j.biochi.2010.02.005] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2009] [Accepted: 02/08/2010] [Indexed: 10/19/2022]
Abstract
Cancers are among the leading causes of death worldwide. Voltage-gated sodium channels, among other ion channels, appear as new molecular players in epithelial cancers. Highly metastatic breast cancer cells express Na(V)1.5, the main isoform expressed in cardiac cells, where the current generated by the flux of sodium ions is responsible for the excitability. Breast cancer cells are not excitable and the protein activity regulates cell invasiveness, through the modulation of activity of acidic cathepsins, a characteristic involved in the metastatic phenotype. Interestingly, it is known that ω-3 LC-PUFA can exert beneficial effects by preventing post-myocardial infarction arrhythmias and by reducing the incidence of metastatic breast cancer. In this review, we compare the effects of some ω-3 LC-PUFA on Na(V)1.5 expressed in both cardiac and MDA-MB-231 breast cancer cells. We propose that some of the effects of ω-3 LC-PUFA act through common mechanisms involved in both diseases.
Collapse
Affiliation(s)
- Ludovic Gillet
- Inserm U921, Nutrition Croissance Cancer, Université François-Rabelais, Faculté de médecine, 10 Bd Tonnellé, 37032 Tours Cedex, France
| | | | | | | | | |
Collapse
|
|
25
|
Abstract
Mechanosensitive ion channels (MSCs) exist in all cells, but mechanosensitivity is a phenotype not a genotype. Specialized mechanoreceptors such as the hair cells of the cochlea require elaborate mechanical impedance matching to couple the channels to the external stress. In contrast, MSCs in nonspecialized cells appear activated by stress in the bilayer local to the channel--within about three lipids. Local mechanical stress can be produced by far-field tension, amphipaths, phase separations, the cytoskeleton, the extracellular matrix, and the adhesion energy between the membrane and a patch pipette. Understanding MSC function requires under standing the stimulus.
Collapse
|
|
26
|
Lundbaek JA, Collingwood SA, Ingólfsson HI, Kapoor R, Andersen OS. Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes. J R Soc Interface 2009; 7:373-95. [PMID: 19940001 DOI: 10.1098/rsif.2009.0443] [Citation(s) in RCA: 239] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Membrane protein function is regulated by the host lipid bilayer composition. This regulation may depend on specific chemical interactions between proteins and individual molecules in the bilayer, as well as on non-specific interactions between proteins and the bilayer behaving as a physical entity with collective physical properties (e.g. thickness, intrinsic monolayer curvature or elastic moduli). Studies in physico-chemical model systems have demonstrated that changes in bilayer physical properties can regulate membrane protein function by altering the energetic cost of the bilayer deformation associated with a protein conformational change. This type of regulation is well characterized, and its mechanistic elucidation is an interdisciplinary field bordering on physics, chemistry and biology. Changes in lipid composition that alter bilayer physical properties (including cholesterol, polyunsaturated fatty acids, other lipid metabolites and amphiphiles) regulate a wide range of membrane proteins in a seemingly non-specific manner. The commonality of the changes in protein function suggests an underlying physical mechanism, and recent studies show that at least some of the changes are caused by altered bilayer physical properties. This advance is because of the introduction of new tools for studying lipid bilayer regulation of protein function. The present review provides an introduction to the regulation of membrane protein function by the bilayer physical properties. We further describe the use of gramicidin channels as molecular force probes for studying this mechanism, with a unique ability to discriminate between consequences of changes in monolayer curvature and bilayer elastic moduli.
Collapse
Affiliation(s)
- Jens A Lundbaek
- Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY 10065, USA.
| | | | | | | | | |
Collapse
|
|
27
|
Protein-induced bilayer perturbations: Lipid ordering and hydrophobic coupling. Biochem Biophys Res Commun 2009; 387:760-5. [DOI: 10.1016/j.bbrc.2009.07.109] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2009] [Accepted: 07/21/2009] [Indexed: 11/20/2022]
|
|
28
|
Singh DK, Rosenhouse-Dantsker A, Nichols CG, Enkvetchakul D, Levitan I. Direct regulation of prokaryotic Kir channel by cholesterol. J Biol Chem 2009; 284:30727-36. [PMID: 19740741 DOI: 10.1074/jbc.m109.011221] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Our earlier studies have shown that channel activity of Kir2 subfamily of inward rectifiers is strongly suppressed by the elevation of cellular cholesterol. The goal of this study is to determine whether cholesterol suppresses Kir channels directly. To achieve this goal, purified prokaryotic Kir (KirBac1.1) channels were incorporated into liposomes of defined lipid composition, and channel activity was assayed by (86)Rb(+) uptake. Our results show that (86)Rb(+) flux through KirBac1.1 is strongly inhibited by cholesterol. Incorporation of 5% (mass cholesterol/phospholipid) cholesterol into the liposome suppresses (86)Rb(+) flux by >50%, and activity is completely inhibited at 12-15%. However, epicholesterol, a stereoisomer of cholesterol with similar physical properties, has significantly less effect on KirBac-mediated (86)Rb(+) uptake than cholesterol. Furthermore, analysis of multiple sterols suggests that cholesterol-induced inhibition of KirBac1.1 channels is mediated by specific interactions rather than by changes in the physical properties of the lipid bilayer. In contrast to the inhibition of KirBac1.1 activity, cholesterol had no effect on the activity of reconstituted KscA channels (at up to 250 microg/mg of phospholipid). Taken together, these observations demonstrate that cholesterol suppresses Kir channels in a pure protein-lipid environment and suggest that the interaction is direct and specific.
