In the absence of Na(+) and K(+) ions the Na,K-ATPase shows a pH-dependent ATP hydrolysis that can be inhibited by ouabain. At pH 7.2 this activity is 5% of the maximal under physiological conditions. It could be inferred that this activity is associated with H(+) transport in both directions across the membrane and facilitates an H-only mode of the sodium pump under such unphysiological conditions. By the analysis of experiments with reconstituted proteoliposomes an overall electroneutral transport mode has been proven. The stoichiometry was determined to be 2 H(+)/2 H(+)/1 ATP and is comparable to what is known from the closely related H,K-ATPase. By time-resolved ATP-concentration jump experiments it was found that at no time was the third, Na(+)-specific binding site of the pump occupied by protons. A modified Post-Albers pump cycle is proposed, with H(+) ions as congeners for Na(+) and K(+), by which all experiments performed can be explained.

KCNN4 channels that provide the driving force for cAMP- and Ca(2+)-induced anion secretion are present in both apical and basolateral membranes of the mammalian colon. However, only a single KCNN4 has been cloned. This study was initiated to identify whether both apical and basolateral KCNN4 channels are encoded by the same or different isoforms. Reverse transcriptase-PCR (RT-PCR), real-time quantitative-PCR (RT-QPCR), and immunofluorescence studies were used to clone and identify tissue-specific expression of KCNN4 isoforms. Three distinct KCNN4 cDNAs that are designated as KCNN4a, KCNN4b, and KCNN4c encoding 425, 424, and 395 amino acid proteins, respectively, were isolated from the rat colon. KCNN4a differs from KCNN4b at both the nucleotide and the amino acid level with distinct 628 bp at the 3'-untranslated region and an additional glutamine at position 415, respectively. KCNN4c differs from KCNN4b by lacking the second exon that encodes a 29 amino acid motif. KCNN4a and KCNN4b/c are identified as smooth muscle- and epithelial cell-specific transcripts, respectively. KCNN4b and KCNN4c transcripts likely encode basolateral (40 kDa) and apical (37 kDa) membrane proteins in the distal colon, respectively. KCNN4c, which lacks the S2 transmembrane segment, requires coexpression of a large conductance K(+) channel beta-subunit for plasma membrane expression. The KCNN4 channel blocker TRAM-34 inhibits KCNN4b- and KCNN4c-mediated (86)Rb (K(+) surrogate) efflux with an apparent inhibitory constant of 0.6 +/- 0.1 and 7.8 +/- 0.4 muM, respectively. We conclude that apical and basolateral KCNN4 K(+) channels that regulate K(+) and anion secretion are encoded by distinct isoforms in colonic epithelial cells.

The Na/K pump, or Na,K-ATPase, is a key enzyme to the homeostasis of osmotic pressure, cell volume, and the maintenance of electrochemical gradients. Its alpha subunit, which holds most of its functions, belongs to a large family of ATPases known as P-type, and to the subfamily IIC, which also includes H,K-ATPases. In this study, we attempt to describe the evolutionary history of IIC ATPases by doing phylogenetic analysis with most of the currently available protein sequences (over 200), and pay special attention to the relationship between their diversity and their osmoregulatory role. We include proteins derived from many completed or ongoing genome projects, many of whose IIC ATPases have not been phylogenetically analyzed previously. We show that the most likely origin of IIC proteins is prokaryotic, and that many of them are present in non-metazoans, such as algae, protozoans or fungi. We also suggest that the pre-metazoan ancestor, represented by the choanoflagellate Monosiga brevicollis, whose genome has been sequenced, presented at least two IIC-type proteins. One of these proteins would have given rise to most current animal IIC ATPases, whereas the other apparently evolved into a lineage that, so far, has only been found in nematodes. We also propose that early deuterostomes presented a single IIC gene, from which all the extant diversity of vertebrate IIC proteins originated by gene and genome duplications.

Since it was discovered 3 decades ago the H,K-ATPase has come to be recognized as the key both to the generation and pharmacologic suppression of gastric acid secretion. Although 30 years of concerted research has answered many questions, it is perhaps not surprising that these efforts have raised many new and crucial issues that await elucidation. These can be divided into 5 broad categories that relate to structure, mechanism, regulation, trafficking, and macromolecular interactions. It is probably safe to predict that the growing sophistication of x-ray crystallographic techniques will yield a picture of the pump's molecular structure in the near future. These insights will, in turn, illuminate the details of the process through which enzymatic hydrolysis is coupled to ion translocation with unprecedented clarity. The gastric parietal cell employs an extremely complicated system of receptors, kinases, and second messengers to maintain tight control over pump function. Upon activation, this cell also performs a massive and elegant membrane trafficking transformation that plays a critical role in the regulatory process. Finally, it is becoming clear that every ion transport protein is a component in a large macromolecular complex whose constituents help to determine all of the transport system's fundamental physiologic properties. These are the major topics that will drive H,K pump research in the future, and it is likely that their resolution will create the foundations for the next generation of therapies aimed at controlling gastric acid secretion and its clinical consequences.

