Conformational Changes Outside the Ion Pathway are required for Transport in a CLC-Type Cl−/H+ Exchanger

Abstract

The CLC proteins catalyze transport of chloride ions (Cl-) through cellular membranes in muscle, kidney, bone, and neurons. While some CLCs are ion channels others are H+-coupled secondary active transporters mediating the stoichiometric exchange of 2 Cl- for 1 H+. The exchange mechanism of the CLCs is unclear. All proposed models postulate that the only conformational changes taking place during transport are the movements of a conserved glutamate's side chain in and out of the Cl- permeation pathway. This hypothesis is supported by structural and functional work. However, others have suggested that regions distal to the Cl- pathway might also be involved in transport.To test whether transport entails only local or also global rearrangements we constrained the movement of helices J, O, P and Q, which do not line the Cl- or H+ pathways in CLC-ec1, a CLC prokaryotic homologue. If exchange involves the relative movement of these helices then these constraints should reduce the transport rate. In a cys-less background we introduced pairs of cysteines at different locations in this 4-helix bundle and Hg2+-crosslinked them. All unreacted proteins mediate Cl-/H+ exchange at rates comparable to that of the WT. Reaction with Hg2+ results in a striking pattern: constraining residues facing the extracellular side has no effect, while targeting residues deeper in to the protein induces progressively a more drastic reduction of activity. Finally, constraints placed close to the intracellular side results in virtually inactive transporters. This reduction is not due to a Hg2+-induced distortion of the Cl- pathway: Cl- binding is preserved as is Cl- transport through a cross-linked doubly ungated mutant.Thus we propose that the transport cycle in CLC-ec1 entails the movement of these helices outside of the Cl- transport pathway.