Collapse
Affiliation(s)
- Dev K Singh
- Department of Medicine, University of Illinois, Chicago, Illinois 60612, USA.
| | | | | | | | | |
Collapse
|
|
29
|
Abstract
To date, most of the major types of Kir channels, Kir2s, Kir3s, Kir4s, and Kir6s, have been found to partition into cholesterol-rich membrane domains and/or to be regulated by changes in the level of membrane cholesterol. Surprisingly, however, in spite of the structural similarities between different Kirs, effects of cholesterol on different types of Kir channels vary from cholesterol-induced decrease in the current density (Kir2 channels) to the loss of channel activity by cholesterol depletion (Kir4 channels) and loss of channel coupling by different mediators (Kir3 and Kir6 channels). Recently, we have gained initial insights into the mechanisms responsible for cholesterol-induced suppression Kir2 channels, but mechanisms underlying cholesterol sensitivity of other Kir channels are mostly unknown. The goal of this review is to present a summary of the current knowledge of the distinct effects of cholesterol on different types of Kir channels in vitro and in vivo.
Collapse
Affiliation(s)
- Irena Levitan
- Department of Medicine, Pulmonary Section, University of Illinois at Chicago, Chicago, IL 60612, USA.
| |
Collapse
|
|
30
|
Identification of a C-terminus domain critical for the sensitivity of Kir2.1 to cholesterol. Proc Natl Acad Sci U S A 2009; 106:8055-60. [PMID: 19416905 DOI: 10.1073/pnas.0809847106] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
A variety of ion channels are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. However, the mechanism underlying cholesterol sensitivity of ion channels is unknown. We have recently shown that an increase in membrane cholesterol suppresses inwardly rectifying K(+) (Kir2) channels that are responsible for maintaining membrane potential in a variety of cell types. Here we show that cholesterol sensitivity of Kir2 channels depends on a specific region of the C terminus of the cytosolic domain of the channel, the CD loop. Within this loop, the L222I mutation in Kir2.1 abrogates the sensitivity of the channel to cholesterol whereas a reverse mutation in the corresponding position in Kir2.3, I214L, has the opposite effect, increasing cholesterol sensitivity. Furthermore, the L222I mutation has a dominant negative effect on cholesterol sensitivity of Kir2.1 WT. Mutations of 2 additional residues in the CD loop in Kir2.1, N216D and K219Q, partially affect the sensitivity of the channel to cholesterol. Yet, whereas these mutations have been shown to affect activation of the channel by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], other mutations outside the CD loop that have been previously shown to affect activation of the channel by PI(4,5)P(2) had no effect on cholesterol sensitivity. Mutations of the lipid-facing residues of the outer transmembrane helix also had no effect. These findings provide insights into the structural determinants of the sensitivity of Kir2 channels to cholesterol, and introduce the critical role of the cytosolic domain in cholesterol dependent channel regulation.
Collapse
|
|
31
|
Søgaard R, Ebert B, Klaerke D, Werge T. Triton X-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABA(A) receptors in Xenopus oocytes. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2009; 1788:1073-80. [PMID: 19366585 DOI: 10.1016/j.bbamem.2009.02.001] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/21/2008] [Revised: 01/16/2009] [Accepted: 02/03/2009] [Indexed: 11/29/2022]
Abstract
Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na(+) and L-type Ca(2+) channels and GABA(A) receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA(A) receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA(A) alpha(1)beta(3)gamma(2S) receptor currents in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 muM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA(A) receptor currents and suppressed allosteric modulation by flunitrazepam at alpha(1)beta(3)gamma(2S) receptors. All effects were independent of the presence of a gamma(2S) subunit in the GABA(A) receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA(A) receptor through changes in lipid bilayer elasticity.
