Functional Study of CBS Domain Interaction during Common Gating of CLC-0 Chloride Channel

Abstract

A pair of tightly interacting cystathionine β-synthetase (CBS) domains serves important regulatory functions in various protein families. Two CBS domains (CBS1 and CBS2) exist in the C-terminal of all CLC channels and appear to mediate most of the cytoplasmic inter-subunit interactions. Previous study in our lab indicates that common gating of CLC-0 is associated with a large conformational change in the C-terminal. How interaction between CBS domains affects common gating remains elusive.Based on three recently reported crystal structures of the C-terminal of CLC channels (CLC-0, CLC-5 and CLC-Ka), we identified a set of residues that likely contribute to CBS interaction. Mutations at most of these positions affected the gating kinetics as well as the equilibrium of common gating. Preliminary data indicate that cysteine mutations at these positions can be modified by thiol-reactive reagents. Furthermore, the kinetics of common gating changed after cysteine modification. These results indicate that CBS domains do play an important role in common gating.The figure shows the time course of NEM modification of L741C (right); the mutation was made in the control channel (left) background that lacked native reactive cysteines.View Large Image | View Hi-Res Image | Download PowerPoint Slide