Ionotropic purinergic receptor 7 (P2X7) channel structure and pharmacology provides insight regarding non-nucleotide agonism.

P2X7 is a member of the Ionotropic Purinergic Receptor (P2X) family. The P2X family of receptors is composed of seven (P2X1-7), ligand-gated, nonselective cation channels. Changes in P2X expression have been reported in multiple disease models. P2Xs have large complex extracellular domains that function as receptors for a variety of ligands, including endogenous and synthetic agonists and antagonists. ATP is the canonical agonist. ATP affinity ranges from nanomolar to micromolar for most P2XRs, but P2X7 has uniquely poor ATP affinity. In many physiological settings, it may be difficult to achieve the millimolar extracellular ATP concentrations needed for P2X7 channel activation; however, channel function is implicated in pain sensation, immune cell function, cardiovascular disease, cancer, and osteoporosis. Multiple high-resolution P2X7 structures have been solved in apo-, ATP-, and antagonist-bound states. P2X7 structural data reveal distinct allosteric and orthosteric antagonist-binding sites. Both allosteric and orthosteric P2X7 antagonists are well documented to inhibit ATP-evoked channel current. However, a growing body of evidence supports P2X7 activation by non-nucleotide agonists, including extracellular histone proteins and human cathelicidin-derived peptides (LL-37). Interestingly, P2X7 non-nucleotide agonism is not inhibited by allosteric antagonists, but is inhibited by orthosteric antagonists. Herein, we review P2X7 function with a focus on the efficacy of available pharmacology on P2X7 channel current activation by non-nucleotide agonists in effort to understand agonist/antagonist efficacy, and consider the impact of these data on the current understanding of P2X7 in physiology and disease given these limitations of P2X7-selective antagonists and incomplete knockout mouse models.

WNK kinase is a vasoactive chloride sensor in endothelial cells.

Endothelial cells (ECs) line the wall of blood vessels and regulate arterial contractility to tune regional organ blood flow and systemic pressure. Chloride (Cl-) is the most abundant anion in ECs and the Cl- sensitive With-No-Lysine (WNK) kinase is expressed in this cell type. Whether intracellular Cl- signaling and WNK kinase regulate EC function to alter arterial contractility is unclear. Here, we tested the hypothesis that intracellular Cl- signaling in ECs regulates arterial contractility and examined the signaling mechanisms involved, including the participation of WNK kinase. Our data obtained using two-photon microscopy and cell-specific inducible knockout mice indicated that acetylcholine, a prototypical vasodilator, stimulated a rapid reduction in intracellular Cl- concentration ([Cl-]i) due to the activation of TMEM16A, a Cl- channel, in ECs of resistance-size arteries. TMEM16A channel-mediated Cl- signaling activated WNK kinase, which phosphorylated its substrate proteins SPAK and OSR1 in ECs. OSR1 potentiated transient receptor potential vanilloid 4 (TRPV4) currents in a kinase-dependent manner and required a conserved binding motif located in the channel C terminus. Intracellular Ca2+ signaling was measured in four dimensions in ECs using a high-speed lightsheet microscope. WNK kinase-dependent activation of TRPV4 channels increased local intracellular Ca2+ signaling in ECs and produced vasodilation. In summary, we show that TMEM16A channel activation reduces [Cl-]i, which activates WNK kinase in ECs. WNK kinase phosphorylates OSR1 which then stimulates TRPV4 channels to produce vasodilation. Thus, TMEM16A channels regulate intracellular Cl- signaling and WNK kinase activity in ECs to control arterial contractility.

High-fat diet induced obesity promotes inflammation, oxidative stress, and hepatotoxicity in female FVB/N mice.

Although obesity and subsequent liver injury are increasingly prevalent in women, female mouse models have generally shown resistance to high-fat diet (HFD)-induced obesity. We evaluated control and HFD-fed male and female FVB/N mice, a strain well-suited to transgenic analyses, for phenotypic, histological, and molecular markers related to control of glucose, lipids, and inflammation in serum, liver, and perigonadal white adipose tissues. Unlike many mouse models, HFD-fed FVB/N females gained more perigonadal and mesenteric fat mass and overall body weight than their male counterparts, with increased hepatic expression of lipogenic PPARγ target genes (Cd36, Fsp27, and Fsp27ß), oxidative stress genes and protein (Nqo1 and CYP2E1), inflammatory gene (Mip-2), and the pro-fibrotic gene Pai-1, along with increases in malondialdehyde and serum ALT levels. Further, inherent to females (independently of HFD), hepatic antioxidant heme oxygenase-1 (HMOX1, HO-1) protein levels were reduced compared to their male counterparts. In contrast, males may have been relatively protected from HFD-induced oxidative stress and liver injury by elevated mRNA and protein levels of hepatic antioxidants BHMT and Gpx2, increased fatty acid oxidation genes in liver and adipocytes (Pparδ), despite disorganized and inflamed adipocytes. Thus, female FVB/N mice offer a valuable preclinical, genetically malleable model that recapitulates many of the features of diet-induced obesity and liver damage observed in human females.

