Separate Ion Pathways in a Cl−/H+ Exchanger

Abstract

CLC-ec1 is a prokaryotic CLC-type Cl−/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl−. A critical glutamate residue, E148, was previously shown to be required for Cl−/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl− transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl− transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl− transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl− and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.

Molecular determinants of common gating of a ClC chloride channel

“…One observation offered tentative support for our hypothesis. In prokaryotic ClC anion/ H þ exchange proteins a glutamate residue, corresponding to E203 in ClC-ec1, forms an essential part of the H þ transport pathway 36 . This residue is present on helix H, which forms the pseudo-symmetrical dimer interface ( Supplementary Fig.…”

“…From comparison with the mechanism proposed by Feng et al 13 for proton transport in ClC antiporters, it is possible that protonation of E232 in this conformation leads to opening of the common gate and H þ transport from the intracellular to the extracellular solution 2,30 . The H þ transport pathway of ClC-1 is unknown, as the intracellular glutamate residue proposed to form the intracellular H þ transport pathway in Cl À /H þ antiporters 36 is not conserved in ClC channels ( Supplementary Fig. S1).…”

CLC-mediated anion transport in plant cells

“…In CLC channels, this glutamate residue is important for gating, whereas in exchangers the same residue is fundamental for the coupling between H C and Cl K . It has been shown that CLC exchangers present an additional glutamate in a position equivalent to position 203 of CLCec-1, which is not present in the channel subtype (Accardi et al 2005). Electrophysiological experiments have shown that the mutation of such glutamate abolishes the coupling with chloride (Accardi et al 2005).…”

CLC Cl − /H + transporters constrained by covalent cross-linking

“…1). The reversal potential (51 mV) of the ''strained'' dimer 207C-207C is slightly lower, but this value still represents respectably tight exchange coupling, especially when compared with variously uncoupled mutants previously described (11,15,16). We conclude, then, that the normal mechanism of Cl Ϫ /H ϩ transport is unperturbed in the covalent-dimer constructs.…”