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.

Figure 1.

Carboxylates in CLC-ec1. (A) Ribbon representation of the CLC-ec1 dimer, view from within the membrane, with extracellular side up. α carbons of the carboxylate residues mutated here are shown for a single subunit as yellow spheres, except for E148 and E203 (red), and E113 (black); gray spheres mark carboxyl residues that are not studied here, and green spheres designate Cl⁻ ions. Also shown are R28 and K131 (blue). (B) Sequence alignment of the PIGG-pen region, with E203 equivalent indicated in red. Genes listed are of human origin except for CLC-0 (Torpedo marmorata), CLC-ec1 (E. coli), CLC-st1 (Salmonella typhimurium), and GEF1 (Saccharomyces cerevisiae).

Figure 2.

Loss of H⁺ coupling in E203Q. (A) Currents mediated by E203Q in symmetrical 300 mM Cl⁻, with or without a 4-unit pH gradient. A 3-s voltage pulse (−100 to +100 mV in 10-mV steps) was applied from a holding potential of 0 mV, followed by a 1-s tail pulse to −100 mV. Dashed line marks zero current. (B) I-V curves, normalized to the current in the same bilayer at −100 mV in symmetrical pH 3.0 and 300 mM Cl⁻. Open circles, wild type, pH 3.0/pH 7; black circles, E203 pH 3/pH 3; red triangles, E203 pH 3.0/pH 7; green squares, E203 pH 7/pH 3; yellow triangles, E203 pH 7.0/pH 7. Gradients are indicated by convention cis/trans sides of planar bilayer, with the trans side defined as zero voltage. (C) Reversal potential, V_rev, as a function of [Cl⁻] for wild type (black circles) and E203Q (red triangles); dashed line is Nernst potential predicted for ideal Cl⁻ selectivity.

Figure 3.

Fluxes of Cl⁻ and H⁺. Liposomes reconstituted with CLC-ec1 (1–5 μg/mg lipid) were assayed for either ³⁶Cl⁻ uptake or Cl⁻-driven H⁺ pumping. Top panels, ³⁶Cl⁻ uptake at pH 7.0 (circles) and pH 4.5 (triangles) for WT (A), E203Q (B), and E148A/E203Q (C). Lower traces show H⁺ flux assays, upward deflection indicating increase in pH of the liposome suspension. Additions of 1 μM valinomycin or FCCP are denoted by * and +, respectively.

Figure 4.

H⁺/Cl⁻ coupling for CLC-ec-1 mutants. Reversal potentials of indicated mutants in presence of (A) a Cl⁻ gradient (300 mM/45 mM) at symmetrical pH 3.0 or (B) a pH gradient (3/7) at symmetrical 300 mM Cl⁻. Dotted lines represent wild-type values under the same conditions. Data for wild type and E148A taken from Accardi et al. (2004); EA/EQ denotes the double mutant E148A/E203Q. Colored bars indicate mutants with H⁺ transport inhibited completely (red) or partially (blue).

Figure 5.

Crystal structure and halide binding. (A) Wild type; (B) E203Q. Top panel, 2F_O-F_C electron density maps (blue) contoured at 1.2 σ, with protein model shown in stick representation. Wild-type map was calculated to 3.3 Å resolution from deposited structure factors (accession no. 1OTS). Bottom panel, anomalous difference density maps (green) of crystals grown in NaBr, determined from data collected at the Br⁻ absorption edge, contoured at 5 σ, with the same wild-type protein model (1OTS) shown for both maps, with subunit A in red and subunit B in blue.

Figure 6.

Bifurcated pathway. CLC dimer, with the following side chains highlighted: E148, E203 (red); S107, Y445 (blue). Cl⁻ ions shown as green spheres. Proposed Cl⁻ and H⁺ pathways are indicated in green and red, respectively. The region between E148 and E203, which the H⁺ must traverse, is indicated by the gray box, with side chains of S107 and Y445 indicated in blue.