Ion permeation through a Cl--selective channel designed from a CLC Cl-/H+ exchanger

Abstract

The CLC family of Cl(-)-transporting proteins includes both Cl(-) channels and Cl(-)/H(+) exchange transporters. CLC-ec1, a structurally known bacterial homolog of the transporter subclass, exchanges two Cl(-) ions per proton with strict, obligatory stoichiometry. Point mutations at two residues, Glu(148) and Tyr(445), are known to impair H(+) movement while preserving Cl(-) transport. In the x-ray crystal structure of CLC-ec1, these residues form putative "gates" flanking an ion-binding region. In mutants with both of the gate-forming side chains reduced in size, H(+) transport is abolished, and unitary Cl(-) transport rates are greatly increased, well above values expected for transporter mechanisms. Cl(-) transport rates increase as side-chain volume at these positions is decreased. The crystal structure of a doubly ungated mutant shows a narrow conduit traversing the entire protein transmembrane width. These characteristics suggest that Cl(-) flux through uncoupled, ungated CLC-ec1 occurs via a channel-like electrodiffusion mechanism rather than an alternating-exposure conformational cycle that has been rendered proton-independent by the gate mutations.

Fig. 1.

Structure of CLC-ec1. Ribbon diagram of WT CLC-ec1 is shown (PDB ID code 1OTS), extracellular side up, with Cl⁻ ions as green spheres. (Left) Rendered in minimally cutaway view to visualize more easily side chains Glu_ex at the outer gate and Tyr_c and Ser_c at the inner gate, as indicated.

Fig. 2.

Unitary Cl⁻ efflux via CLC-ec1 gate mutants. Traces of Cl⁻ appearance (normalized to final Cl⁻ concentration) in a suspension of liposomes reconstituted with various CLC-ec1 constructs, under conditions in which liposomes contain ≈1 transporter on average are shown. Efflux was initiated by valinomycin/FCCP addition, and Cl⁻ concentration was followed by a Ag/AgCl electrode. Liposomes were loaded with 300 mM Cl⁻, and external Cl⁻ typically increased during the time course from 1.1 mM to 1.3 mM. Time courses of Cl⁻ efflux were fit (red) by single exponentials with slow linear leak as described in ref. . Experiment was ended by addition of detergent to disrupt all liposomes, including those devoid of protein (abrupt increase in Cl⁻ at end of red fits). Shown for illustrative purposes are: WT (E/Y), outer gate removed (A/Y), inner gate removed (E/A), and both gates removed (A/A). Unitary turnover rates derived from such traces are reported in Table S1 and Fig. 4.

Fig. 3.

Cl⁻ efflux traces of ungated mutants. Cl⁻ efflux time courses were followed as in Fig. 2, for the indicated CLC-ec1 mutants. (Upper) Variation of the outer gate, with inner gate removed. (Lower) Variation of the inner gate, with outer gate removed.

Fig. 4.

Unitary Cl⁻ transport rates for gate mutants. Cl⁻ efflux traces as in Figs. 2 and 3 were analyzed to derive the unitary transport rates, γ_o, of the indicated mutants. (Upper) Variation of side chain at outer gate with inner gate removed. (Lower) Variation of inner gate with outer gate removed. Mutants with asterisks indicate single-gate mutants, i.e., with one WT gate residue.

Fig. 5.

Conduits through CLC-ec1. The HOLE program was used to visualize Cl⁻ pathways through CLC-ec1 and a doubly ungated mutant. Images made in Pymol show translucent surface representation of CLC-ec1 homodimer, with Cl⁻ ions (green spheres) marking the anion-binding sites and HOLE conduits (red dots). Each conduit shown was synthesized from ≈50 separate HOLE runs; only trajectories that coincided with the Cl⁻-binding sites were selected for display. (Left) WT protein (PDB ID code 1OTS), showing the gate residues Glu_ex and Tyr_c in space-filled representation. (Right) Doubly ungated A/A mutant (PDB ID code 3DET). A third Cl⁻ ion, located at the same position as the Glu_ex side chain of the WT, is crystallographically observed in all mutants with Glu_ex mutated to externally open residues.