CBS Domains Regulate CLC Chloride Channel Gating: Role of the R-Helix Linker
All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-beta-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two alpha-helices and three beta-strands arranged as beta1-alpha1-beta2-beta3-alpha2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form alpha2 of the second CBS domain (CBS2). We demonstrate that interchanging alpha2 between CLH-3a and CLH-3b interchanges their gating properties. The “R-helix” of ClC proteins forms part of the ion conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the “R-helix linker”. C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that alpha2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in alpha2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-like gating and extracellular cysteine reactivity. We postulate that the R-helix linker interacts with CBS2 alpha2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.