hBest1 tagged with the myc epitope at the C terminus in pRK5 was obtained from Jeremy Nathans, Johns Hopkins University (Baltimore, MD). Mutations were made using PCR-based mutagenesis (Quickchanger; Stratagene, La Jolla, CA). hBest1 and pEGFP (Invitrogen, Carlsbad, CA) were transfected into HEK-293 cells (5:1 ratio, 2 μg total DNA per 3.5-cm plate), using a blend of lipids (Fugene-6; Roche Molecular Biochemicals, Indianapolis, IN). Single cells identified by enhanced green fluorescent protein (EGFP) fluorescence were used for whole-cell patch clamp experiments within 72 hours. Fire-polished borosilicate glass patch pipettes were 3 to 5 MΩ. Experiments were conducted at room temperature (20–24°C). Because the liquid junction potentials were small (<2 mV), no correction was made. The standard pipette solution contained (mM) 146 CsCl, 2 MgCl
2, 5 (Ca
2+)-EGTA, 8 HEPES, and 10 sucrose (pH 7.3), adjusted with NMDG (
N-methyl-
d-glucamine). The zero-Ca
2+ pipette solution contained 5 mM EGTA without added Ca
2+, whereas the high-Ca
2+ pipette solution contained a mixture of 5 mM EGTA and 5 mM Ca
2+-EGTA to make solutions with different free [Ca
2+].
36 In the text, high-Ca
2+ solution refers to Ca
2+ concentrations between 600 nM and 4.5 μM. The calculated Ca
2+ concentrations were confirmed in each solution by Fura-2 (Invitrogen-Molecular Probes, Eugene, OR) measurements using a luminescence spectrophotometer (model LS-50B; Perkin Elmer, Wellesley, MA). The standard extracellular solution contained (mM) 140 NaCl, 5 KCl, 2 CaCl
2, 1 MgCl
2, 15 glucose, and 10 HEPES (pH 7.4) with NaOH. This combination of intracellular and extracellular solutions set
E rev (the reversal potential) for Cl
− currents to 0, while cation currents carried by Na
+ or Cs
+ had very positive or negative
E rev, respectively. When Cl
− was replaced with another anion, NaCl was replaced on an equimolar basis with the Na
+ salt of the substitute anion. Osmolarity was adjusted with sucrose to 303 mOsM for all solutions.