Furin cleavage activates the epithelial Na⁺channel by relieving Na⁺self-inhibition

Abstract

Epithelial Na+ channels (ENaC) are inhibited by extracellular Na+, a process referred to as Na+ self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in alpha, or one site in gamma, blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant alpha-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the Na+ self-inhibition response, while channels with a mutant gamma-subunit showed a modestly enhanced Na+ self-inhibition response. Analysis of Na+ self-inhibition at varying [Na+] indicates that channels containing mutant alpha-subunits exhibit an increased Na+ affinity. At the single-channel level, channels with a mutant alpha-subunit had a low open probability (P(o)) in the presence of a high external [Na+] in the patch pipette. P(o) dramatically increased when trypsin was also present, or when a low external [Na+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving Na+ self-inhibition and that activation requires that the alpha-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC P(o) and extracellular [Na+], supporting the notion that Na+ self-inhibition reflects a P(o) reduction due to high extracellular [Na+].

Novel Determinants of Epithelial Sodium Channel Gating within Extracellular Thumb Domains

“…The tight correlation between ENaC P o and the magnitude of Na ϩ self-inhibition validates the concept that Na ϩ self-inhibition is an intrinsic gating event that modulates channel P o (6). The observed changes in the Na ϩ self-inhibition response of…”

“…Our data are consistent with the view that the enhanced Na ϩ self-inhibition observed with the ␣G481M mutant is not due to a large change in Na ϩ binding affinity but primarily reflects allosteric changes subsequent to Na ϩ binding. Mutations Affecting Na ϩ Self-inhibition Alter Open Probability-Because Na ϩ self-inhibition is considered an intrinsic gating event (6,21,22), changes in Na ϩ self-inhibition observed with ␣Gly 481 and ␥Met 438 mutant channels are expected to be associated with parallel changes in channel P o . Unitary Na ϩ currents were obtained from cell-attached patches in oocytes expressing ␣G481M␤␥, ␣␤␥M438V, or ␣G481M/␤␥M438V mENaCs.…”

“…In addition, the N-terminal portion of ECD contains ␥His 239 , a previously identified residue critical for Na ϩ self-inhibition, as well as defined protease cleavage sites (4, 10 -12). Various proteases have been shown to regulate ENaC activity, in part, by interfering with Na ϩ self-inhibition (6,7,13). The resolved high resolution structure of the chicken acidsensing ion channel 1 (cASIC1) revealed an extracellular domain with a highly organized structure characterized by ␤ sheets within a core embedded within peripheral ␣ helices (14).…”

Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft

“…One of the external factors that regulate ENaC is Na ϩ . There are multiple lines of evidence supporting the notion that Na ϩ binding to one or more effector sites within the extracellular domains results in the inhibition of channel activity in an allosteric manner (1,12,13,29,34). Given that allosteric transitions are required for this process, it is not surprising that many substitutions throughout the extracellular regions of ENaC subunits altered the Na ϩ selfinhibition response.…”

“…Na ϩ self-inhibition) ( Fig. 1B) (12,13,29). Of the acidic sites tested, we found that the Trp mutation at ␣Asp-176, ␣Glu-362, and ␣Asp-365 reduced Na ϩ self-inhibition (Fig.…”