Collapse
Affiliation(s)
- Rikke Søgaard
- Institute of Cellular and Molecular Medicine, Panum 12.6, University of Copenhagen, Blegdamsvej 3C, DK-2200, Copenhagen N, Denmark.
| | | | | | | |
Collapse
|
|
32
|
Reeves D, Ursell T, Sens P, Kondev J, Phillips R. Membrane mechanics as a probe of ion-channel gating mechanisms. PHYSICAL REVIEW. E, STATISTICAL, NONLINEAR, AND SOFT MATTER PHYSICS 2008; 78:041901. [PMID: 18999449 PMCID: PMC3496789 DOI: 10.1103/physreve.78.041901] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/10/2008] [Indexed: 05/21/2023]
Abstract
The details of conformational changes undergone by transmembrane ion channels in response to stimuli, such as electric fields and membrane tension, remain controversial. We approach this problem by considering how the conformational changes impose deformations in the lipid bilayer. We focus on the role of bilayer deformations in the context of voltage-gated channels because we hypothesize that such deformations are relevant in this case as well as for channels that are explicitly mechanosensitive. As a result of protein conformational changes, we predict that the lipid bilayer suffers deformations with a characteristic free-energy scale of 10 k{B}T . This free energy is comparable to the voltage-dependent part of the total gating energy, and we argue that these deformations could play an important role in the overall free-energy budget of gating. As a result, channel activity will depend upon mechanical membrane parameters such as tension and leaflet thickness. We further argue that the membrane deformation around any channel can be divided into three generic classes of deformation that exhibit different mechanosensitive properties. Finally, we provide the theoretical framework that relates conformational changes during gating to tension and leaflet thickness dependence in the critical gating voltage. This line of investigation suggests experiments that could discern the dominant deformation imposed upon the membrane as a result of channel gating, thus providing clues as to the channel deformation induced by the stimulus.
Collapse
Affiliation(s)
- Daniel Reeves
- Department of Physics, Brandeis University, Waltham, Massachusetts 02454, USA.
| | | | | | | | | |
Collapse
|
|
33
|
Milescu M, Vobecky J, Roh SH, Kim SH, Jung HJ, Kim JI, Swartz KJ. Tarantula toxins interact with voltage sensors within lipid membranes. J Gen Physiol 2007; 130:497-511. [PMID: 17938232 PMCID: PMC2151668 DOI: 10.1085/jgp.200709869] [Citation(s) in RCA: 98] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2007] [Accepted: 09/27/2007] [Indexed: 11/20/2022] Open
Abstract
Voltage-activated ion channels are essential for electrical signaling, yet the mechanism of voltage sensing remains under intense investigation. The voltage-sensor paddle is a crucial structural motif in voltage-activated potassium (K(v)) channels that has been proposed to move at the protein-lipid interface in response to changes in membrane voltage. Here we explore whether tarantula toxins like hanatoxin and SGTx1 inhibit K(v) channels by interacting with paddle motifs within the membrane. We find that these toxins can partition into membranes under physiologically relevant conditions, but that the toxin-membrane interaction is not sufficient to inhibit K(v) channels. From mutagenesis studies we identify regions of the toxin involved in binding to the paddle motif, and those important for interacting with membranes. Modification of membranes with sphingomyelinase D dramatically alters the stability of the toxin-channel complex, suggesting that tarantula toxins interact with paddle motifs within the membrane and that they are sensitive detectors of lipid-channel interactions.
Collapse
Affiliation(s)
- Mirela Milescu
- Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
| | | | | | | | | | | | | |
Collapse
|
|
34
|
Kelkar DA, Chattopadhyay A. The gramicidin ion channel: A model membrane protein. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2007; 1768:2011-25. [PMID: 17572379 DOI: 10.1016/j.bbamem.2007.05.011] [Citation(s) in RCA: 279] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/10/2006] [Revised: 05/09/2007] [Accepted: 05/10/2007] [Indexed: 02/07/2023]
Abstract
The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid-protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media.
Collapse
Affiliation(s)
- Devaki A Kelkar
- Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | | |
Collapse
|
|
35
|
Adachi S, Nagao T, Ingolfsson HI, Maxfield FR, Andersen OS, Kopelovich L, Weinstein IB. The inhibitory effect of (-)-epigallocatechin gallate on activation of the epidermal growth factor receptor is associated with altered lipid order in HT29 colon cancer cells. Cancer Res 2007; 67:6493-501. [PMID: 17616711 DOI: 10.1158/0008-5472.can-07-0411] [Citation(s) in RCA: 165] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
(-)-Epigallocatechin gallate (EGCG), a major biologically active constituent of green tea, inhibits activation of the epidermal growth factor (EGF) receptor (EGFR) and downstream signaling pathways in several types of human cancer cells, but the precise mechanism is not known. Because several plasma membrane-associated receptor tyrosine kinases (RTK) including EGFR are localized in detergent-insoluble ordered membrane domains, so-called "lipid rafts," we examined whether the inhibitory effect of EGCG on activation of the EGFR is associated with changes in membrane lipid order in HT29 colon cancer cells. First, we did cold Triton X-100 solubility assays. Phosphorylated (activated) EGFR was found only in the Triton X-100-insoluble (lipid raft) fraction, whereas total cellular EGFR was present in the Triton X-100-soluble fraction. Pretreatment with EGCG inhibited the binding of Alexa Fluor 488-labeled EGF to the cells and also inhibited EGF-induced dimerization of the EGFR. To examine possible effects of EGCG on membrane lipid organization, we labeled the cells with the fluorescent lipid analogue 1, 1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, which preferentially incorporates into ordered membrane domains in cells and found that subsequent treatment with EGCG caused a marked reduction in the Triton X-100-resistant membrane fraction. Polyphenon E, a mixture of green tea catechins, had a similar effect but (-)-epicatechin (EC), the biologically inactive compound, did not significantly alter the Triton X-100 solubility properties of the membrane. Furthermore, we found that EGCG but not EC caused dramatic changes in the function of bilayer-incorporated gramicidin channels. Taken together, these findings suggest that EGCG inhibits the binding of EGF to the EGFR and the subsequent dimerization and activation of the EGFR by altering membrane organization. These effects may also explain the ability of EGCG to inhibit activation of other membrane-associated RTKs, and they may play a critical role in the anticancer effects of this and related compounds.
