A conserved mechanism of LRRC8 channel inhibition by distinct drugs

We employed a Leucine Rich Repeat Containing 8 (LRRC8) channel chimera, termed 8C-8A(IL125), to investigate the molecular mechanism of action of the novel volume-sensitive anion channel (VRAC) inhibitor, zafirlukast. 8C-8A(IL125) comprises LRRC8C (8C) and 25 residues from LRRC8A (8A) intracellular loop 1 (IL1) and forms volume-sensitive, structurally defined heptameric channels with normal pharmacological properties. In silico docking and modeling with AlphaFold3 identified a putative zafirlukast binding site comprising the amino (N)-terminal domain (NTD) and inter-subunit fenestrae between transmembrane (TM) helices 1 and 2. Consistent with this model, mutations in NTD, TM1, and TM2 alter 8C-8A(IL125) and heteromeric 8A/8C sensitivity to zafirlukast and the structurally distinct drug pranlukast. Inhibition is not mediated by extracellular pore block or the so-called lipid gate. Mutations or low pH conditions that enhance voltage-dependent inactivation also increase zafirlukast sensitivity. We propose zafirlukast and pranlukast promote channel inactivation through destabilization of the pore.