Functional Interaction Between Caveolin 1 and LRRC8-Mediated Volume-Regulated Anion Channel.

Mikel Rezola,Núria Comes,Xavier Gasull,Aida Castellanos

doi:10.3389/fphys.2021.691045

Abstract

Volume-regulated anion channel (VRAC), constituted by leucine-rich repeat-containing 8 (LRRC8) heteromers, is crucial for volume homeostasis in vertebrate cells. This widely expressed channel has been associated with membrane potential modulation, proliferation, migration, apoptosis, and glutamate release. VRAC is activated by cell swelling and by low cytoplasmic ionic strength or intracellular guanosine 5′-O-(3-thiotriphosphate) (GTP-γS) in isotonic conditions. Despite the substantial number of studies that characterized the biophysical properties of VRAC, its mechanism of activation remains a mystery. Different evidence suggests a possible effect of caveolins in modulating VRAC activity: (1) Caveolin 1 (Cav1)-deficient cells display insignificant swelling-induced Cl– currents mediated by VRAC, which can be restored by Cav1 expression; (2) Caveolin 3 (Cav3) knockout mice display reduced VRAC currents; and (3) Interaction between LRRC8A, the essential subunit for VRAC, and Cav3 has been found in transfected human embryonic kidney 293 (HEK 293) cells. In this study, we demonstrate a physical interaction between endogenous LRRC8A and Cav1 proteins, that is enhanced by hypotonic stimulation, suggesting that this will increase the availability of the channel to Cav1. In addition, LRRC8A targets plasma membrane regions outside caveolae of HEK 293 cells where it associates with non-caveolar Cav1. We propose that a rise in cell membrane tension by hypotonicity would flatten caveolae, as described previously, increasing the amount of Cav1 outside of caveolar structures interacting with VRAC. Besides, the expression of Cav1 in HEK Cav1- cells increases VRAC current density without changing the main biophysical properties of the channel. The present study provides further evidence on the relevance of Cav1 on the activation of endothelial VRAC through a functional molecular interaction.

Highlights

The ability of eukaryotic cells to regulate their volume is crucial for key cellular processes including proliferation, migration, and apoptosis (Hoffmann et al, 2009; Chen et al, 2019)
Since leucine-rich repeatcontaining 8A (LRRC8A) does not target lipid rafts in human embryonic kidney 293 (HEK 293) cells, we suggest that it may interact with the Caveolin 1 (Cav1) located out of raft domains
We describe that HEK Cav1- cells exhibit small swelling-activated Cl− currents consistent with those mediated by volumeregulated anion channel (VRAC), which is in agreement with that found in Cav1deficient cell lines (Trouet et al, 1999, 2001)

Summary

INTRODUCTION

The ability of eukaryotic cells to regulate their volume is crucial for key cellular processes including proliferation, migration, and apoptosis (Hoffmann et al, 2009; Chen et al, 2019). Caveolae are 50–80 nm -shaped invaginations found on the plasma membrane of many vertebrates but abundant in adipocytes, endothelia, fibroblasts, skeletal muscle cells, and myocytes. These structures have been related to functions as diverse as endocytosis, cell adhesion and migration, cholesterol homeostasis, mechanotransduction, and signal transduction (Parton and Simons, 2007). Transfection of caveolin 1 in Cav1-deficient cell lines restores the activity of VRAC (Trouet et al, 1999), and endothelial cells transfected with a mutated form of Cav show an impaired VRAC activity (Trouet et al, 2001) In this context, there is no further evidence on the functional association between Cav and VRAC. The study of the functional effects of the Cav1– LRRC8A interaction can shed some light on the mechanism behind the activation of VRAC currents

MATERIALS AND METHODS

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Findings

DATA AVAILABILITY STATEMENT