Cryo-EM structures of a LRRC8 chimera with native functional properties reveal heptameric assembly

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Abstract

Volume-regulated anion channels (VRACs) mediate volume regulatory Cl^- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional nor exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL1²⁵) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL1²⁵) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL1²⁵) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.

Data availability

Cryo-EM maps and atomic coordinates are deposited to the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) databases. The accession codes are EMD-27770 and 8DXN for class 1; EMD-27771 and 8DXO for class 2; EMD-27772 and 8DXP for class 3; EMD-27773 and 8DXQ for class 4; EMD-27774 and 8DXR for class 5.

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Hirohide Takahashi

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-2553-8806
Toshiki Yamada

Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, United States

Competing interests
No competing interests declared.
Jerod S Denton

Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, United States

Competing interests
No competing interests declared.
Kevin Strange

Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, United States

Competing interests
Kevin Strange, is cofounder and principal scientist of Revidia Therapeutics, Inc..
Erkan Karakas

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, United States

For correspondence
erkan.karakas@vanderbilt.edu

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-6552-3185

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (DK51610)

Jerod S Denton

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.