LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology

Abstract

Swelling-activated VRACs are heterohexameric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryo-electron microscopy (EM) structures of non-native 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 (µ) and insensitive to cell swelling with normal µ. Combining low µ with swelling modestly activates LRRC8A allowing characterization of pore properties. VRACs are strongly inhibited by 10 mM 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, a LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ~90% by 10 mM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly µ-dependent. At normal intracellular µ, LRRC8A+LRRC8C heterohexamers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular µ. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage-dependence under low µ conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heterohexameric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homohexameric channels.

Allosteric modulation of LRRC8 channels by targeting their cytoplasmic domains

“…Insight into the structural basis of VRAC modulation was obtained from cryo-EM structures of LRRC8A-sybody complexes. Irrespective of the fact that LRRC8A homomers are not found under physiological conditions, such assemblies form functional anion channels, although with compromised activation properties 18,23,24 . Moreover, the observed strong potentiation of LRRC8A activity by the sybodies Sb4 and Sb5 and its inhibition by Sb3 further emphasize an equivalent modulatory role of binders also in the context of homomeric channels (Fig.…”

“…The general architecture of LRRC8 channels was revealed from homomeric structures of the LRRC8A [18][19][20][21] and LRRC8D 22 subunits. Although 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 also observed in heteromeric proteins 18 .…”

LRRC8A is essential for volume‐regulated anion channel in smooth muscle cells contributing to cerebrovascular remodeling during hypertension

“… 38 Accumulating evidence from the structure of LRRC8A and other electrophysiological studies indicated that heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog are crucial for VRAC activity. 37 , 38 , 39 , 40 However, some other studies show that LRRC8A is dispensable for VRAC activity, 15 , 16 suggesting that LRRC8A alone is not sufficient for volume regulation in response to hypotonic stimulation and VRAC activity in all cell types. The controversy of these studies led us to investigate whether LRRC8A is necessary for VRAC functions in cerebrovascular smooth muscle cells.…”

“…In 2014, the studies from two laboratories identified LRRC8A as an essential component of VRAC 13,14 . It has been demonstrated that LRRC8A homohexamers poorly recapitulated VRAC electrophysiological properties 38 . Accumulating evidence from the structure of LRRC8A and other electrophysiological studies indicated that heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog are crucial for VRAC activity 37–40 .…”

“…It has been demonstrated that LRRC8A homohexamers poorly recapitulated VRAC electrophysiological properties 38 . Accumulating evidence from the structure of LRRC8A and other electrophysiological studies indicated that heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog are crucial for VRAC activity 37–40 . However, some other studies show that LRRC8A is dispensable for VRAC activity, 15,16 suggesting that LRRC8A alone is not sufficient for volume regulation in response to hypotonic stimulation and VRAC activity in all cell types.…”