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

Abstract

CLC Cl(-)/H(+) exchangers are homodimers with Cl(-)-binding and H(+)-coupling residues contained within each subunit. It is not known whether the transport mechanism requires conformational rearrangement between subunits or whether each subunit operates as a separate exchanger. We designed various cysteine substitution mutants on a cysteine-less background of CLC-ec1, a bacterial CLC exchanger of known structure, with the aim of covalently linking the subunits. The constructs were cross-linked in air or with exogenous oxidant, and the cross-linked proteins were reconstituted to assess their function. In addition to conventional disulfides, a cysteine-lysine cross-bridge was formed with I(2) as an oxidant. The constructs, all of which contained one, two, or four cross-bridges, were functionally active and kinetically competent with respect to Cl(-) turnover rate, Cl(-)/H(+) exchange stoichiometry, and H(+) pumping driven by a Cl(-) gradient. These results imply that large quaternary rearrangements, such as those known to occur for "common gating" in CLC channels, are not necessary for the ion transport cycle and that it is therefore likely that the transport mechanism is carried out by the subunits working individually, as with "fast gating" of the CLC channels.

Fig. 1.

Cl⁻/H⁺ coupling in the Cys-less transporter. Cys-less CLC-ec1 was inserted into planar lipid bilayers, and the resulting currents were recorded under voltage-clamp conditions. (A) Representative families of currents, with voltages from −100 to 100 mV in 10-mV steps, with either Cl⁻ (300 mM/17 mM) (Left) or pH gradient (3.0/7.0) (Right) across the bilayer. Dashed lines mark zero-current level. (B) Current–voltage curves measured 200 ms before the end of the pulse interval for the gradient of Cl⁻ (open points) or H⁺ (filled points). Reversal potentials are marked by arrows. (C) Variation of reversal potential, V_r, with Cl⁻ or pH gradients. For Cl⁻ gradients at symmetrical pH 3.0 (filled points), one side of the membrane was held at 300 mM KCl while KCl was varied on the opposite side. For pH gradients at symmetrical 300 mM KCl (open points), one side of the membrane was held at pH 3.0 while the opposite-side pH was varied. Gradients are reported as Nernst equilibrium potentials of each ion. Dashed lines represent the previously published measurements for wild-type protein (1).

Fig. 2.

Design of intersubunit cross-links. (Left) “Side view” of the dimeric transporter, with extracellular side up. (Right) “Head-on” cross-interface view of a single subunit obtained after removing one subunit and rotating the other by 90^o, as indicated. Wild-type positions that, when cysteine-substituted, fail to form cross-links well are shown in yellow. Positions capable of fully cross-linking with a partner on the opposite subunit are shown in blue (207/207), green (230/249), and red (216/433).

Fig. 3.

Analysis of cross-linking by SDS/PAGE. The indicated CLC-ec1 variants were run on 10–12% SDS gels under nonreducing conditions. Lanes marked “DTT” are samples in which reducing conditions were maintained throughout the protein preparation. (A) Spontaneous cross-linking of 230C/249C. The molecular masses of standards are indicated in kilodaltons. (B) Spontaneous partial cross-linking for 207C driven to completion by 50 μM CuP. (C) Cysteine–lysine cross-linking. Shown are partial cross-linking of 433C at a concentration of I₂ (3 μM) slightly below the protein concentration (4 μM) and complete cross-linking by excess I₂. For the negative control lanes, CuP or H₂O₂ was used as an oxidant, and K216M was used as the mutant. (D) Straitjacketing by multiple cross-links in the triple mutant 230C/249C/433/C.

Fig. 4.

Mechanism for Cys–Lys cross-link by I₂-driven oxidative amination.

Fig. 5.

Proton pumping by cross-linked transporters. CLC Cys-less and cross-linked variants indicated were reconstituted into liposomes and tested for Cl⁻-driven H⁺ pumping. Upward deflection indicates pH rise of the liposome suspension accompanying uphill proton influx.

Fig. 6.

Cl⁻/H⁺ coupling of cross-linked transporters. Reversal potentials were determined on the indicated CLC exchangers under symmetrical Cl⁻ pH gradients (4.0/7.0), as in Fig. 1. Each point represents the average ± SE of at least five independent measurements. The gray horizontal bar represents the range of values found for wild-type protein.

Fig. 7.

Unitary Cl⁻ turnover rate. (A) Traces from Cl⁻ electrode show the release of Cl⁻ into the liposome suspension. Arrows mark the addition of Vln/FCCP at the beginning of the experiment and 50 mM octylglucose at the end. Raw traces are shown (from the top to the bottom traces) for Cys-less, 433C, 230C/249C, 207C/207C, triple, and control with no protein reconstituted. All mutants were assayed after full cross-linking. (B) Turnover rates γ (filled bars) and f_o (open bars) for the indicated CLCs, each representing mean ± SE of five measurements.