Fluxes of H+ or Cl− were followed by continuous recording, with ion-specific electrodes, in suspensions of liposomes reconstituted with CLC-ec1. Voltage from the electrodes was fed to an Orion 701A high-sensitivity pH meter (Ebay.com) and digitized at 5–10 Hz by a DI-70 datalogger (DATAQ Instruments). Inward proton pumping driven by an outward Cl− gradient was assayed as described previously (Accardi and Miller, 2004 (link)), using a glass pH electrode to follow H+ uptake in a lightly buffered suspension. After thawing, liposomes (2.5 μg/mg protein density) loaded with 300 mM KCl, 25 mM CPi, pH 4.8, were extruded 21 times through a 400-nm membrane filter (Nguitragool and Miller, 2006 (link)) and were then centrifuged through Sephadex G-50 (100-μl sample per 1.5 ml column) equilibrated with proton-pumping buffer (PPB), 290 mM K-isethionate, 10 mM KCl, 2 mM citrate, pH 5.2, and diluted 10-fold into PPB in a 2-ml stirred cell fitted with a pH electrode. Proton uptake was initiated by addition of 1 μM valinomycin (Vln) and collapsed by FCCP (2 μM). Proton efflux experiments were set up analogously, using liposomes loaded with 300 mM KCl, 25 mM citrate/25 mM MES, pH 4.5, and suspended in 300 mM KCl, 1 mM citrate/MES pH 6.5.
Net Cl− efflux was similarly followed with Ag/AgCl electrodes in a stirred cell temperature-controlled to 25°C. Electrodes were constructed from silver wire cleaned overnight in concentrated HNO3 and coated with AgCl by immersion in Clorox bleach or 0.1 M FeCl3 solution. Liposomes reconstituted with 0.03–4 μg/mg CLC-ec1, and loaded with 300 mM KCl, 25 mM citrate-NaOH, pH 4.5, were extruded and centrifuged through Sephadex G-50 equilibrated in Cl− dump-buffer (CDB), 300 mM K-isethionate, 1 mM KCl, 25 mM citrate, pH 4.5. The sample containing 1.2 mg lipid was added, and KCl efflux was evoked by Vln/FCCP. After 1–3 min, 50 mM octylglucoside detergent was added to release all trapped Cl−. The electrode voltage signal, V(t), zeroed before initiating the efflux, was converted to the increase in Cl− concentration, Δc(t), above the initial concentration c(0) by: and α, an electrode-imperfection factor (of unknown origin) determined by calibrating with 75 μM Cl− at the beginning of each experiment, falls in the range 0.93 ± 0.03. This time course was fit to a two-component relaxation, one for the fraction (1 − fo) of liposomes containing transporters, the other for the fraction (fo) devoid of protein: where ΔcT, the total concentration of Cl− released in the experiment (determined directly by detergent addition), typically reflects an increase of 0.15–0.2 mM over the 1 mM Cl− present before the efflux. Here, kt and kL are the rate constants for Cl− flux through the transporter and for the background leak through the liposome membrane, respectively. This background leak was measured in separate experiments on protein-free liposomes to be 5.7 ± 0.5 × 10−4 s−1, typically 50-fold lower than the transporter-mediated rate constant. For reasons explained in the text, we report the inverse of kt as the useful transporter-mediated kinetic parameter, denoted the “average time constant,” 〈τ〉. Experiments were temperature controlled at 25°C.
Net Cl− efflux was similarly followed with Ag/AgCl electrodes in a stirred cell temperature-controlled to 25°C. Electrodes were constructed from silver wire cleaned overnight in concentrated HNO3 and coated with AgCl by immersion in Clorox bleach or 0.1 M FeCl3 solution. Liposomes reconstituted with 0.03–4 μg/mg CLC-ec1, and loaded with 300 mM KCl, 25 mM citrate-NaOH, pH 4.5, were extruded and centrifuged through Sephadex G-50 equilibrated in Cl− dump-buffer (CDB), 300 mM K-isethionate, 1 mM KCl, 25 mM citrate, pH 4.5. The sample containing 1.2 mg lipid was added, and KCl efflux was evoked by Vln/FCCP. After 1–3 min, 50 mM octylglucoside detergent was added to release all trapped Cl−. The electrode voltage signal, V(t), zeroed before initiating the efflux, was converted to the increase in Cl− concentration, Δc(t), above the initial concentration c(0) by: and α, an electrode-imperfection factor (of unknown origin) determined by calibrating with 75 μM Cl− at the beginning of each experiment, falls in the range 0.93 ± 0.03. This time course was fit to a two-component relaxation, one for the fraction (1 − fo) of liposomes containing transporters, the other for the fraction (fo) devoid of protein: where ΔcT, the total concentration of Cl− released in the experiment (determined directly by detergent addition), typically reflects an increase of 0.15–0.2 mM over the 1 mM Cl− present before the efflux. Here, kt and kL are the rate constants for Cl− flux through the transporter and for the background leak through the liposome membrane, respectively. This background leak was measured in separate experiments on protein-free liposomes to be 5.7 ± 0.5 × 10−4 s−1, typically 50-fold lower than the transporter-mediated rate constant. For reasons explained in the text, we report the inverse of kt as the useful transporter-mediated kinetic parameter, denoted the “average time constant,” 〈τ〉. Experiments were temperature controlled at 25°C.