The N terminus of α-ENaC mediates ENaC cleavage and activation by furin

Epithelial Na⁺ channels comprise three homologous subunits (α, β, and γ) that are regulated by alternative splicing and proteolytic cleavage. Here, we determine the basis of the reduced Na⁺ current (I_Na) that results from expression of a previously identified, naturally occurring splice variant of the α subunit (α-ENaC), in which residues 34-82 are deleted (α_Δ34-82). α_Δ34-82-ENaC expression with WT β and γ subunits in Xenopus oocytes produces reduced basal I_Na, which can largely be recovered by exogenous trypsin. With this α_Δ34-82-containing ENaC, both α and γ subunits display decreased cleavage fragments, consistent with reduced processing by furin or furin-like convertases. Data using MTSET modification of a cysteine, introduced into the degenerin locus in β-ENaC, suggest that the reduced I_Na of α_Δ34-82-ENaC arises from an increased population of uncleaved, near-silent ENaC, rather than from a reduced open probability spread uniformly across all channels. After treatment with brefeldin A to disrupt anterograde trafficking of channel subunits, I_Na in oocytes expressing α_Δ34-82-ENaC is reestablished more slowly than that in oocytes expressing WT ENaC. Overnight or acute incubation of oocytes expressing WT ENaC in the pore blocker amiloride increases basal ENaC proteolytic stimulation, consistent with relief of Na⁺ feedback inhibition. These responses are reduced in oocytes expressing α_Δ34-82-ENaC_. We conclude that the α-ENaC N terminus mediates interactions that govern the delivery of cleaved and uncleaved ENaC populations to the oocyte membrane.