LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology

Swelling-activated volume-regulated anion channels (VRACs) are heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryoelectron microscopy (cryo-EM) structures of nonnative LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c(-/-) HCT116 cells are poorly activated by low intracellular ionic strength (mu) and insensitive to cell swelling with normal mu. Combining low mu with swelling modestly activates LRRC8A, allowing characterization of pore properties. VRACs are strongly inhibited by 10 mu M 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1oxo-1-H-inden-5-yl)oxy]butanoic acid (DCPIB) in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, an LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited similar to 90% by 10 mu M DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly mu dependent. At normal intracellular mu, LRRC8A + LRRC8C heteromers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular mu. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage dependence under low mu conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heteromeric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homomeric channels.

Automated summary

Swelling-activated volume-regulated anion channels (VRACs) are heteromeric channels comprising LRRC8A and LRRC8 paralogs. Cryo-EM structures of LRRC8A homohexamers have limitations in mimicking VRAC function, while LRRC8A + LRRC8C heteromers exhibit strong DCPIB sensitivity that is mu dependent. The LRRC8A R103F mutation eliminates voltage dependence under low intracellular mu conditions.