Daniel M. Collier

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 SO42−, 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− < SO42− < 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 (αH418A and βR388A) 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 α-, β-, and γENaC, 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 (pH50 = 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 γENaC. 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.

Regulation of Epithelial Sodium Channel Trafficking by Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)

Background: The epithelial Na+ channel ENaC functions as a pathway for Na+ absorption across epithelia. Results: PCSK9 reduced ENaC expression at the cell surface by enhancing its proteasomal degradation. Conclusion: PCSK9 inhibits ENaC-mediated Na+ absorption. Significance: These findings provide new insights into mechanisms that regulate Na+ homeostasis and blood pressure. 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 αγβ 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-, β-, 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 βENaC. 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 βENaC 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 αγβ orientation (listed clockwise when viewed from the top).

Identification of ENaC inter-subunit Cl- inhibitory residues suggests a trimeric αγβ 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-, β-, 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 βENaC. 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 βENaC 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 αγβ 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.

Intersubunit conformational changes mediate epithelial sodium channel gating

Residues forming interfaces between the three ENaC subunits participate in conformational changes required for transition between open and closed states.

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

Background: The epithelial Na+ channel (ENaC) functions as a pathway for Na+ absorption across epithelia. Results: Seven acidic residues in the extracellular domain of γENaC and one in βENaC are required for regulation by acidic pH. Conclusion: The ENaC extracellular domains function as sensors to detect changes in extracellular pH. Significance: These findings provide new insights into mechanisms that regulate Na+ homeostasis and blood pressure. 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. γE455 is conserved in βENaC (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 βE446A 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 β- and γENaC that are required for regulation by acidic pH.