Targeting ATP12A, a Nongastric Proton Pump α Subunit, for Idiopathic Pulmonary Fibrosis Treatment.

Idiopathic pulmonary fibrosis (IPF) is a pathological condition of unknown etiology that results from injury to the lung and an ensuing fibrotic response that leads to the thickening of the alveolar walls and obliteration of the alveolar space. The pathogenesis is not clear, and there are currently no effective therapies for IPF. Small airway disease and mucus accumulation are prominent features in IPF lungs, similar to cystic fibrosis lung disease. The ATP12A gene encodes the α-subunit of the nongastric H+, K+-ATPase, which functions to acidify the airway surface fluid and impairs mucociliary transport function in patients with cystic fibrosis. It is hypothesized that the ATP12A protein may play a role in the pathogenesis of IPF. The authors' studies demonstrate that ATP12A protein is overexpressed in distal small airways from the lungs of patients with IPF compared with normal human lungs. In addition, overexpression of the ATP12A protein in mouse lungs worsened bleomycin induced experimental pulmonary fibrosis. This was prevented by a potassium competitive proton pump blocker, vonoprazan. These data support the concept that the ATP12A protein plays an important role in the pathogenesis of lung fibrosis. Inhibition of the ATP12A protein has potential as a novel therapeutic strategy in IPF treatment.

Proton pump inhibitors decrease eotaxin-3/CCL26 expression in patients with chronic rhinosinusitis with nasal polyps: Possible role of the nongastric H,K-ATPase.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by tissue eosinophilia that is associated with poor prognosis. Recent findings that proton pump inhibitors (PPIs) directly modulate the expression of eotaxin-3, an eosinophil chemoattractant, in patients with eosinophilic diseases suggest therapeutic potential for PPIs in those with CRSwNP. OBJECTIVE: We assessed the effect of type 2 mediators, particularly IL-13 and eotaxin-3, on tissue eosinophilia and disease severity in patients with chronic rhinosinusitis (CRS). Further investigation focused on PPI suppression of eotaxin-3 expression in vivo and in vitro, with exploration of underlying mechanisms. METHODS: Type 2 mediator levels in nasal tissues and secretions were measured by using a multiplex immunoassay. Eotaxin-3 and other chemokines expressed in IL-13-stimulated human sinonasal epithelial cells (HNECs) and BEAS-2B cells with or without PPIs were assessed by using ELISA, Western blotting, real-time PCR, and intracellular pH imaging. RESULTS: Nasal tissues and secretions from patients with CRSwNP had increased IL-13, eotaxin-2, and eotaxin-3 levels, and these were positively correlated with tissue eosinophil cationic protein levels and radiographic scores in patients with CRS (P < .05). IL-13 stimulation of HNECs and BEAS-2B cells dominantly induced eotaxin-3 expression, which was significantly inhibited by PPIs (P < .05). Patients with CRS taking PPIs also showed lower in vivo eotaxin-3 levels compared with those without PPIs (P < .05). Using intracellular pH imaging and altering extracellular K+, we found that IL-13 enhanced H+,K+-exchange, which was blocked by PPIs and the mechanistically unrelated H,K-ATPase inhibitor, SCH-28080. Furthermore, knockdown of ATP12A (gene for the nongastric H,K-ATPase) significantly attenuated IL-13-induced eotaxin-3 expression in HNECs. PPIs also had effects on accelerating IL-13-induced eotaxin-3 mRNA decay. CONCLUSION: Our results demonstrated that PPIs reduce IL-13-induced eotaxin-3 expression by airway epithelial cells. Furthermore, mechanistic studies suggest that the nongastric H,K-ATPase is necessary for IL-13-mediated epithelial responses, and its inhibitors, including PPIs, might be of therapeutic value in patients with CRSwNP by reducing epithelial production of eotaxin-3.

A link between fertility and K+ homeostasis: role of the renal H,K-ATPase type 2.

