Charge Transport in the ClC-type Chloride-Proton Anti-porter from Escherichia coli

Gernot Kieseritzky,Ernst-Walter Knapp

doi:10.1074/jbc.m110.163246

Abstract

The first chloride transporter identified in the superfamily of ClC chloride channels was from Escherichia coli (EClC) (Accardi, A., and Miller, C. (2004) Nature 427, 803-807). Pathways, energetics, and mechanism of proton and chloride translocation and their coupling are up to now unclear. To bridge the hydrophobic gap of proton transport, we modeled four stable buried waters into both subunits of the WT EClC structure. Together they form a "water wire" connecting Glu-203 with the chloride at the central site, which in turn connects to Glu-148, the hypothetical proton exit site. Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu-203 all the way downstream onto Glu-148. We demonstrated by electrostatic and quantum chemical computations that protonation of the central chloride is energetically feasible. We characterized all chloride occupancies and protonation states possibly relevant for the proton-chloride transport cycle in EClC and constructed a working model. Accordingly, EClC evolves through states involving up to two excess protons and between one and three chlorides, which was required to fulfill the experimentally observed 2:1 stoichiometry. We show that the Y445F and E203H mutants of EClC can operate similarly, thus explaining why they exhibit almost WT activity levels. The proposed mechanism of coupled chloride-proton transport in EClC is consistent with available experimental data and allows predictions on the importance of specific amino acids, which may be probed by mutation experiments.

Highlights

On the other hand, replacing Tyr-445 with alanine causes a complete breakdown of PT, whereas the chloride transport is not impaired (4, 7). We infer from these facts that Tyr-445 serves two fundamental functions in WT EClC; (i) it defines the exact geometry of the central chloride binding site Cl(2) and facilitates chloride binding by providing a strong H-bond, and (ii) it creates a narrow, well defined hydrophobic pore for the linear water wire keeping the protonation energy of the waters at a high level to allow the exit of protons against large proton concentration
Summary and Conclusion—As shown in Fig. 6B the total energy balance of the discussed transport cycle is zero, as we set the proton and chloride concentration to be equal on both sides of the membrane
Under conditions where the chloride concentration is higher in the periplasmic lumen than inside the bacterial cell, the exchange cycle depicted in Fig. 6A would effectively pump protons into the periplasmic lumen driven by the translocation of chlorides into the bacterial cell

Summary

Introduction

Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu203 all the way downstream onto Glu-148. Any such string refers to an EClC structure with geometry-optimized hydrogen and buried water positions in the presence of the corresponding chlorides and protons in the transport pathways.

Results

Conclusion