Collapse
Affiliation(s)
- Seiji Adachi
- Herbert Irving Comprehensive Cancer Center and Department of Medicine, Columbia University Medical Center, New Yourk, NY 10032-2704, USA
| | | | | | | | | | | | | |
Collapse
|
|
36
|
Bruno MJ, Koeppe RE, Andersen OS. Docosahexaenoic acid alters bilayer elastic properties. Proc Natl Acad Sci U S A 2007; 104:9638-43. [PMID: 17535898 PMCID: PMC1887599 DOI: 10.1073/pnas.0701015104] [Citation(s) in RCA: 118] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
At low micromolar concentrations, polyunsaturated fatty acids (PUFAs) alter the function of many membrane proteins. PUFAs exert their effects on unrelated proteins at similar concentrations, suggesting a common mode of action. Because lipid bilayers serve as the common "solvent" for membrane proteins, the common mechanism could be that PUFAs adsorb to the bilayer/solution interface to promote a negative-going change in lipid intrinsic curvature and, like other reversibly adsorbing amphiphiles, increase bilayer elasticity. PUFA adsorption thus would alter the bilayer deformation energy associated with protein conformational changes involving the protein/bilayer boundary, which would alter protein function. To explore the feasibility of such a mechanism, we used gramicidin (gA) analogues of different lengths together with bilayers of different thicknesses to assess whether docosahexaenoic acid (DHA) could exert its effects through a bilayer-mediated mechanism. Indeed, DHA increases gA channel appearance rates and lifetimes and decreases the free energy of channel formation. The appearance rate and lifetime changes increase with increasing channel-bilayer hydrophobic mismatch and are not related to differing DHA bilayer absorption coefficients. DHA thus alters bilayer elastic properties, not just lipid intrinsic curvature; the elasticity changes are important for DHA's bilayer-modifying actions. Oleic acid (OA), which has little effect on membrane protein function, exerts no such effects despite OA's adsorption coefficient being an order of magnitude greater than DHA's. These results suggest that DHA (and other PUFAs) may modulate membrane protein function by bilayer-mediated mechanisms that do not involve specific protein binding but rather changes in bilayer material properties.
Collapse
Affiliation(s)
- Michael J. Bruno
- *Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021; and
| | - Roger E. Koeppe
- Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701
| | - Olaf S. Andersen
- *Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021; and
- To whom correspondence should be addressed. E-mail:
| |
Collapse
|
|
37
|
Chattopadhyay A, Rawat SS. Wavelength-selective fluorescence in ion channels formed by gramicidin A in membranes. J CHEM SCI 2007. [DOI: 10.1007/s12039-007-0020-x] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
|
|
38
|
Gottlieb PA, Suchyna TM, Sachs F. Properties and Mechanism of the Mechanosensitive Ion Channel Inhibitor GsMTx4, a Therapeutic Peptide Derived from Tarantula Venom. CURRENT TOPICS IN MEMBRANES 2007; 59:81-109. [PMID: 25168134 DOI: 10.1016/s1063-5823(06)59004-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
Mechanosensitive ion channels (MSCs) are found in all types of cells ranging from Escherichia coli to morning glories to humans. They seem to fall into two families: those in specialized receptors, such as the hair cells of the cochlea, and those in cells not clearly differentiated for sensory duty. The physiological function of the channels in nonspecialized cells has not been demonstrated, although their activity has been demonstrated innumerable times in vitro. The only specific reagent to block MSCs isGsMTx4, a 4-kDa peptide isolated from tarantula venom. Despite being isolated from venom, it is nontoxic to mice. GsMTx4 is specific for an MSC subtype, the nonselective cation channels that may be members of the transient receptor potential (TRP) family. GsMTx4 acts as a gating modifier, increasing the energy of the open state relative to the closed state. The mirror image D enantiomer of GsMTx4 is equally active, so mode of action is not via the traditional lock and key model. GsMTx4 probably acts in the boundary lipid of the channel by changing local curvature and mechanically stressing the channel toward the closed state. Despite the lack of definitive physiological data on the function of the cationic MSCs, GsMTx4 may prove useful as a drug or lead compound that can affect physiological processes. These processes would be those driven by mechanical stress, such as blood vessel autoregulation, stress-induced contraction of smooth muscle, and Ca(2+) loading in muscular dystrophy.
