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

Abstract

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) 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). α-ENaC expression with WT β and γ subunits in oocytes produces reduced basal I, which can largely be recovered by exogenous trypsin. With this α-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 of α-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 in oocytes expressing α-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-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.

A call for an independent inquiry into the origin of the SARS-CoV-2 virus

“…In fact, the assertion that the FCS in SARS-CoV-2 has an unusual, nonstandard amino acid sequence is false. The amino acid sequence of the FCS in SARS-CoV-2 also exists in the human ENaC α subunit ( 16 ), where it is known to be functional and has been extensively studied ( 17 , 18 ). The FCS of human ENaC α has the amino acid sequence RRAR'SVAS ( Fig.…”

“…ENaC is an epithelial sodium channel, expressed on the apical surface of epithelial cells in the kidney, colon, and airways ( 19 , 20 ), that plays a critical role in controlling fluid exchange. The ENaC α subunit has a functional FCS ( 17 , 18 ) that is essential for ion channel function ( 19 ) and has been characterized in a variety of species. The FCS sequence of human ENaC α ( 20 ) is identical in chimpanzee, bonobo, orangutan, and gorilla ( SI Appendix , Fig.…”

Proliferative regulation of alveolar epithelial type 2 progenitor cells by human Scnn1d gene

“…The effects of the extended cytosolic NH2-terminal tail for the δ 802 -ENaC are unknown. The N-terminal tails of α, β, and γ-ENaC subunits are involved in endocytosis-related channel activity 53, 54, PIP2 binding and hydrolysis 55, 56, intracellular gating 57, 58, interaction with CFTR 31, and proteolysis 14, 59, 60. By comparing of δ 1 βγ and δ 2 βγ channels in oocytes, it was found that δ 2 channels had an increased single channel conductance and activity 29.…”

Osmotic Adaptation by Na+-Dependent Transporters and ACE2: Correlation with Hemostatic Crisis in COVID-19

“…This near-identical furin site enhances SARS-CoV-2 cleavage for replication [104,106] and implies competition between it and the hosts' αENaC in endosomes for furin-mediated cleavage. Furin is a physiological activator of ENaC [108]. In the absence of furin, ENaC is cleaved on different sites [118,119] by extracellular proteases, including channel-activating protease (CAP1) [120], CAP2/TMPRSS4, CAP3/matriptase, and plasmin [119] (reviewed in [55]), resulting in overactive ENaC.…”