Ion-binding properties of the ClC chloride selectivity filter

Abstract

The ClC channels are members of a large protein family of chloride (Cl-) channels and secondary active Cl- transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl- ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl- channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction.

Figure 1

Structure and ion-binding properties of the EcClC selectivity filter. (A) View of a ribbon representation of the EcClC dimer from within the membrane. The subunits are colored in green and blue. The ions are represented as red spheres. The region of the selectivity filter in one subunit is indicated by a transparent gray box. (B) Selectivity filter of wtEcClC (closed) and the EcClC mutant E148Q (open) viewed from the dimer interface. The protein backbone is shown as a ribbon, with selected residues as sticks. The N-terminal ends of α-helices are colored in cyan. The ions are represented as red spheres. The Br⁻ anomalous difference density (contoured at 6σ) is shown superimposed (red). The path for sampling the anomalous difference density is shown as gray lines (open). Aqueous cavities from the extracellular solution (out) and intracellular solution (in) are shown as cyan mesh. The ion-binding sites are labeled. (A) and (B) were prepared with DINO (www.dino3d.org). (C) One-dimensional anomalous difference electron density in the selectivity filter at high Br⁻ concentration. The density (ρ) is plotted in units of its standard deviation. The filter position is shown relative to S_cen. The curve for the ‘open conformation' is colored in blue, the curve for the ‘closed conformation' in red.

Figure 2

Structure and function of a mutant with a single ion-binding site. (A) Stereo view of the selectivity filter of the mutant S107A/E148Q/Y445A (TRPL). The data were collected from a crystal grown in 100 mM Br⁻. The view is the same as in Figure 1B. A 2F_o–F_c electron density map calculated at 3.5 Å resolution and contoured at 1σ is shown superimposed on the refined structure. The Br⁻ anomalous difference density (red), which was calculated from the same data set, is contoured at 4.5σ. Mutated residues are labeled. (B) EcClC-mediated ³⁶Cl⁻ uptake into liposomes. The averages over three data points and the standard deviations are shown. All measurements were carried out at pH 5. The protein was reconstituted at a ratio of 5 μg/mg of lipid. Time courses from vesicles containing wt EcClC and TRPL are shown in blue and red, respectively. Uptake from vesicles without protein is shown in green. (C) Anomalous difference electron density in the selectivity filter of TRPL at high Br⁻ concentration (250 mM). Electron density of the ‘open conformation' is shown for comparison.

Figure 3

Binding affinity of Br⁻ to the EcClC selectivity filter. (A) Anomalous difference density in the ‘open conformation' at different Br⁻ concentrations. The values refer to the ion concentration in the medium. The traces at different ion concentration have unique colors, with their respective Br⁻ concentration in mM shown in the same color. The filter position is shown relative to S_cen. (B) Anomalous difference density in the ‘closed conformation' at different Br⁻ concentrations. (C) Br⁻ affinity in the ‘open conformation'. The averages of the peak heights of the electron densities were obtained as described in Table II. The standard deviation (for n=4) or the range of values (for n=2) is shown as bars. Data for S_int, S_cen and S_ext are colored in green, red and blue, respectively. Curves show a fit of the averaged density to equation (1). Curves, data points and errors were scaled by subtracting ρ_min and by subsequently dividing by (ρ_max−ρ_min). (D) Br⁻ affinity in the ‘closed conformation'. The data were obtained as described in (C) and is colored in cyan for S_int and orange for S_cen.

Figure 4

Br⁻/Cl⁻ selectivity in the EcClC selectivity filter. (A) Anomalous difference density in the ‘open conformation' at different Br⁻/Cl⁻ ratios. The total halide ion concentration in solution is in all cases 250 mM. The data sets are colored in unique colors and labeled with the respective Br⁻ ratio. The filter position is shown relative to S_cen. (B) Anomalous difference density in the ‘closed conformation' at different Br⁻/Cl⁻ ratios. The Br⁻ ion concentration at 100% Br⁻ is 400 mM; in all other cases the total halide concentration is 250 mM. (C) Br⁻/Cl⁻ selectivity in the ‘open conformation'. The averages of the peak heights of the electron densities were obtained as described in Table II. The standard deviation (for n=4) or the range of values (for n=2) is shown as bars. Data for S_int, S_cen and S_ext are colored in green, red and blue, respectively. Curves show a fit of the averaged density to equation (2). Curves, data points and errors were scaled by dividing by (ρ_max). (D) Br⁻ affinity in the ‘closed conformation'. The data were obtained as described in (C) and are colored in cyan for S_int and orange for S_cen.

Figure 5

Two models for ion conduction. Schematic drawing of ion conduction in a single ion pore and a multiple-ion pore. (A) Single-ion pore: The selectivity filter binds only one ion at a time. During permeation the ion enters the selectivity filter from the solution and diffuses between the different binding sites of the channels until it dissociated from the filter. (B) Multiple-ion pore: The selectivity filter binds multiple ions, which permeate in a single file when additional ions enter the filter. The filter is depicted in its open state; the ions are drawn as spheres.