Diabetic Endothelial Cell Glycogen Synthase Kinase 3ß Activation Induces VCAM1 Ectodomain Shedding.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been found to be associated with poor cardiovascular disease (CVD) outcomes, supporting VCAM1's role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1's release or its interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane-bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs). Still, the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from an inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3ß, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3ß activation in control ECs increased ADAM10/17 and VCAM1. A GSK3ß inhibitor reduced active GSK3ß and VCAM1 ectodomain shedding. These findings suggest diabetic ECs with elevated GSK3ß activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3ß inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease, particularly in diabetes, holds significant therapeutic potential.

Identification of cell division cycle protein 20 in various forms of acute and chronic kidney injury in mice.

Tubular epithelial cell fate following exposure to various types of injurious stimuli can be decided at distinct cell cycle checkpoints. One such checkpoint occurs during mitosis, known as the spindle assembly checkpoint, and is tightly regulated through the actions of cell division cycle protein 20 (CDC20). Due to our paucity of knowledge about the role of CDC20 in the kidney, the present study was designed to investigate the expression levels and distribution of CDC20 within the kidney and how pharmacological inhibition of CDC20 function affects kidney recovery using various rodent models of kidney injury. CDC20 is normally detected in distal tubules, but upon injury by either cisplatin administration or ureter obstruction, CDC20 accumulation is considerably elevated. Blockade of CDC20 activity using a selective pharmacological inhibitor, Apcin, lowered serum creatinine, tubular damage, and DNA injury following acute kidney injury compared with vehicle-treated mice. In unilateral ureteral obstruction, Apcin reduced tissue kidney injury molecule-1 levels, sirius red staining, and tubulointerstitial α-smooth muscle actin staining in the tissue. The findings in the present study demonstrated that elevations in CDC20 levels in the kidney are associated with kidney injury and that inhibition of CDC20 can alleviate and reverse some of the pathological effects on the architecture and function of kidney.NEW & NOTEWORTHY To our knowledge, this is the first study to characterize the expression and localization of cell division cycle 20 protein (CDC20) in normal and acute, and chronically injured kidneys. Tubular epithelial cell damage was markedly reduced through the administration of a selective inhibitor of CDC20, Apcin. This study provides new evidence that CDC20 can be induced in damaged kidney cells and negatively impact the recovery of the kidney following acute kidney injury.

Extracellular histone proteins activate P2XR7 channel current.

Extracellular histone proteins are elevated in circulation after injury or activation of the innate immune response. In resistance-size arteries, extracellular histone proteins increased endothelial cell (EC) Ca2+ influx and propidium iodide (PI) labeling, but paradoxically decreased vasodilation. These observations could be explained by the activation of an EC resident non-selective cation channel. We tested the hypothesis that the ionotropic purinergic receptor 7 (P2XR7), a non-selective cation channel associated with cationic dye uptake, is activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). Selective P2XR7 antagonists, AZ10606120, A438079, GW791343, and AZ11645373, inhibited ATP-evoked P2XR7 currents but did not inhibit histone-evoked P2XR7 currents. As previously reported with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca2+. These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents in a heterologous expression system. These results provide insight into a new allosteric mechanism of P2XR7 activation by histone proteins.

Myogenic tone contributes to the regulation of permeability in mesenteric microvessels.

The microvascular endothelium plays a key role in regulating solute permeability in the gut, but the contribution of vascular smooth muscle to barrier function is unknown. We sought to determine the role of vascular smooth muscle and its myogenic tone in the vascular barrier to solutes in mesenteric microvessels. We determined vascular permeability to 4.4 kDa and 70 kDa dextrans in isolated mouse mesenteric arteries at increasing pressure increments. The myogenic response was simultaneously monitored using video edge-detection of vessel diameter and wall thickness. We expressed permeability as the apparent permeability coefficient, or the solute flux per second normalized to surface area and concentration gradient. We compared the effects of myogenic tone, L-type calcium channel blockade, calcium elimination, and endothelial removal on the permeability of each dextran. We found arteries resisted changes in 4.4 kDa and 70 kDa dextran permeability coefficients at intravascular pressures associated with myogenic tone. Manipulations that reduced or eliminated myogenic tone (L-type calcium channel blockade or calcium elimination) caused vasodilation and increased permeability coefficients. Thus, the maintenance of a reactive mesenteric vascular smooth muscle layer and its myogenic tone prevents increases in vascular permeability that would otherwise occur with increasing pressure. Conditions that impact vascular tone, such as trauma, stroke, or major surgery could diminish the gut-vascular barrier against dissemination of the microbiome.