Collapse
Affiliation(s)
- Philip A Gottlieb
- The Department of Physiology and Biophysics, Center for Single Molecule Biophysics, SUNY at Buffalo, Buffalo, New York 14214
| | - Thomas M Suchyna
- The Department of Physiology and Biophysics, Center for Single Molecule Biophysics, SUNY at Buffalo, Buffalo, New York 14214
| | - Frederick Sachs
- The Department of Physiology and Biophysics, Center for Single Molecule Biophysics, SUNY at Buffalo, Buffalo, New York 14214
| |
Collapse
|
|
39
|
Székely A, Kitajka K, Panyi G, Márián T, Gáspár R, Krasznai Z. Nutrition and immune system: certain fatty acids differently modify membrane composition and consequently kinetics of KV1.3 channels of human peripheral lymphocytes. Immunobiology 2007; 212:213-27. [PMID: 17412288 DOI: 10.1016/j.imbio.2007.01.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2006] [Revised: 11/21/2006] [Accepted: 01/16/2007] [Indexed: 10/23/2022]
Abstract
Potassium (K(+)) channels of human peripheral lymphocytes play a considerable role in the signalling processes required for immune responses. Modification of the fatty acid composition of the membrane influences the functions of various membrane enzymes and ion channels. We set out to establish how the incorporation of fatty acids with different carbon chain lengths and degrees of unsaturation into the cell membrane influences the function of K(V)1.3 channels of lymphocytes, thereby potentially modifying the immune responses of the cells. The incorporation of the fatty acids into the cell membrane was monitored by gas chromatography. Whole-cell patch-clamp experiments demonstrated that the polyunsaturated linoleic acid, arachidonic acid and docosahexaenoic acid all decreased the activation and inactivation time constants of the K(V)1.3 channels, but did not affect the voltage-dependence of steady-state activation and steady-state inactivation of the channels. Treatment with the saturated palmitic acid, stearic acid and the monounsaturated oleic acid did not result in significant changes in the biophysical parameters of K(V)1.3 gating studied. We conclude that the incorporation of fatty acids unsaturated to different degrees into the cell membrane of lymphocytes influenced the rate of gating transitions but not the equilibrium distribution of the channels between different states. This effect depended on the degree of unsaturation and the chain length of the fatty acids: no effects of saturated and monounsaturated fatty acids (16:0, 18:0 and 18:1) were observed whereas treatment with polyunsaturated fatty acids (18:2, 20:4 and 22:6) resulted in significant changes in the channel kinetics.
Collapse
Affiliation(s)
- Andrea Székely
- Faculty of Medicine, Department of Biophysics and Cell Biology, Medical and Health Science Centre, University of Debrecen, H-4032 Debrecen, Nagyerdei krt, 98, Hungary
| | | | | | | | | | | |
Collapse
|
|
40
|
Bowman CL, Gottlieb PA, Suchyna TM, Murphy YK, Sachs F. Mechanosensitive ion channels and the peptide inhibitor GsMTx-4: history, properties, mechanisms and pharmacology. Toxicon 2007; 49:249-70. [PMID: 17157345 PMCID: PMC1852511 DOI: 10.1016/j.toxicon.2006.09.030] [Citation(s) in RCA: 140] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
Sensing the energy from mechanical inputs is ubiquitous--and perhaps the oldest form of biological energy transduction. However, the tools available to probe the mechanisms of transduction are far fewer than for the chemical and electric field sensitive transducers. The one pharmacological tool available for mechansensitive ion channels (MSCs) is a peptide (GsMTx-4) isolated from venom of the tarantula, Grammostola spatulata, that blocks cationic MSCs found in non-specialized eukaryotic tissues. In this review, we summarize the current knowledge of GsMTx-4, and discuss the inevitable crosstalk between the MSC behavior and the mechanical properties of the cell cortex.
Collapse
Affiliation(s)
- Charles L Bowman
- Center for Single Molecule Biophysics and The Department of Physiology and Biophysics, SUNY at Buffalo, Buffalo, NY 14214, USA.
| | | | | | | | | |
Collapse
|
|
41
|
Kelkar DA, Chattopadhyay A. Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2007; 1768:1103-13. [PMID: 17321493 DOI: 10.1016/j.bbamem.2007.01.016] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/04/2006] [Revised: 01/18/2007] [Accepted: 01/22/2007] [Indexed: 11/25/2022]
Abstract
The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.
