The effect of Clin on current mediated by NBCe1-B, NBCe1-A, and NBCe2-C activity was analyzed in transiently transfected HEK-293 cells using whole-cell current recording at room temperature, exactly as detailed previously (16), and varying the pipette Cl concentration between 5 and 140 mM. Patch pipettes had a resistance of 5 to 7 megohms when filled with CsCl-based pipette solution. The cell capacitance was between 10 and 18 pF. The pipette solutions contained 2 mM MgSO4, 1 mM adenosine 5′-triphosphate, 0.5 mM EGTA, 10 mM Hepes, and a mixture of CsCl and Cs-gluconate to yield Cl concentrations of 5, 10, 20, 30, 40, and 140 mM. pH was adjusted to 7.3 with CsOH. The Hepes-buffered bath solution contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM Hepes (pH 7.4 with NaOH). The HCO3-buffered solution was prepared by replacing 25 mM NaCl with equimolar amount of NaHCO3, and the solution was equilibrated with 5% CO2/95% O2. The current was recorded by 400-ms rapid alteration of membrane potential from −60 to +60 mV every 2 s from a holding potential of 0 mV. Before current recording, the junction potential at each pipetted solution Cl concentration was offset to 0 using the Axopatch 200B amplifier. The current recorded at +60 mV was used to calculate current density as picoampere/picofarad. Axopatch 200B patch-clamp amplifier, Digidata -1440A, and pClamp 10 software (Molecular Devices) were used for data acquisition and analysis. The currents were filtered at 1 kHz and sampled at 10 kHz.

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