Renal K(+) retention is activated during pregnancy through a mechanism unknown to date. Here, we showed that the renal stimulation of H,K-ATPase type 2 (HKA2), whose expression was recently identified to be progesterone-dependent, is part of the mechanism favoring K(+) accumulation during gestation. Moreover, investigation of the gestational phenotype of HKA2-null mice compared to their wild-type (WT) littermate revealed a decrease in fertility (gestation was successful in 33 % of HKA2-null mice vs. 83 % of WT mice) and in litter size (6.5 ± 0.6 and 7.8 ± 0.4 fetuses per litter, respectively). We also observed that urinary K(+) excretion decreased by 20 % and plasma K(+) concentration rose slightly (11 %) in WT mice during gestation (relative to basal conditions). In contrast, the renal excretion of K(+) and plasma K(+) levels in HKA2-null mice remained constant during gestation, whereas fecal K(+) excretion increased. As a consequence, HKA2-null mice did not accumulate K(+) in their extracellular compartment as efficiently as WT mice did. Finally, the link between inefficient K(+) balance adaptations and gestational complications was established when we observed that these complications could be reversed with an increased K(+) uptake. Altogether, these results define a novel physiological role for the HKA2 transporter and uncover a link between K(+) metabolism and fertility.

Postnatal regulation of X,K-ATPases in rat skin and conserved lateroapical polarization of Na,K-ATPase in vertebrate epidermis.

Development of epidermis creates stratified epithelium with different sets of ion-transporting enzymes in its layers. We have characterized expression of Na,K- and H,K-ATPase α and ß subunits and FXYD isoforms in rat skin. Maturation of rat skin from newborn to adult is associated with an increase in FXYD4 and a decrease of Na,K-ATPase α1-isoform, ATP1B4 and FXYD6 transcripts. Na,K-ATPase of rat epidermis is represented predominantly by α1 and ß3 isoforms. Keratinization is associated with the loss of the Na,K-ATPase α-subunit and an enrichment of αng. Na,K-ATPase α1 is abundant in the innermost layer, stratum basale, where it is lacking in basal membranes, thus indicating lateroapical polarization of Na,K-ATPase. Immunocytochemical detection of Na,K-ATPase in Xenopus laevis skin shows that cellular and subcellular localization of the enzyme has a pattern highly similar to that of mammals: basolateral in glandular epithelium and lateroapical in epidermis.

Eutherian-Specific Functions of BetaM Acquired through Atp1b4 Gene Co-Option in the Regulation of MyoD Expression.

Vertebrate ATP1B4 genes represent a rare instance of orthologous gene co-option, resulting in radically different functions of the encoded BetaM proteins. In lower vertebrates, BetaM is a Na, K-ATPase ß-subunit that is a component of ion pumps in the plasma membrane. In placental mammals, BetaM lost its ancestral role and, through structural alterations of the N-terminal domain, became a skeletal and cardiac muscle-specific protein of the inner nuclear membrane, highly expressed during late fetal and early postnatal development. We previously determined that BetaM directly interacts with the transcriptional co-regulator SKI-interacting protein (SKIP) and is implicated in the regulation of gene expression. This prompted us to investigate a potential role for BetaM in the regulation of muscle-specific gene expression in neonatal skeletal muscle and cultured C2C12 myoblasts. We found that BetaM can stimulate expression of the muscle regulatory factor (MRF), MyoD, independently of SKIP. BetaM binds to the distal regulatory region (DRR) of MyoD, promotes epigenetic changes associated with activation of transcription, and recruits the SWI/SNF chromatin remodeling subunit, BRG1. These results indicate that eutherian BetaM regulates muscle gene expression by promoting changes in chromatin structure. These evolutionarily acquired new functions of BetaM might be very essential and provide evolutionary advantages to placental mammals.

Structural evolution and tissue-specific expression of tetrapod-specific second isoform of secretory pathway Ca2+-ATPase.

