Synergistic Substrate binding in the CLC-Ec1 Exchanger

Abstract

Secondary active transporters couple the electrochemical gradients of two or more substrates to catalyze their stoichiometric exchange across biological membranes. In canonical models of alternating access exchange binding of different molecules is mutually exclusive or competitive. CLC-ec1 is a structurally known H+/Cl- exchanger of the CLC family and it has served as a guide in understanding the basic functional properties of CLC channels and transporters. Two glutamates (E148, E203) define the external and internal end points of the H+ permeation pathway. However the coupling mechanism of these exchangers is still obscure. We used Isothermal Titration Calorimetry to probe the coupling between H+/Cl- binding and transport by measuring the enthalpy of Cl- binding in different buffers. We found that binding of Cl- and H+ is synergistic: binding of 2 Cl- to CLC-ec1 induces the binding of 1 H+. Thus, the stoichiometries of binding and transport are equal but have opposite sign. Cl- binding induces the uptake of 1 H+ from the buffer in the E203Q mutant despite the fact that this mutant is unable to transport H+. In contrast, Cl- and H+ binding become decoupled in the E148A mutant. Thus, while both glutamates are essential for coupling movement of H+ and Cl- only E148 controls their coupled binding while protonation of E203 takes place in a Cl--independent manner. Decoupling H+ and Cl- fluxes by mutating residues that impair Cl- binding leads to a parallel disruption of their binding synergism, strongly suggesting that this step is critical for the transport process.In conclusion, our results show that during the exchange cycle binding of Cl- at one side of the membrane induces binding of H+ at the other side. This, implies that the transport cycle of CLC-ec1 is drastically different from conventional alternating access mechanisms.