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SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.

The SARS-CoV-2 3a protein is a putative ion channel implicated in virus life cycle and pathogenesis. We recently expressed, purified, and reconstituted 3a into lipid nanodiscs to solve its structure by cryo-EM to 2.1Å resolution. In this chapter, we describe methods we developed in order to facilitate the study of this protein in other laboratories. We emphasize factors that enabled rapid progression from gene sequence to reconstituted protein (3 weeks in the case of 3a) and provide general observations and tips for adapting these protocols to other membrane proteins of interest.

The SARS-CoV-2 3a protein is a putative ion channel implicated in virus life cycle and pathogenesis. We recently expressed, purified, and reconstituted 3a into lipid nanodiscs to solve its structure by cryo-EM to 2.1Å resolution. In this chapter, we describe methods we developed in order to facilitate the study of this protein in other laboratories. We emphasize factors that enabled rapid progression from gene sequence to reconstituted protein (3 weeks in the case of 3a) and provide general observations and tips for adapting these protocols to other membrane proteins of interest.

Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here, we present single-particle cryo-electron microscopy structures of Mus musculus LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.

Hypoosmotic conditions activate volume-regulated anion channels in vertebrate cells. These channels are formed by leucine-rich repeat-containing protein 8 (LRRC8) family members and contain LRRC8A in homo- or hetero-hexameric assemblies. Here, we present single-particle cryo-electron microscopy structures of Mus musculus LRRC8A in complex with the inhibitor DCPIB reconstituted in lipid nanodiscs. DCPIB plugs the channel like a cork in a bottle - binding in the extracellular selectivity filter and sterically occluding ion conduction. Constricted and expanded structures reveal coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by internal stimuli. Conformational and symmetry differences between LRRC8A structures determined in detergent micelles and lipid bilayers related to reorganization of intersubunit lipid binding sites demonstrate a critical role for the membrane in determining channel structure. These results provide insight into LRRC8 gating and inhibition and the role of lipids in the structure of an ionic-strength sensing ion channel.