Secretory pathway Ca-ATPases are less characterized mammalian calcium pumps than plasma membrane Ca-ATPases and sarco-endoplasmic reticulum Ca-ATPases. Here we report analysis of molecular evolution, alternative splicing, tissue-specific expression and subcellular localization of the second isoform of the secretory pathway Ca-ATPase (SPCA2), the product of the ATP2C2 gene. The primary structure of SPCA2 from rat duodenum deduced from full-length transcript contains 944 amino acid residues, and exhibits 65% sequence identity with known SPCA1. The rat SPCA2 sequence is also highly homologous to putative human protein KIAA0703, however, the latter seems to have an aberrant N-terminus originating from intron 2. The tissue-specificity of SPCA2 expression is different from ubiquitous SPCA1. Rat SPCA2 transcripts were detected predominantly in gastrointestinal tract, lung, trachea, lactating mammary gland, skin and preputial gland. In the newborn pig, the expression profile is very similar with one remarkable exception: porcine bulbourethral gland gave the strongest signal. Upon overexpression in cultured cells, SPCA2 shows an intracellular distribution with remarkable enrichment in Golgi. However, in vivo SPCA2 may be localized in compartments that differ among various tissues: it is intracellular in epidermis, but enriched in plasma membranes of the intestinal epithelium. Analysis of SPCA2 sequences from various vertebrate species argue that ATP2C2 gene radiated from ATP2C1 (encoding SPCA1) during adaptation of tetrapod ancestors to terrestrial habitats.

Microcystin-LR aerosol induces inflammatory responses in healthy human primary airway epithelium.

Harmful algal blooms plague bodies of freshwater globally. These blooms are often composed of outgrowths of cyanobacteria capable of producing the heptapeptide Microcystin-LR (MC-LR) which is a well-known hepatotoxin. Recently, MC-LR has been detected in aerosols generated from lake water. However, the risk for human health effects due to MC-LR inhalation exposure have not been extensively investigated. In this study, we exposed a fully differentiated 3D human airway epithelium derived from 14 healthy donors to MC-LR-containing aerosol once a day for 3 days. Concentrations of MC-LR ranged from 100 pM to 1 µM. Although there were little to no detrimental alterations in measures of the airway epithelial function (i.e. cell survival, tissue integrity, mucociliary clearance, or cilia beating frequency), a distinct shift in the transcriptional activity was found. Genes related to inflammation were found to be upregulated such as C-C motif chemokine 5 (CCL5; log2FC = 0.57, p = 0.03) and C-C chemokine receptor type 7 (CCR7; log2FC = 0.84, p = 0.03). Functionally, conditioned media from MC-LR exposed airway epithelium was also found to have significant chemo-attractive properties for primary human neutrophils. Additionally, increases were found in the concentration of secreted chemokine proteins in the conditioned media such as CCL1 (log2FC = 5.07, p = 0.0001) and CCL5 (log2FC = 1.02, p = 0.046). These results suggest that MC-LR exposure to the human airway epithelium is capable of inducing an inflammatory response that may potentiate acute or chronic disease.

As We Drink and Breathe: Adverse Health Effects of Microcystins and Other Harmful Algal Bloom Toxins in the Liver, Gut, Lungs and Beyond.

Freshwater harmful algal blooms (HABs) are increasing in number and severity worldwide. These HABs are chiefly composed of one or more species of cyanobacteria, also known as blue-green algae, such as Microcystis and Anabaena. Numerous HAB cyanobacterial species produce toxins (e.g., microcystin and anatoxin-collectively referred to as HAB toxins) that disrupt ecosystems, impact water and air quality, and deter recreation because they are harmful to both human and animal health. Exposure to these toxins can occur through ingestion, inhalation, or skin contact. Acute health effects of HAB toxins have been well documented and include symptoms such as nausea, vomiting, abdominal pain and diarrhea, headache, fever, and skin rashes. While these adverse effects typically increase with amount, duration, and frequency of exposure, susceptibility to HAB toxins may also be increased by the presence of comorbidities. The emerging science on potential long-term or chronic effects of HAB toxins with a particular emphasis on microcystins, especially in vulnerable populations such as those with pre-existing liver or gastrointestinal disease, is summarized herein. This review suggests additional research is needed to define at-risk populations who may be helped by preventative measures. Furthermore, studies are required to develop a mechanistic understanding of chronic, low-dose exposure to HAB toxins so that appropriate preventative, diagnostic, and therapeutic strategies can be created in a targeted fashion.

Isolation and characterization of BetaM protein encoded by ATP1B4--a unique member of the Na,K-ATPase ß-subunit gene family.

ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a ß-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase ß-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was also characterized by SELDI-TOF mass spectrometry before and after deglycosylation. This allowed us to determine that the carbohydrate moiety of BetaM has molecular mass 5.9kDa and consists of short high-mannose type N-glycans. The results of direct analysis of the purified native eutherian BetaM protein provide first insights into structural properties underlying its entirely new evolutionarily acquired functions.

