Splice variation alters CBS domain structure and gating of a C. elegans ClC channel

Abstract

Eukaryotic CLCs have large cytoplasmic C‐termini containing two Cystathionine‐β‐Synthase (CBS) domains. The role of CBS domains in ClC channel gating and structure is unclear. clh‐3 encodes two splice variants, CLH‐3a and CLH‐3b, of a C. elegans ClC channel that exhibit striking differences in sensitivity to depolarizing voltages, pH and Cl−. Mutagenesis studies have shown that the C‐terminus of CLH‐3b gives rise to its unique gating properties. Splice variation of the C‐terminus includes the last six amino acids in the second CBS domain (CBS2). Mutating these amino acids to those present in CLH‐3a gives rise to CLH‐3a gating properties. Similarly, CLH‐3b gating properties are induced in CLH‐3a by mutating the last six amino acids of CBS2 to those present in CLH‐3b. To define how these amino acids alter CBS2 structure, we developed homology models based on crystal structures of CBS domains in vertebrate CLCs using Modeller and Rosetta software. Our models show that the six amino acid splice variation is part of the last α‐helix of CBS2. Sampling 10,000 conformations with Rosetta demonstrated that the splice variation in CLH‐3b has a higher probability of forming a helical turn. Thus, the last α‐helix of CBS2 in CLH‐3a is shorter compared to CLH‐3b. We are currently carrying out modeling and mutagenesis studies to determine how CBS2 splice variation alters the channel dimer‐dimer interface and gating.