Gastric acid secretion is a complex process that requires hormonal, neuronal, or calcium-sensing receptor activation for insertion of pumps into the apical surface of the parietal cell. Activation of any or all these pathways causes the parietal cell to secrete concentrated acid with a pH at or close to 1. This acidic fluid combines with enzymes that are secreted from neighbouring chief cells and passes out of the gland up through a mucous gel layer covering the surface of the stomach producing a final intragastric pH of less than 4 during the active phase of acid secretion. Defects in either the mucosal barrier or in the regulatory mechanisms that modulate the secretory pathways will result in erosion of the barrier and ulcerations of the stomach or esophagus. The entire process of acid secretion relies on activation of the catalytic cycle of the gastric H+,K+-ATPase, resulting in the secretion of acid into the parietal cell canaliculus, with K+ being the important and rate-limiting ion in this activation process. In addition to K+ as a rate limiter for acid production, Cl- secretion via an apical channel must also occur. In this review we present a discussion of the mechanics of acid secretion and a discussion of recently identified transporter proteins and receptors. Included is a discussion of some of the recent candidates for the apical K' recycling channel, as well as two recently identified apical proteins (NHE-3, PAT-1), and the newly characterized calcium-sensing receptor (CaSR). We hope that this review will give additional insight into the complex process of acid secretion.

The effect of point mutation in the sequence 316TWLE319, which occurs in the extracellular loop flanking the third (M3) and the fourth (M4) transmembrane segment (L3/4) of the Na+,K+-ATPase alpha-subunit, was examined. Mutation of Glu319 to Asp yielded an enzyme with full activity, whereas substituting Glu319 to Ala resulted in a severe loss of activity. A negative charge was introduced along the sequence, one residue at a time, from Thr316 to Leu318 (by E-scanning) in the mutant construct with Glu319 already mutated to Gln. The activity that had been reduced to 60% by the mutation of Glu319 to Gln was restored upon the introduction of a negative charge by E-scanning. When Leu318 was replaced by Glu in a series of scanning experiments, the K+ sensitivity of the ATPase activity was lowered. The lowering of K+ sensitivity was further demonstrated when a mutation of Leu318 to Glu was introduced into the wild-type enzyme. Furthermore, mutants with Leu318 to Gln, Arg, and Phe displayed lower K+ sensitivity similar to that of Leu318 to Glu mutant. Leu318 may be in access path for K+, and any substitution at this position may interfere with access of K+ from outside the cell.

P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolution is available only for one species, the SR Ca-ATPase. However, biochemical and biophysical studies provide an abundance of details on the function of this class of ion pumps. The aim of this review is to summarize the results of preferentially biophysical investigations of the three best-studied ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to compare functional properties to recent structural insights with the aim of contributing to the understanding of their structure-function relationship.

Epithelial Na(+) channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na(+) selectivity, unitary Na(+) conductance (gamma(Na)), and amiloride sensitivity of delta-ENaC are strikingly different from those of alpha-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of delta- and alpha-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li(+) and Na(+) (P(Li)/P(Na)) for the swap alpha chimeras co-expressed with betagamma-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of P(Li)/P(Na) for the swap delta constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the gamma(Na) of alpha-ENaC but decreased the gamma(Na) of delta-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (K(i)(amil)) was observed in the alpha chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of K(i)(amil) was obtained in the chimeric delta constructs with substitution of the H2 and pre-H2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the P(Li)/P(Na) ratio, single channel Na(+) conductance, and amiloride sensitivity of alpha- and delta-ENaCs.

The mechanism of proton pumping by P-type plasma membrane H(+)-ATPases is not well clarified. Site-directed mutagenesis studies suggest that Asp684, situated in transmembrane segment M6, is involved in coordination of proton(s) in plant plasma membrane H(+)-ATPase. This hypothesis is supported by atomic models of H(+)-ATPases built on the basis of the crystal structure of the related SERCA1a Ca(2+)-ATPase. However, more biochemical, genetic, and structural studies are required before we will be able to understand the nature of the proton binding site(s) in P-type H(+)-ATPases and the mechanism of action of these pumps.

The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase alpha-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His(6) tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [gamma-(32)P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in (32)P incorporation into either the His(6) tag or glutathione S-transferase fusion proteins. The MgATP-induced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 microm eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.