Basic Mechanisms of Ion Channel Function

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DESCRIPTION (provided by applicant): This research is aimed at understanding in chemical detail the fundamental mechanisms by which proteins belonging to the "CLC family of Cl- channels" work. CLC channels are widespread throughout the biological world and are essential to the proper functioning of mammalian cell membranes in many different physiological contexts. Mutations or disruptions of any of the nine CLC isoforms inhabiting the human genome lead to numerous diseases, from skeletal muscle myotonias to compromised renal control of blood pressure to improper reabsorption of bone. As a result of recent work we now know that the CLC family includes isoforms that operate by fundamentally different Cl- transport mechanisms; while the long-studied muscle type CLCs are passive channels, a bacterial homolog was surprisingly found to be a secondary active transporter in which uphill Cl- transport is coupled to downhill H+ transport. I propose to investigate the ramifications of this perplexing discovery by carrying out a detailed structure-function study of the bacterial CLC homolog. We will investigate the mechanism of proton coupling to Cl- transport, will attempt to transform, by directed mutagenesis, the active transporter into a true channel, and will work to clone, express, and reconstitute a set of prokaryotic CLCs carrying a long c-terminal domain.