Chemicals and other materials were of reagent quality. Expression, His-tag cleavage, purification, and liposome reconstitution of CLC-ec1, the product of Escherichia coli gene clcA (accession P37019), were performed as previously described (Accardi et al., 2004 (link)), except that for samples used in liposome fluxes, the final purification step was gel filtration on Superdex 200 rather than anion exchange chromatography on Poros HQ, which was used exclusively for planar lipid bilayer experiments. All preparations (typically 1–10 mg/ml in 5–20 mM decylmaltoside) were checked by overloaded SDS-PAGE to be free of His-tagged and other contaminating bands. Point mutants were constructed by conventional PCR methods and were fully sequenced. All mutants reported expressed well (1–3 mg/liter culture) and gave gel filtration profiles identical to the wild-type homodimer.
Liposomes were formed within 1 d of protein preparation by 30-h dialysis of micellar solutions containing E. coli polar lipid (Avanti, 20 mg/ml), detergent (Chaps, 35 mM), and protein (0.03–50 μg/mg lipid). Protein concentration is reported throughout as protein/lipid weight ratio, denoted “protein density.” Liposomes used for planar bilayer recording were prepared at a protein density of 50 μg/mg in 450 mM KCl, 25 mM KH2PO4, 22.5 mM K3-citrate, 2.5 mM citric acid, pH 7.5. Liposomes used for flux measurements were formed with protein at 0.03–5 μg/mg, 300 mM KCl, and buffered with 25 mM citrate for Cl− flux experiments or 25 mM citrate/ 25 mM phosphate (CPi) for H+ flux experiments, adjusted with NaOH to the desired pH in the range 4.5–5.5. (Some experiments used 75 mM glutamate as buffer, with similar results.) After dialysis, liposomes were stored in aliquots at −80°C until the day of use.
Liposomes were formed within 1 d of protein preparation by 30-h dialysis of micellar solutions containing E. coli polar lipid (Avanti, 20 mg/ml), detergent (Chaps, 35 mM), and protein (0.03–50 μg/mg lipid). Protein concentration is reported throughout as protein/lipid weight ratio, denoted “protein density.” Liposomes used for planar bilayer recording were prepared at a protein density of 50 μg/mg in 450 mM KCl, 25 mM KH2PO4, 22.5 mM K3-citrate, 2.5 mM citric acid, pH 7.5. Liposomes used for flux measurements were formed with protein at 0.03–5 μg/mg, 300 mM KCl, and buffered with 25 mM citrate for Cl− flux experiments or 25 mM citrate/ 25 mM phosphate (CPi) for H+ flux experiments, adjusted with NaOH to the desired pH in the range 4.5–5.5. (Some experiments used 75 mM glutamate as buffer, with similar results.) After dialysis, liposomes were stored in aliquots at −80°C until the day of use.