Collapse
Affiliation(s)
- Devaki A Kelkar
- Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
| | | |
Collapse
|
|
42
|
Andersen OS, Bruno MJ, Sun H, Koeppe RE. Single-molecule methods for monitoring changes in bilayer elastic properties. Methods Mol Biol 2007; 400:543-570. [PMID: 17951759 DOI: 10.1007/978-1-59745-519-0_37] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/25/2023]
Abstract
Membrane-spanning proteins perturb the organization and dynamics of the adjacent bilayer lipids. For example, when the hydrophobic length (l) of a bilayer-spanning protein differs from the average thickness (d0) of the host bilayer, the bilayer thickness will vary locally in the vicinity of the protein in order to "match" the length of the protein's hydrophobic exterior to the thickness of the bilayer hydrophobic core. Such bilayer deformations incur an energetic cost, the bilayer deformation energy (DeltaG0def), which will vary as a function of the protein shape, the protein-bilayer hydrophobic mismatch (d0 - l), the lipid bilayer elastic properties, and the lipid intrinsic curvature (c0). Thus, if the membrane protein conformational changes underlying protein function involve the protein/bilayer interface, the ensuing changes in DeltaG0def (DeltaDeltaG0def) will contribute to the overall free-energy change of the conformational changes (DeltaG0tot)-meaning that the host lipid bilayer will modulate protein function. For a given protein, (DeltaDeltaG0def) varies as a function of the bilayer geometric properties (thickness and intrinsic curvature) and the elastic (bending and compression) moduli, which vary as a function of changes in lipid composition or with the adsorption of amphiphiles at the bilayer/solution interface. To understand how changes in bilayer properties modulate the function of bilayer-spanning proteins, single-molecule methods have been developed to probe changes in bilayer elastic properties using gramicidins as molecular force transducers. Different approaches to measuring the deformation energy are described: (1) measurements of changes in channel lifetimes and appearance rates as the lipid bilayer thickness or channel length are varied, (2) measurements of the equilibrium distribution among channels of different lengths, formed by homo- and heterodimers between gramicidin subunits of different lengths, and (3) measurements of the ratio of the appearance rates of heterodimer channels relative to parent homodimer channels formed by gramicidin subunits of different lengths.
Collapse
Affiliation(s)
- Olaf S Andersen
- Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY, USA
| | | | | | | |
Collapse
|
|
43
|
Artigas P, Al'aref SJ, Hobart EA, Díaz LF, Sakaguchi M, Straw S, Andersen OS. 2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms: the role of bilayer material properties. Mol Pharmacol 2006; 70:2015-26. [PMID: 16966478 DOI: 10.1124/mol.106.026070] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
2,3-Butanedione monoxime (BDM) is widely believed to act as a chemical phosphatase. We therefore examined the effects of BDM on the cystic fibrosis transmembrane regulator (CFTR) Cl(-) channel, which is regulated by phosphorylation in a complex manner. In guinea pig ventricular myocytes, forskolin-activated whole-cell CFTR currents responded biphasically to external 20 mM BDM: a rapid approximately 2-fold current activation was followed by a slower (tau approximately 20 s) inhibition (to approximately 20% of control). The inhibitory response was abolished by intracellular dialysis with the phosphatase inhibitor microcystin, suggesting involvement of endogenous phosphatases. The BDM-induced activation was studied further in Xenopus laevis oocytes expressing human epithelial CFTR. The concentration for half-maximal BDM activation (K(0.5)) was state-dependent, approximately 2 mM for highly and approximately 20 mM for partially phosphorylated channels, suggesting a modulated receptor mechanism. Because BDM modulates many different membrane proteins with similar K(0.5) values, we tested whether BDM could alter protein function by altering lipid bilayer properties rather than by direct BDM-protein interactions. Using gramicidin channels of different lengths (different channel-bilayer hydrophobic mismatch) as reporters of bilayer stiffness, we found that BDM increases channel appearance rates and lifetimes (reduces bilayer stiffness). At 20 mM BDM, the appearance rates increase approximately 4-fold (for the longer, 15 residues/monomer, channels) to approximately 10-fold (for the shorter, 13 residues/monomer channels); the lifetimes increase approximately 50% independently of channel length. BDM thus reduces the energetic cost of bilayer deformation, an effect that may underlie the effects of BDM on CFTR and other membrane proteins; the state-dependent changes in K(0.5) are consistent with such a bilayer-mediated mechanism.
Collapse
Affiliation(s)
- Pablo Artigas
- Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, 1230 York Ave., New York, NY 10021, USA.