Extracellular histones induce calcium signals in the endothelium of resistance-sized mesenteric arteries and cause loss of endothelium-dependent dilation.

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca2+ but not depletion of intracellular Ca2+ stores prevented histone-induced Ca2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. NEW & NOTEWORTHY We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca2+ indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca2+ signaling. We found that histones induce an influx of Ca2+ in ECs that does not cause vasodilation but instead causes Ca2+ overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.

Intersubunit conformational changes mediate epithelial sodium channel gating.

The epithelial Na(+) channel (ENaC) functions as a pathway for Na(+) absorption in the kidney and lung, where it is crucial for Na(+) homeostasis and blood pressure regulation. However, the basic mechanisms that control ENaC gating are poorly understood. Here we define a role in gating for residues forming interfaces between the extracellular domains of the three ENaC subunits. Using cysteine substitution combined with chemical cross-linking, we determined that residues located at equivalent positions in the three subunits (αK477, ßE446, and γE455) form interfaces with residues in adjacent subunits (ßV85, γV87, and αL120, respectively). Cross-linking of these residues altered ENaC activity in a length-dependent manner; long cross-linkers increased ENaC current by increasing its open probability, whereas short cross-linkers reduced ENaC open probability. Cross-linking also disrupted ENaC gating responses to extracellular pH and Na(+), signals which modulate ENaC activity during shifts in volume status. Introduction of charged side chains at the interfacing residues altered ENaC activity in a charge-dependent manner. Current increased when like charges were present at both interfacing residues, whereas opposing charges reduced current. Together, these data indicate that conformational changes at intersubunit interfaces participate in ENaC transitions between the open and closed states; movements that increase intersubunit distance favor the open state, whereas the closed state is favored when the distance is reduced. This provides a mechanism to modulate ENaC gating in response to changing extracellular conditions that threaten Na(+) homeostasis.

Identification of extracellular domain residues required for epithelial Na+ channel activation by acidic pH.

A growing body of evidence suggests that the extracellular domain of the epithelial Na(+) channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na(+) self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ(E455) is conserved in ßENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with ß(E446A) generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na(+) self-inhibition. Together, the data identify eight extracellular domain residues in human ß- and γENaC that are required for regulation by acidic pH.

Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9).

The epithelial Na(+) channel (ENaC) is critical for Na(+) homeostasis and blood pressure control. Defects in its regulation cause inherited forms of hypertension and hypotension. Previous work found that ENaC gating is regulated by proteases through cleavage of the extracellular domains of the α and γ subunits. Here we tested the hypothesis that ENaC is regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), a protease that modulates the risk of cardiovascular disease. PCSK9 reduced ENaC current in Xenopus oocytes and in epithelia. This occurred through a decrease in ENaC protein at the cell surface and in the total cellular pool, an effect that did not require the catalytic activity of PCSK9. PCSK9 interacted with all three ENaC subunits and decreased their trafficking to the cell surface by increasing proteasomal degradation. In contrast to its previously reported effects on the LDL receptor, PCSK9 did not alter ENaC endocytosis or degradation of the pool of ENaC at the cell surface. These results support a role for PCSK9 in the regulation of ENaC trafficking in the biosynthetic pathway, likely by increasing endoplasmic reticulum-associated degradation. By reducing ENaC channel number, PCSK9 could modulate epithelial Na(+) absorption, a major contributor to blood pressure control.

N-glycosylation of acid-sensing ion channel 1a regulates its trafficking and acidosis-induced spine remodeling.

Acid-sensing ion channel-1a (ASIC1a) is a potential therapeutic target for multiple neurological diseases. We studied here ASIC1a glycosylation and trafficking, two poorly understood processes pivotal in determining the functional outcome of an ion channel. We found that most ASIC1a in the mouse brain was fully glycosylated. Inhibiting glycosylation with tunicamycin reduced ASIC1a surface trafficking, dendritic targeting, and acid-activated current density. N-glycosylation of the two glycosylation sites, Asn393 and Asn366, has differential effects on ASIC1a biogenesis. Maturation of Asn393 increased ASIC1a surface and dendritic trafficking, pH sensitivity, and current density. In contrast, glycosylation of Asn366 was dispensable for ASIC1a function and may be a rate-limiting step in ASIC1a biogenesis. In addition, we revealed that acidosis reduced the density and length of dendritic spines in a time- and ASIC1a-dependent manner. ASIC1a N366Q, which showed increased glycosylation and dendritic targeting, potentiated acidosis-induced spine loss. Conversely, ASIC1a N393Q, which had diminished dendritic targeting and inhibited ASIC1a current dominant-negatively, had the opposite effect. These data tie N-glycosylation of ASIC1a with its trafficking. More importantly, by revealing a site-specific effect of acidosis on dendritic spines, our findings suggest that these processes have an important role in regulating synaptic plasticity and determining long-term consequences in diseases that generate acidosis.

Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture.

The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of α-, ß-, and γENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl(-) inhibitory sites in ENaC. One is formed by residues in the thumb domain of αENaC and the palm domain of ßENaC. Mutation of residues at this interface decreased Cl(-) inhibition and decreased Na(+) self-inhibition. The second site is formed by residues at the interface of the thumb domain of ßENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl(-) inhibition yet had no effect on Na(+) self-inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had little or no effect on Cl(-) inhibition or Na(+) self-inhibition. The data demonstrate that Cl(-) inhibits ENaC activity by two distinct Na(+)-dependent and Na(+)-independent mechanisms that correspond to the two functional Cl(-) inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl(-) inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl(-) inhibition, the data support a model in which ENaC subunits assemble in an αÎ³ß orientation (listed clockwise when viewed from the top).

The Drosophila gene CheB42a is a novel modifier of Deg/ENaC channel function.

Degenerin/epithelial Na(+) channels (DEG/ENaC) represent a diverse family of voltage-insensitive cation channels whose functions include Na(+) transport across epithelia, mechanosensation, nociception, salt sensing, modification of neurotransmission, and detecting the neurotransmitter FMRFamide. We previously showed that the Drosophila melanogaster Deg/ENaC gene lounge lizard (llz) is co-transcribed in an operon-like locus with another gene of unknown function, CheB42a. Because operons often encode proteins in the same biochemical or physiological pathway, we hypothesized that CHEB42A and LLZ might function together. Consistent with this hypothesis, we found both genes expressed in cells previously implicated in sensory functions during male courtship. Furthermore, when coexpressed, LLZ coprecipitated with CHEB42A, suggesting that the two proteins form a complex. Although LLZ expressed either alone or with CHEB42A did not generate ion channel currents, CHEB42A increased current amplitude of another DEG/ENaC protein whose ligand (protons) is known, acid-sensing ion channel 1a (ASIC1a). We also found that CHEB42A was cleaved to generate a secreted protein, suggesting that CHEB42A may play an important role in the extracellular space. These data suggest that CHEB42A is a modulatory subunit for sensory-related Deg/ENaC signaling. These results are consistent with operon-like transcription of CheB42a and llz and explain the similar contributions of these genes to courtship behavior.

Extracellular chloride regulates the epithelial sodium channel.

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.

Extracellular protons regulate human ENaC by modulating Na+ self-inhibition.

The epithelial Na(+) channel, ENaC, is exposed to a wide range of proton concentrations in the kidney, lung, and sweat duct. We, therefore, tested whether pH alters ENaC activity. In Xenopus oocytes expressing human alpha-, beta-, and gammaENaC, amiloride-sensitive current was altered by protons in the physiologically relevant range (pH 8.5-6.0). Compared with pH 7.4, acidic pH increased ENaC current, whereas alkaline pH decreased current (pH(50) = 7.2). Acidic pH also increased ENaC current in H441 epithelia and in human primary airway epithelia. In contrast to human ENaC, pH did not alter rat ENaC current, indicating that there are species differences in ENaC regulation by protons. This resulted predominantly from species differences in gammaENaC. Maneuvers that lock ENaC in a high open-probability state ("DEG" mutation, proteolytic cleavage) abolished the effect of pH on human ENaC, indicating that protons alter ENaC current by modulating channel gating. Previous work showed that ENaC gating is regulated in part by extracellular Na(+) ("Na(+) self-inhibition"). Based on several observations, we conclude that protons regulate ENaC by altering Na(+) self-inhibition. First, protons reduced Na(+) self-inhibition in a dose-dependent manner. Second, ENaC regulation by pH was abolished by removing Na(+) from the extracellular bathing solution. Third, mutations that alter Na(+) self-inhibition produced corresponding changes in ENaC regulation by pH. Together, the data support a model in which protons modulate ENaC gating by relieving Na(+) self-inhibition. We speculate that this may be an important mechanism to facilitate epithelial Na(+) transport under conditions of acidosis.