Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis

Alexandr V Ilyaskin,Christoph Korbmacher,Alexei Diakov

doi:10.1016/j.jbc.2021.100404

Alexandr V Ilyaskin, Christoph Korbmacher + Show 1 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100404

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Abstract

Mice lacking connexin 30 (Cx30) display increased epithelial sodium channel (ENaC) activity in the distal nephron and develop salt-sensitive hypertension. This indicates a functional link between Cx30 and ENaC, which remains incompletely understood. Here, we explore the effect of Cx30 on ENaC function using the Xenopus laevis oocyte expression system. Coexpression of human Cx30 with human αβγENaC significantly reduced ENaC-mediated whole-cell currents. The size of the inhibitory effect on ENaC depended on the expression level of Cx30 and required Cx30 ion channel activity. ENaC inhibition by Cx30 was mainly due to reduced cell surface ENaC expression resulting from enhanced ENaC retrieval without discernible effects on proteolytic channel activation and single-channel properties. ENaC retrieval from the cell surface involves the interaction of the ubiquitin ligase Nedd4-2 with PPPxY-motifs in the C-termini of ENaC. Truncating the C- termini of β- or γENaC significantly reduced the inhibitory effect of Cx30 on ENaC. In contrast, mutating the prolines belonging to the PPPxY-motif in γENaC or coexpressing a dominant-negative Xenopus Nedd4 (xNedd4-CS) did not significantly alter ENaC inhibition by Cx30. Importantly, the inhibitory effect of Cx30 on ENaC was significantly reduced by Pitstop-2, an inhibitor of clathrin-mediated endocytosis, or by mutating putative clathrin adaptor protein 2 (AP-2) recognition motifs (YxxФ) in the C termini of β- or γ-ENaC. In conclusion, our findings suggest that Cx30 inhibits ENaC by promoting channel retrieval from the plasma membrane via clathrin-dependent endocytosis. Lack of this inhibition may contribute to increased ENaC activity and salt-sensitive hypertension in mice with Cx30 deficiency.

Highlights

The epithelial sodium channel (ENaC) belongs to the ENaC/ degenerin family of ion channels
Inward currents elicited by Ca2+ and Mg2+ removal were not detectable in AS connexin 38 (Cx38) injected control oocytes and were much higher in oocytes coinjected with against Cx38 (AS Cx38) and complementary RNA (cRNA) for human connexin 30 (Cx30) than those in noninjected oocytes expressing endogenous Cx38 (Fig. 1B)
We investigated the functional interaction of human αβγENaC with human Cx30 heterologously expressed in X. laevis oocytes

Summary

Introduction

The epithelial sodium channel (ENaC) belongs to the ENaC/ degenerin family of ion channels. ENaC is expressed in the apical membrane of principal cells in the aldosterone-sensitive distal nephron (ASDN). The mechanisms involved in mediating the stimulatory effect of aldosterone on ENaC are highly complex and probably include increased forward trafficking of ENaC [11, 12] and reduced channel retrieval resulting in increased channel expression at the cell surface. In addition to aldosterone and several other hormones, numerous extracellular and intracellular factors including regulatory proteins can modulate ENaC activity in the ASDN. They include proteases, which activate ENaC by cleaving the channel at specific sites in the extracellular domains of the α- and γsubunit. This releases inhibitory domains and probably causes a conformational change leading to channel activation [31, 32]

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