Impact of Comorbidities on SARS-CoV-2 Viral Entry-Related Genes.

Viral entry mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are an important aspect of virulence. Proposed mechanisms involve host cell membrane-bound angiotensin-converting enzyme 2 (ACE2), type II transmembrane serine proteases (TTSPs), such as transmembrane serine protease isoform 2 (TMPRSS2), lysosomal endopeptidase Cathepsin L (CTSL), subtilisin-like proprotein peptidase furin (FURIN), and even potentially membrane bound heparan sulfate proteoglycans. The distribution and expression of many of these genes across cell types representing multiple organ systems in healthy individuals has recently been demonstrated. However, comorbidities such as diabetes and cardiovascular disease are highly prevalent in patients with Coronavirus Disease 2019 (COVID-19) and are associated with worse outcomes. Whether these conditions contribute directly to SARS-CoV-2 virulence remains unclear. Here, we show that the expression levels of ACE2, TMPRSS2 and other viral entry-related genes, as well as potential downstream effector genes such as bradykinin receptors, are modulated in the target organs of select disease states. In tissues, such as the heart, which normally express ACE2 but minimal TMPRSS2, we found that TMPRSS2 as well as other TTSPs are elevated in individuals with comorbidities compared to healthy individuals. Additionally, we found the increased expression of viral entry-related genes in the settings of hypertension, cancer, or smoking across target organ systems. Our results demonstrate that common comorbidities may contribute directly to SARS-CoV-2 virulence and we suggest new therapeutic targets to improve outcomes in vulnerable patient populations.

Properties of a cryptic lysyl oxidase from haloarchaeon Haloterrigena turkmenica.

BACKGROUND: Lysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic. METHODS: LOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting. RESULTS: Cultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+ at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in H. turkmenica cells. CONCLUSION: H. turkmenica contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic. SIGNIFICANCE: This is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.

Evolutionary diversification of the BetaM interactome acquired through co-option of the ATP1B4 gene in placental mammals.

ATP1B4 genes represent a rare instance of orthologous vertebrate gene co-option that radically changed properties of the encoded BetaM proteins, which function as Na,K-ATPase subunits in lower vertebrates and birds. Eutherian BetaM has lost its ancestral function and became a muscle-specific resident of the inner nuclear membrane. Our earlier work implicated BetaM in regulation of gene expression through direct interaction with the transcriptional co-regulator SKIP. To gain insight into evolution of BetaM interactome we performed expanded screening of eutherian and avian cDNA libraries using yeast-two-hybrid and split-ubiquitin systems. The inventory of identified BetaM interactors includes lamina-associated protein LAP-1, myocyte nuclear envelope protein Syne1, BetaM itself, heme oxidases HMOX1 and HMOX2; transcription factor LZIP/CREB3, ERGIC3, PHF3, reticulocalbin-3, and ß-sarcoglycan. No new interactions were found for chicken BetaM and human Na,K-ATPase ß1, ß2 and ß3 isoforms, indicating the uniqueness of eutherian BetaM interactome. Analysis of truncated forms of BetaM indicates that residues 72-98 adjacent to the membrane in nucleoplasmic domain are important for the interaction with SKIP. These findings demonstrate that evolutionary alterations in structural and functional properties of eutherian BetaM proteins are associated with the increase in its interactome complexity.

Role of homologous ASP334 and GLU319 in human non-gastric H,K- and Na,K-ATPases in cardiac glycoside binding.

Cardiac steroids inhibit Na,K-ATPase and the related non-gastric H,K-ATPase, while they do not interact with gastric H,K-ATPase. Introducing an arginine, the residue present in the gastric H,K-ATPase, in the second extracellular loop at the corresponding position 334 in the human non-gastric H,K-ATPase (D334R mutation) rendered it completely resistant to 2mM ouabain. The corresponding mutation (E319R) in alpha1 Na,K-ATPase produced a approximately 2-fold increase of the ouabain IC(50) in the ouabain-resistant rat alpha1 Na,K-ATPase and a large decrease of the ouabain affinity of human alpha1 Na,K-ATPase, on the other hand this mutation had no effect on the affinity for the aglycone ouabagenin. These results provide a strong support for the orientation of ouabain in its biding site with its sugar moiety interacting directly with the second extracellular loop.

