Figure 3. Monomeric CLC mutant in phospholipid membranes.

a. Glutaraldehyde crosslinking of wildtype ClC-ec1 and WW mutant in liposomes. Glutaraldehyde treatment was as in Fig 2, except that protein was incorporated into PC/PG liposomes, and gel was silver-stained. b. Passive Cl⁻ efflux from reconstituted liposomes for wildtype ClC-ec1 and WW mutant. Traces show release of Cl⁻ from liposomes loaded with 300 mM Cl⁻ into the extraliposomal solution (containing 1 mM Cl⁻) initiated by 0.5 μM valinomycin (downward arrow), normalized to the level of complete release upon disrupting liposomes with 50 mM octylglucoside (upward arrow). Unitary turnover calculated on a per-subunit basis from the initial rate of Cl⁻ release¹⁴ was: 290 ± 30 s⁻¹ for wildtype, 160 ± 9 s⁻¹ for WW (mean ± s.e.m, N=9). c. Cl⁻-driven H⁺ pumping against a pH gradient. Liposomes loaded with 300 mM Cl⁻ pH 5.0, were suspended in 1 mM Cl⁻, pH 5.2, and transport was initiated by valinomycin and terminated by FCCP (arrows), while pH of suspension was recorded. Upward deflection represents uptake of H⁺ into liposomes.