Proton Transport and Conformational Changes in H+/CL- Exchangers

Abstract

CLC transporters localize to the membranes of intracellular compartments, such as lysosomes and endosomes, where they mediate a variety of physiological roles by exchanging 2 Cl- ions for 1 H+. Very little is known about the mechanisms underlying the transport process and many questions are still unanswered, in particular the H+ pathway has not been identified and which conformational changes the transporters undergo is still not clear. To elucidate these important aspects we decided to investigate how protons translocate through the protein by studying the deuterium kinetic isotope effect and to probe the conformational changes by measuring the temperature dependence of the transport rate of the human transporter CLC-5 and the bacterial homologue CLC-ec1. We found that both CLC-5 and CLC-ec1 have similar thermodynamic profile. The transport rate in deuterium is decreased by ∼20-40%, suggesting a H+ movement through a hydrogen-bonded pathway, possibly formed by water. Both transporters also have a similar and modest temperature dependence, suggesting that the proteins undergo limited conformational changes. Interestingly, we observed unaltered apparent activation enthalpy of transport when Cl- or H+ binding, coupling or transporter gating are impaired, while the transport rates are affected. We hypothesize that H+ movement through a CLC transporter takes place along a series of hydrogen bond formed by water molecules and that only limited conformational changes occur during the transport cycle. Finally, we propose a transport mechanism where several rate-limiting steps with similar apparent activation enthalpies are involved, instead of a single rate-limiting step mechanism.