Characterization of hampin/MSL1 as a node in the nuclear interactome.

Hampin, homolog of Drosophila MSL1, is a partner of histone acetyltransferase MYST1/MOF. Functions of these proteins remain poorly understood beyond their participation in chromatin remodeling complex MSL. In order to identify new proteins interacting with hampin, we screened a mouse cDNA library in yeast two-hybrid system with mouse hampin as bait and found five high-confidence interactors: MYST1, TPR proteins TTC4 and KIAA0103, NOP17 (homolog of a yeast nucleolar protein), and transcription factor GC BP. Subsequently, all these proteins were used as baits in library screenings and more new interactions were found: tumor suppressor RASSF1C and spliceosome component PRP3 for KIAA0103, ring finger RNF10 for RASSF1C, and RNA polymerase II regulator NELF-C for MYST1. The majority of the observed interactions was confirmed in vitro by pull-down of bacterially expressed proteins. Reconstruction of a fragment of mammalian interactome suggests that hampin may be linked to diverse regulatory processes in the nucleus.

Evolution of Na,K-ATPase beta m-subunit into a coregulator of transcription in placental mammals.

Change in gene functions (gene cooption) is one of the key mechanisms of molecular evolution. Genes can acquire new functions via alteration in properties of encoded proteins and/or via changes in temporal or spatial regulation of expression. Here we demonstrate radical changes in the functions of orthologous ATP1B4 genes during evolution of vertebrates. Expression of ATP1B4 genes is brain-specific in teleost fishes, whereas it is predominantly muscle-specific in tetrapods. The encoded beta m-proteins in fish, amphibian, and avian species are beta-subunits of Na,K-ATPase located in the plasma membrane. In placental mammals beta m-proteins lost their ancestral functions, accumulate in nuclear membrane of perinatal myocytes, and associate with transcriptional coregulator Ski-interacting protein (SKIP). Through interaction with SKIP, eutherian beta m acquired new functions as exemplified by regulation of TGF-beta-responsive reporters and by augmentation of mRNA levels of Smad7, an inhibitor of TGF-beta signaling. Thus, orthologous vertebrate ATP1B4 genes represent an instance of gene cooption that created fundamental changes in the functional properties of the encoded proteins.

Loss of acidification of anterior prostate fluids in Atp12a-null mutant mice indicates that nongastric H-K-ATPase functions as proton pump in vivo.

The physiological functions of nongastric (colonic) H-K-ATPase (gene symbol Atp12a), unlike those of Na-K-ATPase and gastric H-K-ATPase, are poorly understood. It has been suggested that it pumps Na+ more efficiently than H+; however, so far, there is no direct evidence that it pumps H+ in vivo. Previously, we found that the nongastric H-K-ATPase alpha-subunit is expressed in apical membranes of rodent anterior prostate epithelium, in a complex with the Na-K-ATPase beta1-subunit. Here we report the effects of Atp12a gene ablation on polarization of the beta1-subunit and secretory function of the anterior prostate. In nongastric H-K-ATPase-deficient prostate, the Na-K-ATPase alpha-subunit resided exclusively in basolateral membranes; however, the beta1-subunit disappeared from apical membranes, demonstrating that beta1 is an authentic subunit of nongastric H-K-ATPase in vivo and that apical localization of beta1 in the prostate is completely dependent on its association with the nongastric H-K-ATPase alpha-subunit. A remarkable reduction in acidification of anterior prostate fluids was observed: pH 6.38 +/- 0.14 for wild-type mice and 6.96 +/- 0.10 for homozygous mutants. These results show that nongastric H-K-ATPase is required for acidification of luminal prostate fluids, thereby providing a strong in vivo correlate of previous functional expression studies demonstrating that it operates as a proton pump.

Role of the self-association of beta subunits in the oligomeric structure of Na+/K+-ATPase.

