The ClC-3 Cl-/H+ antiporter becomes uncoupled at low extracellular pH

Abstract

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl(-)/H(+) antiport current (I(ClC-3)) in HEK293 cells. The outward rectification and time dependence of I(ClC-3) closely resemble an endogenous HEK293 cell acid-activated Cl(-) current (ICl(acid)) seen at extracellular pH <or= 5.5. ICl(acid) was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. ICl(acid) was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (E(rev)) of I(ClC-3) between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl(-) and H(+) transport become uncoupled at low pH. At pH 4.0 E(rev) was completely Cl(-) dependent (55.8 +/- 2.3 mV/decade). Several findings linked ClC-3 with native ICl(acid); 1) RNA interference directed at ClC-3 message reduced native ICl(acid); 2) removal of the extracellular "fast gate" (E224A) produced large currents that were pH-insensitive; and 3) wild-type I(ClC-3) and ICl(acid) were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10-500 microm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type I(ClC-3) and from native ICl(acid) by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native ICl(acid). Low pH uncouples Cl(-)/H(+) transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl(-)/H(+) antiporters and perhaps for pH regulation in highly acidic intracellular compartments.

FIGURE 1.

Activation of ICl_acid in wild-type and ClC-3 null VSM cells. Representative current tracings at pH 7.35 and 4.0 for wild-type (A) and null (B) cells (HP = −40 mV, TP = −100 to +100 mV in 20-mV increments). C, I/V relationships at pH 7.35 and pH 4.0. *, significantly different from the voltage-matched current at pH 7.35 (p < 0.05). No significant differences were noted between baseline wild-type and null currents at pH 7.35 (p < 0.05). Error bars indicate ±S.E.

FIGURE 2.

ICl_acid in HEK293 cells. Representative tracings from Ad-eGFP (A and B) and Ad-ClC-3 expressing (C and D) cells at the indicated pH. HP = −40 mV, TP = −100 to +100 mV in 20-mV increments. E, I/V relationships for Ad-eGFP- and Ad-ClC-3-infected cells at the indicated pH. *, significantly different from current at pH 7.35 for a given TP (p < 0.05). Error bars indicate ±S.E.

FIGURE 3.

The effect of lowering pH on whole cell currents. The I/V relationship for Ad-eGFP (A and C) and Ad-ClC-3 expressing cells (B and D) at the indicated pH values. Representative whole cell currents are shown in C and D. Error bars indicate ±S.E.

FIGURE 4.

Shifts in the reversal potential of I_ClC-3 in response to changes in extracellular pH. A, substitution of extracellular Cl⁻ with SCN⁻ increases the size of the current. B, raw data tracings of ramp currents (−40 to +40 mV) at different pH values (5.2 to 8.2) with either Cl⁻ (left) or SCN⁻ (right) as the dominant extracellular anion. All recordings are from the same cell. C, comparison of average E_rev of ramp currents for Cl⁻ and SCN⁻ at varying pH (n = 5 for Cl⁻ and n = 4 for SCN⁻, two cells were assessed under both conditions). *, significantly different from pH 7.2 (p < 0.05).

FIGURE 5.

RNAi knockdown of ClC-3 suppresses native ICl_acid. I/V relationships for HEK293 cells under the indicated conditions. Wild-type ClC-3 is expressed using plasmid. *, significantly different from pH 7.35 for a given condition. #, significantly different from scramble current at pH 4 (p < 0.05).

FIGURE 6.

Effect of low pH on currents from HEK293 cells expressing E224A ClC-3 plasmid. RNAi was used to suppress endogenous ClC-3. A, whole cell currents from a representative cell at pH 7.35 and 4.0. The I/V relationships are shown in B. There were no significant differences between currents at 7.35 and pH 4.0 at any TP (p < 0.05). Currents at pH 7.35 were previously published (8) but are provided for comparison.

FIGURE 7.

ClC-3 current induced by Ad-ClC-3 at pH 7.35 is inhibited by a 5-min incubation in 100 μm MTSES. This effect is reversed by a 5-min incubation in 500 μm DTT. *, significantly different from Ad-CIC-3. #, significantly different from MTSES (p < 0.05). Error bars indicate ±S.E.

FIGURE 8.

MTSES inhibits native Icl_acid. A, I/V relationships for currents recorded at pH 4.0 following incubation in varying concentrations of MTSES at pH 7.35. MTSES was washed off as the bath was changed to pH 4.0. B, reversal of the effect of MTSES (500 μm) by DTT (500 μm). Representative raw currents for these experiments are shown in C. The sequence of bath solution exchanges used to create the I/V relationships in B was as follows: 1) control, pH 7.35; 2) effect of reducing pH to 4.0; 3) return to pH 7.35 and incubation in MTSES; 4) change to pH 4 with wash-off of MTSES; 5) return to pH 7.35 and incubation in DTT; and 6) change to pH 4 after DTT. Error bars indicate ±S.E.

FIGURE 9.

The effect of MTSES (500 μm) on cells expressing the C161_P188del plasmid. RNAi was used to suppress endogenous ClC-3. Raw current traces from a representative HEK293 cell expressing the C161_P188del mutant are shown at pH 7.35 during incubation in MTSES at pH 7.35 (A) and after changing to pH 4.0 (B). The I/V relationship for these experiments are shown in C. Error bars indicate ±S.E.

FIGURE 10.

Time course of the increase in current elicited by pulsing from −40 to +80 mV following application of pH 4.0 buffer (bars at top) and washout of this effect after returning to pH 7.35. The cells all started in pH 7.35 buffer and returned to pH 7.35 immediately after the effect of pH 4.0 was stable. In control HEK293 cells (open circles) the response to pH changes is very brisk. In C161_P188del expressing cells the changes in current magnitude are much slower. Depolarizing pulses were applied every 5 s. Error bars indicate ±S.E.