Testing the Limits of Stoichiometric Exchange in a CLC-Type Transporter

The CLC transporters mediate the stoichiometric exchange of 2 Cl-: 1 H+ in prokaryotes and in eukaryotes. The CLC Cl- transport pathway is defined by 3 anionic binding sites, Sext, Scen and Sint which can be occupied either by Cl- ions or by a conserved glutamate, Gluex. Three conformation of the pathway have been observed where (i) the protonated Gluex is out of the pathway and the 3 sites are occupied by Cl-, (ii) Gluex occupies the Sext while Cl- ions are bound to Scen and Sint, and (iii) Gluex in Scen sandwiched by two Cl- ions in Sext and Sint. While the role of Scen and Sext as well as that of Gluex as the external gate is well documented, the rate-limiting step regulating the exchange of ions between Sint and Scen remain poorly understood. Here we investigate (i) how robust is the coupling stoichiometry of the CLCs and (ii) the role of Sint in the transport cycle.To this end we reconstituted CLC-ec1 in proteoliposomes and used Cl- and H+ flux assays to measure transport. To test the robustness of the stoichiometry of coupled transport we are measuring simultaneous Cl- and H+ fluxes under extreme Cl- gradients and at voltages as high as 180 mV. To identify the role of Sint in transport we measured H+ transport driven by voltage at different symmetrical Cl- concentrations. As expected H+ transport increases when [Cl-] is raised from 0.02 to ∼0.3 M. Interestingly, we found that H+ transport decreases for [Cl-] >0.3 M and is drastically inhibited at 1 M [Cl-]. This result suggests that at high [Cl-] Gluex becomes “sandwiched” between Cl- ions in Sext and Scen, indicating that the internal site does play a role in the transport cycle.