| | | | | | | | | | | | | |
Collapse
|
|
44
|
Chen TY, Takeuchi H, Kurahashi T. Odorant inhibition of the olfactory cyclic nucleotide-gated channel with a native molecular assembly. ACTA ACUST UNITED AC 2006; 128:365-71. [PMID: 16940558 PMCID: PMC2151561 DOI: 10.1085/jgp.200609577] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Human olfaction comprises the opposing actions of excitation and inhibition triggered by odorant molecules. In olfactory receptor neurons, odorant molecules not only trigger a G-protein–coupled signaling cascade but also generate various mechanisms to fine tune the odorant-induced current, including a low-selective odorant inhibition of the olfactory signal. This wide-range olfactory inhibition has been suggested to be at the level of ion channels, but definitive evidence is not available. Here, we report that the cyclic nucleotide-gated (CNG) cation channel, which is a key element that converts odorant stimuli into electrical signals, is inhibited by structurally unrelated odorants, consistent with the expression of wide-range olfactory inhibition. Interestingly, the inhibitory effect was small in the homo-oligomeric CNG channel composed only of the principal channel subunit, CNGA2, but became larger in channels consisting of multiple types of subunits. However, even in the channel containing all native subunits, the potency of the suppression on the cloned CNG channel appeared to be smaller than that previously shown in native olfactory neurons. Nonetheless, our results further showed that odorant suppressions are small in native neurons if the subsequent molecular steps mediated by Ca2+ are removed. Thus, the present work also suggests that CNG channels switch on and off the olfactory signaling pathway, and that the on and off signals may both be amplified by the subsequent olfactory signaling steps.
Collapse
Affiliation(s)
- Tsung-Yu Chen
- Center for Neuroscience and Department of Neurology, University of California, Davis, CA 95616, USA.
| | | | | |
Collapse
|
|
45
|
Shlyonsky VG, Markin VS, Andreeva I, Pedersen SE, Simon SA, Benos DJ, Ismailov II. Role of membrane curvature in mechanoelectrical transduction: Ion carriers nonactin and valinomycin sense changes in integral bending energy. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2006; 1758:1723-31. [PMID: 17069752 DOI: 10.1016/j.bbamem.2006.09.016] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/21/2006] [Revised: 09/13/2006] [Accepted: 09/15/2006] [Indexed: 10/24/2022]
Abstract
We describe the phenomenon of mechanoelectrical transduction in macroscopic lipid bilayer membranes modified by two cation-selective ionophores, valinomycin and nonactin. We found that bulging these membranes, while maintaining the membrane tension constant, produced a marked supralinear increase in specific carrier-mediated conductance. Analyses of the mechanisms involved in mechanoelectrical transduction induced by the imposition of a hydrostatic pressure gradient or by an amphipathic compound chlorpromazine reveal similar changes in the charge carrier motility and carrier reaction rates at the interface(s). Furthermore, the relative change in membrane conductance was independent of membrane diameter, but was directly proportional to the square of membrane curvature, thus relating the observed phenomena to the bilayer bending energy. Extrapolated to biological membranes, these findings indicate that ion transport in cells can be influenced simply by changing shape of the membrane, without a change in membrane tension.
Collapse
Affiliation(s)
- V Gh Shlyonsky
- Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | | | | | | | | | | | | |
Collapse
|
|
46
|
Pashkovskaya AA, Lukashev EP, Antonov PE, Finogenova OA, Ermakov YA, Melik-Nubarov NS, Antonenko YN. Grafting of polylysine with polyethylenoxide prevents demixing of O-pyromellitylgramicidin in lipid membranes. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2006; 1758:1685-95. [PMID: 16901462 DOI: 10.1016/j.bbamem.2006.06.011] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/13/2006] [Revised: 05/19/2006] [Accepted: 06/06/2006] [Indexed: 01/24/2023]
Abstract
Both natural and synthetic polycations can induce demixing of negatively charged components in artificial and possibly in natural membranes. This process can result in formation of clusters (binding of several components to a polycation chain) and/or domains (aggregation of clusters and formation of a separate phase enriched in some particular component). In order to distinguish between these two phenomena, a model lipid membrane system containing ion channels, formed by a negatively charged peptide, O-pyromellitylgramicidin, and polycations of different structures was used. Microelectrophoresis of liposomes, changes in boundary potential of planar bilayers, the shape of compression curves and potentials of lipid and lipid/peptide monolayers were used to monitor the electrostatic factors in polymer adsorption to the membrane and peptide-polymer interactions. The synthesized PEO-grafted polylysine, PLL-PEO20000, did not induce peptide demixing monitored by stabilization of the gramicidin channels, in contrast to parent polylysine (PLL). Both polymers were shown to bind effectively to negatively charged liposomes and lipid monolayers, suggesting that the ineffectiveness of PLL-PEO20000 was not due to reduction of its binding. It was hypothesized that PLL-PEO20000 could not induce domain formation due to steric hindrance of long PEO chains preventing lateral fusion of clusters. Another copolymer, PLL-PEO4000, having four PEO chains of 4000 Da, exhibited intermediate effect between PLL and PLL-PEO20000, which shows the importance of the copolymer architecture for the effect on the lateral distribution of OPg channels. The model system can be relevant to regulation of lateral organization of ion channels and other components in natural membrane systems.
