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.
Highlights
Members of the LRRC8 family form heteromeric assemblies, which function as volumeregulated anion channels
Together our results provide a set of specific modulators of LRRC8 channels and they emphasize the importance of the leucine-rich repeat (LRR) domains as regulatory units that modulate channel activity by allosteric mechanisms
From the pool of sybodies enriched against the full-length LRRC8A channel, we subsequently focused on a subset of binders that target its cytoplasmic domain
Summary
Members of the LRRC8 family form heteromeric assemblies, which function as volumeregulated anion channels These modular proteins consist of a transmembrane pore and cytoplasmic leucine-rich repeat (LRR) domains. The general architecture of LRRC8 channels was revealed from homomeric structures of the LRRC8A18–21 and LRRC8D22 subunits Such homomeric assemblies are not observed in a cellular context, the subunits form hexamers and, in case of LRRC8A, they function as ion channels with compromised activation properties[18,23,24]. With respect to their structure, homomeric LRRC8A channels appear to exhibit general features that are observed in heteromeric proteins[18]. Together our results provide a set of specific modulators of LRRC8 channels and they emphasize the importance of the LRR domains as regulatory units that modulate channel activity by allosteric mechanisms
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
