Abstract
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.
Highlights
ENaC is a member of the DEG/ENaC family of ion channels
These findings suggest that the ENaC extracellular domain might regulate epithelial Naϩ transport by sensing and integrating diverse signals in the extracellular environment
When ClϪ was replaced by SO42Ϫ, amiloride-sensitive ENaC current increased by 2.4-fold (Fig. 1, A and B)
Summary
A convergence of data indicate that ENaC gating is modulated by a variety of molecules that bind to or modify its extracellular domains, including proteases (10 –12), Naϩ [13,14,15], protons [16], and the divalent cations Zn2ϩ and Ni2ϩ [17, 18]. These findings suggest that the ENaC extracellular domain might regulate epithelial Naϩ transport by sensing and integrating diverse signals in the extracellular environment. The functional role of ClϪ binding to ASIC1a is unknown, it supports the hypothesis that extracellular ClϪ might regulate the activity of DEG/ENaC ion channels
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