Large transient capacitive currents in wild-type lysosomal Cl-/H+ antiporter ClC-7 and residual transport activity in the proton glutamate mutant E312A

Abstract

ClC-7 is a lysosomal 2 Cl-/1 H+ antiporter of the CLC protein family, which comprises Cl- channels and other Cl-/H+ antiporters. Mutations in ClC-7 and its associated β subunit Ostm1 lead to osteopetrosis and lysosomal storage disease in humans and mice. Previous studies on other mammalian CLC transporters showed that mutations of a conserved, intracellularly located glutamate residue, the so-called proton glutamate, abolish steady-state transport activity but increase transient capacitive currents associated with partial reactions of the transport cycle. In contrast, we observed large, transient capacitive currents for the wild-type ClC-7, which depend on external pH and internal, but not external, Cl-. Very similar transient currents were observed for the E312A mutant of the proton glutamate. Interestingly, and unlike in other mammalian CLC transporters investigated so far, the E312A mutation strongly reduces, but does not abolish, stationary transport currents, potentially explaining the intermediate phenotype observed in the E312A mouse line.

Figure 1.

Structure and function of the ClC-7/Ostm1 complex. (A) Dimeric architecture of the ClC-7/Ostm1 complex (Protein Data Bank accession no. 7JM7) viewed from the membrane plane (extracellular side above and cytoplasmic side below). The two subunits of Ostm1 are shown in blue, whereas the two subunits of ClC-7 are shown in green and gray. The proton glutamate, E312 in ClC-7, is shown in blue, the gating glutamate, E245, is shown in red. Anions bound at S_cen and S_int are represented as magenta spheres. (B) Cartoon illustrating the physiological role of ClC-7 in lysosomes. The coupled Cl⁻/H⁺ exchange mediated by ClC-7, together with the action of other proteins, establish the acidic lysosomal pH. The present study measures currents resulting from ClC-7 redirected to the plasma membrane by mutation of di-leucine lysosomal targeting motifs. If ClC-7 retains the same topology and voltage dependence in lysosomes and at the plasma membrane, then lysosomal ClC-7 directs Cl⁻ toward the cytosol. Proof of this concept awaits direct measurements of transport currents in lysosomes.

Figure S1.

Representative measurements of steady-state currents from ClC-7 in different extracellular solutions as indicated. Currents were elicited with test pulses of 1-s duration (the voltage protocol is shown as inset).

Figure 2.

Transient currents of ClC-7. (A) Representative transient currents from ClC-7 in control conditions (left), without extracellular Cl⁻ (middle), and after wash (right). The voltage protocol is shown as inset. Currents were elicited with test pulses of 5-ms duration, and linear leak and capacitive artifacts were subtracted through a P/N voltage protocol (see Materials and methods). (B) Charge measured upon return to the holding voltage (off charge), plotted as a function of the preceding step voltage (Q(V)) from the same cell in different extracellular solutions. Lines represent fits with a Boltzmann function (see Materials and methods). (C) Box plots overlaid on original data show mean values of the voltage of half-maximal activation (V_1/2) derived from the analysis shown in B in different ionic conditions. (D) Box plots overlaid on original data show mean values of the apparent gating charge (z).

Figure S2.

Correlation between steady-state current and off charge of the transient inward currents. (A) For the WT, the correlation is between steady-state current elicited by a voltage step to 100 mV measured after 500 ms and off charge after a 5-ms voltage step to 200 mV (r²= 0.56; slope, 640 ± 140 s⁻¹). (B) For E312A, the correlation is between steady-state current measured at the end of the 100-ms r² test pulse to 160 mV and the off charge after the same voltage step (r² = 0.77; slope, 137 ± 15 s⁻¹).

Figure 3.

Cl⁻ dependence of transient and steady-state currents of mutant E312A. (A and B) Representative currents from the mutant E312A in control conditions and without extracellular Cl⁻ with test pulses of 5-ms duration (A) and 100-ms duration (B). Voltage protocols in A and B are shown as insets. (C) Charge measured upon return to the holding voltage (off charge) for the measurements shown in B with a test pulse duration of 5 ms. Lines represent fit with a Boltzmann function (see Materials and methods). (D) Box plots overlaid on original data show mean values of the voltage of half-maximal activation (V_1/2) for E312A in control conditions and without extracellular Cl⁻. (E) Box plots overlaid on original data show mean values of the apparent gating charge (z).

Figure 4.

pH dependence of transient and steady-state currents of mutant E312A. (A) Representative current traces of the mutant E312A in control conditions and at pH 8.3 and pH 6.3. Currents were elicited with test pulses of 100-ms duration (the voltage protocol is shown as inset). (B) Mean values of the voltage of half-maximal activation (V_1/2) at pH 8.3 and pH 6.3 extracted as explained in Fig. 2. For comparison, the mean value at pH 7.3 shown in Fig. 3 D is included here as a dashed line. (C) Mean values of the voltage of the apparent gating charge (z) at pH 8.3 and pH 6.3 obtained as in Fig. 2. For comparison, the mean value at pH 7.3 shown in Fig. 3 E is included here as a dashed line.

Figure S3.

Effect of the P/N protocol on currents from nontransfected cells in solutions of different ionic composition. (A) Upper traces are representative recordings from the same cell in solutions at pH 7.3, 8.3, and 6.3 and without Cl⁻ with application of the standard voltage protocol (shown as inset) and without P/N subtraction. (B) Same recordings as in A, but with application of the P/N protocol.

Figure S4.

Effect of the P/N protocol on currents from E312A. (A) Representative recordings from E312A without P/N subtraction in standard solution, without Cl⁻, and back to standard solution. The voltage protocol is shown as inset. (B) Same recordings as in A, but with application of the P/N protocol. (C) Averaged current-voltage relationship for E312A (n = 8) and mock-transfected cells (n = 12) obtained from measurements without application of the P/N protocol.

Figure 5.

Dependence of transient capacitive currents on the duration of the activating pulse for WT and the R760Q mutation. (A) Three representative recordings with duration of the activating pulse of 30, 220, and 330 ms. Transient capacitive currents are shown as insets on a magnified timescale. The voltage protocol is shown as inset. (B) Mean values of the normalized charge (Q_norm) associated with the transient capacitive currents of WT ClC-7 calculated as in Fig. 2. Normalization was performed with the data point at 100 ms (n = 4). (C) Representative recordings of steady-state current from the mutant R760Q with duration of the activating pulse of 30, 220, and 330 ms. Transient capacitive currents are shown as insets on a magnified timescale. (D) Mean values of the charge associated with the transient capacitive currents of the R760Q mutant calculated as for the WT (n = 3).

Figure S5.

Effect of the P/N protocol on currents from E245A. (A) Representative recordings from E245A without P/N subtraction in standard solution and without Cl⁻. The voltage protocol is shown as inset. (B) Same recordings as in A, but with application of the P/N protocol.