The two subunits of Na(+)/K(+)-ATPase that are essential for function are alpha and beta. Previous cross-linking studies on the oligomeric structure of the membrane-bound enzyme identified alpha,beta and alpha,alpha associations, but only the former and not the latter could be detected after solubilization. To study the possibility of direct beta,beta association, the purified membrane enzyme and a trypsin-digested enzyme that occludes cations and contains an essentially intact beta and fragments of alpha were subjected to oxidative cross-linking in the presence of Cu(2+)-phenanthroline. Resolution of products on polyacrylamide gels, N-terminal analysis and reactivity with anti-beta antibody showed that, in addition to previously identified products (e.g. alpha,alpha and alpha,beta dimers), a beta,beta dimer, most likely linked through intramembrane Cys(44) residues of two chains, is also formed. This dimer was also noted when digitonin-solubilized intact enzyme, and the trypsin-digested enzyme solubilized with digitonin or polyoxyethylene 10-laurylether were subjected to cross-linking, indicating that the detected beta,beta association was not due to random collisions. In the digested enzyme, K(+) but not Na(+) enhanced beta,beta dimer formation. The alternative cross-linking of beta-Cys(44) to a Cys residue of a transmembrane alpha-helix was antagonized specifically by K(+) or Na(+). The findings (i) indicate the role of beta,beta association in maintaining the minimum oligomeric structure of (alpha,beta)(2), (ii) provide further support for conformation-dependent flexibilities of the spatial relations of the transmembrane helices of alpha and beta and (iii) suggest the possibility of significant differences between the quaternary structures of the P-type ATPases that do and do not contain a beta subunit.

Human nongastric H+-K+-ATPase: transport properties of ATP1al1 assembled with different beta-subunits.

To investigate whether nongastric H+-K+-ATPases transport Na+ in exchange for K+ and whether different beta-isoforms influence their transport properties, we compared the functional properties of the catalytic subunit of human nongastric H+-K+-ATPase, ATP1al1 (AL1), and of the Na+-K+-ATPase alpha1-subunit (alpha1) expressed in Xenopus oocytes, with different beta-subunits. Our results show that betaHK and beta1-NK can produce functional AL1/beta complexes at the oocyte cell surface that, in contrast to alpha1/beta1 NK and alpha1/betaHK complexes, exhibit a similar apparent K+ affinity. Similar to Na+-K+-ATPase, AL1/beta complexes are able to decrease intracellular Na+ concentrations in Na+-loaded oocytes, and their K+ transport depends on intra- and extracellular Na+ concentrations. Finally, controlled trypsinolysis reveals that beta-isoforms influence the protease sensitivity of AL1 and alpha1 and that AL1/beta complexes, similar to the Na+-K+-ATPase, can undergo distinct K+-Na+- and ouabain-dependent conformational changes. These results provide new evidence that the human nongastric H+-K+-ATPase interacts with and transports Na+ in exchange for K+ and that beta-isoforms have a distinct effect on the overall structural integrity of AL1 but influence its transport properties less than those of the Na+-K+-ATPase alpha-subunit.

Betam, a structural member of the X,K-ATPase beta subunit family, resides in the ER and does not associate with any known X,K-ATPase alpha subunit.

betam, a muscle-specific protein, is structurally closely related to the X,K-ATPase beta subunits, but its intrinsic function is not known. In this study, we have expressed betam in Xenopus oocytes and have investigated its biosynthesis and processing as well as its putative role as a chaperone of X,K-ATPase alpha subunits, as a regulator of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), or as a Ca(2+)-sensing protein. Our results show that betam is stably expressed in the endoplasmic reticulum (ER) in its core glycosylated, partially trimmed form. Both full-length betam, initiated at Met(1), and short betam species, initiated at Met(89), are detected in in vitro translations as well as in Xenopus oocytes. betam cannot associate with and stabilize Na,K-ATPase (NK), or gastric and nongastric H,K-ATPase (HK) alpha isoforms. betam neither assembles stably with SERCA nor is its trypsin sensitivity or electrophoretic mobility influenced by Ca(2+). A mutant, in which the distinctive Glu-rich regions in the betam N-terminus are deleted, remains stably expressed in the ER and can associate with, but not stabilize X,K-ATPase alpha subunits. On the other hand, a chimera in which the ectodomain of betam is replaced with that of beta1 NK associates efficiently with alpha NK isoforms and produces functional Na,K-pumps at the plasma membrane. In conclusion, our results indicate that betam exhibits a cellular location and functional role clearly distinct from the typical X,K-ATPase beta subunits.