Channel-like slippage modes in the human anion/proton exchanger ClC-4

Abstract

The ClC family encompasses two classes of proteins with distinct transport functions: anion channels and transporters. ClC-type transporters usually mediate secondary active anion-proton exchange. However, under certain conditions they assume slippage mode behavior in which proton and anion transport are uncoupled, resulting in passive anion fluxes without associated proton movements. Here, we use patch clamp and intracellular pH recordings on transfected mammalian cells to characterize exchanger and slippage modes of human ClC-4, a member of the ClC transporter branch. We found that the two transport modes differ in transport mechanisms and transport rates. Nonstationary noise analysis revealed a unitary transport rate of 5 x 10(5) s(-1) at +150 mV for the slippage mode, indicating that ClC-4 functions as channel in this mode. In the exchanger mode, unitary transport rates were 10-fold lower. Both ClC-4 transport modes exhibit voltage-dependent gating, indicating that there are active and non-active states for the exchanger as well as for the slippage mode. ClC-4 can assume both transport modes under all tested conditions, with exchanger/channel ratios determined by the external anion. We propose that binding of transported anions to non-active states causes transition from slippage into exchanger mode. Binding and unbinding of anions is very rapid, and slower transitions of liganded and non-liganded states into active conformations result in a stable distribution between the two transport modes. The proposed mechanism results in anion-dependent conversion of ClC-type exchanger into an anion channel with typical attributes of ClC anion channels.

Figure 1.

External anions modify the anion/proton transport stoichiometry. (A) Pulse protocol and representative ClC-4 whole cell current traces in standard Cl⁻-based intracellular and extracellular solutions. (B) Time courses of intracellular pH changes at the same cell at various test potentials. Red lines show linear fits to obtain ΔpH/Δt. (C) Current–voltage dependences measured for various external anions with Cl⁻ intracellular solution. Cells were sequentially perfused with different extracellular anions, and current amplitudes were normalized to currents determined in Cl⁻-based external solution (n = 4–6). (D) Rates of intracellular pH change in the same cells as in C normalized to values obtained in Cl⁻-based external solution. (E) Relative current to proton flux ratio for Cl⁻, Br⁻, I⁻, NO₃⁻, and SCN⁻ at +90 mV. (F) Absolute anion to proton transport stoichiometry for Cl⁻, NO₃⁻, and SCN⁻ determined at +90 mV (n = 4–7).

Figure 2.

External Cl⁻ promotes coupled anion–proton exchange. (A) Representative intracellular pH time course at +90 mV in a ClC-4 expressing cell perfused with extracellular solutions containing 30 mM SCN⁻ and variable concentrations of Cl⁻. Solid lines represent linear fits. (B) Normalized current–voltage relationships from the same cell at extracellular solutions containing 30 mM SCN⁻ and variable concentrations of Cl⁻. (C) Variation of normalized current amplitudes and normalized proton fluxes by increasing concentrations of Cl⁻ at +90 mV (n = 4–5). Lines represent fits with standard one-site binding function (Michaelis-Menten functions). (D) Ratios of unitary transport rates of H⁺-coupled and uncoupled ClC-4 currents for different [Cl⁻] with constant [SCN⁻] at +90 mV (n = 3).

Figure 3.

Unitary properties of ClC-4 in intracellular I⁻ and extracellular SCN⁻. (A) Averaged power spectrum of ClC-4 currents from 30 sweeps after correction for background noise at −115 or +135 mV. Lines represent fits with a second-order Lorentzian function. Blue crosses depict a representative power spectrum from a nontransfected HEK293 cell in extracellular SCN⁻. (B) Representative time course of ClC-4 mean currents and variances used for nonstationary noise analysis. (C) Current–variance plot (top panel) and linear transformation of the data (bottom panel) for the cell given in B. Red lines represent the fit with standard parabolic function (top panel) or a linear fit (bottom panel). (D) Representative ClC-4 current responses to a voltage ramp between −300 and +400 mV. Current amplitudes were normalized to the value at +150 mV.

Figure 4.

Effects of filtering on ClC-4 current noise. (A) Effects of low-pass filtering (3 and 10 kHz) on the amplitude of simulated single channels with different lengths: 10 µs (mimicking transporter mode), 100 µs (flickering ion channel), and 1 ms (ion channel). (B) Simulated single-channel events with 50% open probability and different mean open lifetimes (1 ms and 100 µs) before and after low-pass filtering with 1-kHz cutoff frequency. (C) Predicted effects of the low-pass filter frequency on the standard deviation of simulated single-channel traces with mean open lifetimes of 10 µs, 100 µs, and 1 ms. (D) Dependence of experimentally determined ClC-4 unitary current amplitude on the low-pass filter cutoff frequency. Nonstationary noise analysis was performed after offline filtering of macroscopic ClC-4 currents. The effects of low-pass filtering on simulated unitary events of 100 and 200 µs are given as solid lines.

Figure 5.

Cl⁻ reduces the number of channels detected by noise analysis. (A) Representative whole cell currents and current variances obtained in a cell stably expressing ClC-4 sequentially perfused with extracellular solution containing 100% SCN⁻, 50% SCN⁻ plus 50% gluconate, and 50% SCN⁻ plus 50% Cl⁻. An intracellular I⁻-based solution was used. (B) Current–variance plots of the data in A. Lines represent fits with standard parabolic function. (C) Linear transformation of the data in B. Note the different slope resulting when 50% of SCN⁻ anions were substituted with 50% Cl⁻ or gluconate. Lines represent linear fits to the data.

Figure 6.

Anion/proton transport stoichiometry of ClC-4 increases with reduced apparent anion binding affinity. (A) Representative current–voltage relationship from a cell perfused with extracellular solutions containing various SCN⁻ concentrations. Current–voltage relationships were obtained by applying 100-ms voltage ramps from −50 to +135 mV. (B) Dependence of the macroscopic current amplitude at +130 mV on the external anion concentration for various external anions (n = 4–7). Lines represent fits with Michaelis-Menten equation to obtain apparent saturation constants. (C) Relative anion/proton coupling as determined in Fig. 1 F plotted against the measured apparent Michaelis constant for various external anions.

Figure 7.

Voltage-dependent gating of ClC-4. (A) Schematic representation of voltage clamp protocols as well as predicted ClC-4 whole cell current and the proton currents for gated and not-gated exchanger function. Voltage steps were repetitively applied with T_P and T_I denoting pulse and interpulse duration. Q_P gives transported charge during a single depolarizing voltage step. (B) Representative intracellular pH recordings from ClC-4–expressing cells stimulated with different voltage protocols. The top panel compares intracellular alkalinization elicited by two protocols with identical total pulse () and total interpulse () durations, (Tshort) or (Tlong), respectively. The bottom panel compares intracellular alkalinization elicited by protocols that differ in total pulse () and total interpulse () durations but exhibit equal total transported charge (). For “Q short,” T_P and T_I equal 1.5 ms, whereas “Q long” denotes a pulse protocol with and In the protocol “Q₂ = 1.5 QT,” and resulting in total transported charge, which is 1.5 times larger compared with pulse protocols “Q short” and “Q long.” (C) Statistical analysis of pH responses in several cells (n = 4–6).