Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains

Abstract

Members of the LRRC8 family form heteromeric assemblies, which function as volume-regulated anion channels. These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. Despite their known molecular architecture, the mechanism of activation and the role of the LRR domains in this process has remained elusive. Here we address this question by generating synthetic nanobodies, termed sybodies, which target the LRR domain of the obligatory subunit LRRC8A. We use these binders to investigate their interaction with homomeric LRRC8A channels by cryo-electron microscopy and the consequent effect on channel activation by electrophysiology. The five identified sybodies either inhibit or enhance activity by binding to distinct epitopes of the LRR domain, thereby altering channel conformations. In combination, our work provides a set of specific modulators of LRRC8 proteins and reveals the role of their cytoplasmic domains as regulators of channel activity by allosteric mechanisms.

Properties, Structures, and Physiological Roles of Three Types of Anion Channels Molecularly Identified in the 2010’s

“…This is thought to occur due to altered electrostatic interactions between subunits via a mechanism, which could also be caused by mechanical stress ( Strange et al, 2019 ). Consistent with this paradigm, a recent study demonstrated that synthetic proteinaceous nanobodies (called sybodies) targeting different LRR epitopes profoundly modulate activation of the native channel by low ionic strength ( Deneka et al, 2021 ).…”

“…The uniquely arranged LRR region contains numerous charged amino acids at the contacting surfaces between adjacent subunits ( Deneka et al, 2018 ; Kasuya et al, 2018 ; Kefauver et al, 2018 ; Kern et al, 2019 ; Nakamura et al, 2020 ). Such arrangements may explain the well-known activation by low ionic strength of the native VSOR/VRAC ( Cannon et al, 1998 ; Voets et al, 1999 ; Sabirov et al, 2000 ; Deneka et al, 2021 ) and purified LRRC8 proteins in lipid bilayers ( Syeda et al, 2016 ). This is thought to occur due to altered electrostatic interactions between subunits via a mechanism, which could also be caused by mechanical stress ( Strange et al, 2019 ).…”

Recent Advances in the Structural Biology of the Volume-Regulated Anion Channel LRRC8

“…The overall architecture and subunit folding are the same in all of the LRRC8 structures that have been determined ( Deneka et al, 2021 , 2018 ; Gunasekar et al, 2022 ; Kasuya et al, 2018 ; Kefauver et al, 2018 ; Kern et al, 2019 ; Nakamura et al, 2020 ). The overall architecture of LRRC8 assembles into a hexamer ( Figure 1A ).…”

“…Due to recent advances in cryo-EM, the first structure of LRRC8 was reported in 2018, only 4 years after the identification of LRRC8 as the molecular entity of VRAC ( Deneka et al, 2018 ). Since then, six studies reported the LRRC8 structures to date ( Kasuya et al, 2018 ; Kefauver et al, 2018 ; Kern et al, 2019 ; Nakamura et al, 2020 ; Deneka et al, 2021 ; Gunasekar et al, 2022 ). In this review, we describe the structural and functional features of the LRRC8 protein family that were elucidated by those studies and discuss the possible directions of further research in this field.…”

“…In 2020, the overall structure of HsLRRC8D homo-hexamer in detergents was determined by cryo-EM ( Nakamura et al, 2020 ), revealing the N-terminal helix involved in ion permeation. In 2021, the overall structures of MmLRRC8A homo-hexamer in complex with five different sybodies were determined by cryo-EM ( Deneka et al, 2021 ). Sybodies are synthetic single-domain antibodies that bind to specific regions of target proteins ( Zimmermann et al, 2018 ).…”