LRRC8 N termini influence pore properties and gating of volume-regulated anion channels (VRACs)

Pingzheng Zhou,Maya M Polovitskaya,Thomas J Jentsch

doi:10.1074/jbc.ra118.002853

Abstract

Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology. VRACs were recently discovered to be formed by heteromers of leucine-rich repeat–containing 8 (LRRC8) proteins. However, the structural determinants of VRAC permeation and gating remain largely unknown. We show here that the short stretch preceding the first LRRC8 transmembrane domain determines VRAC conductance, ion permeability, and inactivation gating. Substituted-cysteine accessibility studies revealed that several of the first 15 LRRC8 residues are functionally important and exposed to a hydrophilic environment. Substituting glutamate 6 with cysteine decreased the amplitudes of swelling-activated ICl,vol currents, strongly increased iodide-over-chloride permeability, and markedly shifted the voltage dependence of channel inactivation. Importantly, these effects were reversed by 2-sulfonatoethyl methanethiosulfonate, which restores the negative charge at this amino acid position. Cd2+-mediated blocking of ICl,vol in cysteine variants suggested that the LRRC8 N termini come close together in the multimeric channel complex and might form part of the pore. We propose a model in which the N termini of the LRRC8 subunits line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway.

Highlights

Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology
Green florescent protein (GFP)-LRRC8A or LRRC8A-green florescent protein (GFP) or with LRRC8A and either GFP-LRRC8C or LRRC8C-GFP resulted in fluorescence at the outer cell membrane (Fig. 1A and Fig. S1, A–C)
Because N-terminal addition of GFP to LRRC8A or LRRC8C interfered with ionic currents but not protein localization, leucine-rich repeat– containing 8 (LRRC8) N termini might be critically involved in forming or regulating the pore of VRAC channels

Summary

Introduction

Volume-regulated anion channels (VRACs) are crucial for cell volume regulation and have various roles in physiology and pathology. We show here that the short stretch preceding the first LRRC8 transmembrane domain determines VRAC conductance, ion permeability, and inactivation gating. Substituting glutamate 6 with cysteine decreased the amplitudes of swelling-activated ICl,vol currents, strongly increased iodideover-chloride permeability, and markedly shifted the voltage dependence of channel inactivation. These effects were reversed by 2-sulfonatoethyl methanethiosulfonate, which restores the negative charge at this amino acid position. Cd2؉mediated blocking of ICl,vol in cysteine variants suggested that the LRRC8 N termini come close together in the multimeric channel complex and might form part of the pore. VRACs seem to be ubiquitously expressed in vertebrate cells They transport chloride and various molecules, including organic osmolytes, neurotransmitters, and drugs (1, 4 –7). VRAC may play roles in signal transduction, cell migration, apoptosis, tumor drug resistance, and stroke (1, 5, 8)

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Conclusion