Collapse
Affiliation(s)
- A A Pashkovskaya
- A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russian Federation
| | | | | | | | | | | | | |
Collapse
|
|
47
|
Oz M. Receptor-independent actions of cannabinoids on cell membranes: Focus on endocannabinoids. Pharmacol Ther 2006; 111:114-44. [PMID: 16584786 DOI: 10.1016/j.pharmthera.2005.09.009] [Citation(s) in RCA: 126] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2005] [Accepted: 09/30/2005] [Indexed: 01/08/2023]
Abstract
Cannabinoids are a structurally diverse group of mostly lipophilic molecules that bind to cannabinoid receptors. In fact, endogenous cannabinoids (endocannabinoids) are a class of signaling lipids consisting of amides and esters of long-chain polyunsaturated fatty acids. They are synthesized from lipid precursors in plasma membranes via Ca(2+) or G-protein-dependent processes and exhibit cannabinoid-like actions by binding to cannabinoid receptors. However, endocannabinoids can produce effects that are not mediated by these receptors. In pharmacologically relevant concentrations, endocannabinoids modulate the functional properties of voltage-gated ion channels including Ca(2+) channels, Na(+) channels, various types of K(+) channels, and ligand-gated ion channels such as serotonin type 3, nicotinic acetylcholine, and glycine receptors. In addition, modulatory effects of endocannabinoids on other ion-transporting membrane proteins such as transient potential receptor-class channels, gap junctions and transporters for neurotransmitters have also been demonstrated. Furthermore, functional properties of G-protein-coupled receptors for different types of neurotransmitters and neuropeptides are altered by direct actions of endocannabinoids. Although the mechanisms of these effects are currently not clear, it is likely that these direct actions of endocannabinoids are due to their lipophilic structures. These findings indicate that additional molecular targets for endocannabinoids exist and that these targets may represent novel sites for cannabinoids to alter either the excitability of the neurons or the response of the neuronal systems. This review focuses on the results of recent studies indicating that beyond their receptor-mediated effects, endocannabinoids alter the functions of ion channels and other integral membrane proteins directly.
Collapse
Affiliation(s)
- Murat Oz
- National Institute on Drug Abuse, NIH/DHHS, Intramural Research Program, Cellular Neurobiology Branch, 5500 Nathan Shock Drive, Baltimore MD, 21224, USA.
| |
Collapse
|
|
48
|
McIntosh TJ, Simon SA. ROLES OF BILAYER MATERIAL PROPERTIES IN FUNCTION AND DISTRIBUTION OF MEMBRANE PROTEINS. ACTA ACUST UNITED AC 2006; 35:177-98. [PMID: 16689633 DOI: 10.1146/annurev.biophys.35.040405.102022] [Citation(s) in RCA: 186] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Structural, compositional, and material (elastic) properties of lipid bilayers exert strong influences on the interactions of water-soluble proteins and peptides with membranes, the distribution of transmembrane proteins in the plane of the membrane, and the function of specific membrane channels. Theoretical and experimental studies show that the binding of either cytoplasmic proteins or extracellular peptides to membranes is regulated by the presence of charged lipids and that the sorting of transmembrane proteins into or out of membrane microdomains (rafts) depends on several factors, including bilayer material properties governed by the presence of cholesterol. Recent studies have also shown that bilayer material properties modify the permeability of membrane pores, formed either by protein channels or by cell-lytic peptides.
Collapse
Affiliation(s)
- Thomas J McIntosh
- Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
| | | |
Collapse
|
|
49
|
Sobko AA, Kotova EA, Antonenko YN, Zakharov SD, Cramer WA. Lipid Dependence of the Channel Properties of a Colicin E1-Lipid Toroidal Pore. J Biol Chem 2006; 281:14408-16. [PMID: 16556601 DOI: 10.1074/jbc.m513634200] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 A, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.
Collapse
Affiliation(s)
- Alexander A Sobko
- A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russia
| | | | | | | | | |
Collapse
|
|
50
|
Sinclair J, Olofsson J, Phil J, Orwar O. Stabilization of high-resistance seals in patch-clamp recordings by laminar flow. Anal Chem 2006; 75:6718-22. [PMID: 16465721 DOI: 10.1021/ac0346611] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The formation of a high-resistance electrical seal between a cell membrane and a glass micropipet tip is essential in patch-clamp experiments. We have studied the electrical properties and the mechanical stability of the seal using a microfluidic chip generating laminar flow in open volumes. We show that, by using fluid flow (1-10 mm/s) acting along the symmetry axis of the cell-pipet, seals of a higher mechanical stability with increased resistances can be achieved, allowing up to 100% longer recording times and over 40% decreased noise levels (Irms). These improved properties are beneficial for high-sensitivity patch-clamp recordings, in particular, in longtime studies of ion channel receptor systems that are relevant in biosensor applications of the technique. Furthermore, these observations support the combination of patch-clamp with microfluidic devices, for example, for rapid solution exchange around a single cell sensor for high-throughput electrophysiology and for highly resolved kinetic studies.
Collapse
Affiliation(s)
- Jon Sinclair
- Department of Physical Chemistry and Microtechnology Centre, Chalmers University of Technology, SE-412 96 Göteborg, Sweden
| | | | | | | |
Collapse
|