Extracellular chloride regulates ENaC gating

Abstract

The extracellular domain of the epithelial sodium ion channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We tested whether chloride alters ENaC activity. In Xenopus oocytes expressing human αβγENaC, amiloride sensitive current was increased when chloride was replaced with sulfate (238% increase). Substitution of chloride with iodide or bromide resulted in an 11.6% increase and a 17.7% decrease in amiloride sensitive current, respectively. We hypothesized that chloride might regulate ENaC activity by altering channel gating. Consistent with this hypothesis, we found that changes in extracellular chloride had little effect on ENaC activity in the presence of the DEG mutation in the β subunit (βS520K) that locks the channel in the open state. We found that the response to chloride was altered by mutations in the extracellular domain that are homologous to the chloride binding site of the chicken ASIC1 crystal structure (2QTS). Mutation of αH418A and βR388A, both independently and jointly, decreased the response to extracellular chloride, while γH396A had little effect. Our results demonstrate that chloride regulates ENaC activity, providing a potential mechanism by which changes in extracellular chloride might modulate epithelial Na+ transport.