Proton block of the CLC-5 Cl-/H+ exchanger

Abstract

CLC-5 is a H(+)/Cl(-) exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dent's disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH. The mechanistic origins of both phenomena are currently not well understood. It has been proposed that rectification arises from the voltage dependence of a H(+) transport step, and that inhibition of CLC-5 currents by low extracellular pH is a result of a reduction in the driving force for exchange caused by a pH gradient. We show here that the pH dependence of CLC-5 currents arises from H(+) binding to a single site located halfway through the transmembrane electric field and driving the transport cycle in a less permissive direction, rather than a reduction in the driving force. We propose that protons bind to the extracellular gating glutamate E211 in CLC-5. It has been shown that CLC-5 becomes severely uncoupled when SCN(-) is the main charge carrier: H(+) transport is drastically reduced while the rate of anion movement is increased. We found that in these conditions, rectification and pH dependence are unaltered. This implies that H(+) translocation is not the main cause of rectification. We propose a simple transport cycle model that qualitatively accounts for these findings.

Figure 1.

Inhibition of CLC-5 currents by extracellular H⁺’s. (A) CLC-5 currents recorded from the same oocyte at various pH_ex. Scale bars represent 1 µA and 10 ms. (B) Current–voltage plots for the currents shown in A. Squares, pH 9; circles, pH 7.5; triangles, pH 6.0; diamonds, pH 4.5. Solid lines hold no theoretical meaning. (C) Bar representation of the currents recorded at various voltages and pHs: black bar, I(pH 8, +70 mV); white bar, I(pH 7, +90 mV); hatched gray bar, I(pH 6, +110 mV). (D) Plot of normalized currents at +60 (filled circles) and +110 (open circles) mV as a function of pH. Solid lines are fits to Eq. 3 as described in Results, with K = 8.1 × 10⁻⁷, I_max = 1.02 and I_min = 0.27 at +60 mV, and K = 2.4 × 10⁻⁶, I_max = 0.99 and I_min = 0.33 at +110 mV. (E) Plot of the mean values of the pK obtained as in D, versus voltage. Solid line is a fit to pK(V) = pK(0) + zFV/RT, where pK(V) is the pK at voltage V, pK(0) = 6.6 is the pK extrapolated at V = 0, z = 0.51 is the fraction of the membrane potential acting on the blocking ion, and F, R, and T have their usual meanings.

Figure 2.

Proton inhibition of CLC-5 currents in low extracellular Cl⁻. (A) Plot of normalized currents at +60 (filled circles) and +110 (open circles) mV as a function of pH. Solid lines are fits to Eq. 3 as described in Results, with K = 7.1 × 10⁻⁷, I_max = 0.98 and I_min = 0.25 at +60 mV, and K = 2.1 × 10⁻⁶, I_max = 0.97 and I_min = 0.24 at +110 mV. Dotted lines represent the fits at +110 (gray) and +60 (black) mV at 100 mM [Cl⁻]_ex. (B) Plot of the mean values of the pK obtained as in A, versus voltage. Solid line is a fit to pK(V) = pK(0) + zFV/RT, where pK(V) is the pK at voltage V, pK(0) = 6.7 is the pK extrapolated at V = 0, z = 0.56 is the fraction of the membrane potential acting on the blocking ion, and F, R, and T have their usual meanings.

Figure 3.

Inhibition of CLC-5 currents in SCN⁻. (A) CLC-5 currents recorded from the same oocyte in various pH_ex. Scale bars represent 2 µA and 10 ms. (B) Current–voltage plots for the currents shown in A. Squares, pH 9; circles, pH 7.5; triangles, pH 6.0; diamonds, pH 4.5. Solid lines hold no theoretical meaning. (C) Plot of normalized currents at +60 (filled circles) and +110 (open circles) mV as a function of pH. Solid lines are fits to Eq. 3 as described in Results, with K = 6.0 × 10⁻⁷, I_max = 1.04 and I_min = 0.25 at +60 mV, and K = 2.1 × 10⁻⁶, I_max = 1.05 and I_min = 0.31 at +110 mV. Dotted lines represent the fits at +110 (gray) and +60 (black) mV at 100 mM [Cl⁻]_ex. (D) Plot of the mean values of the pK obtained as in C, versus voltage. Solid line is a fit to pK(V) = pK(0) + zFV/RT, where pK(V) is the pK at voltage V, pK(0) = 6.9 is the pK extrapolated at V = 0, z = 0.63 is the fraction of the membrane potential acting on the blocking ion, and F, R, and T have their usual meanings.

Figure 4.

Simplified transport model for CLC-5. (A) Coupled transport in Cl⁻. (B) Uncoupled transport mode in SCN⁻. Green circles, Cl⁻ ions; red circles, H⁺; yellow circles, SCN⁻ ions. The model is described in the Discussion.