Bestrophin-1 Cl
− channel activity was measured as previously described.
19,24 Briefly, conventional whole-cell recordings were made using an Axon Multiclamp 700B amplifier, with command potentials generated by a computer using pClamp 9 software and a Digidata 1320 interface (all Molecular Devices, Wokingham, Berkshire, UK). The extracellular bath solution contained (mM) 140 NaCl, 2 CaCl
2, 1 MgCl
2, 10 glucose, 30 mannitol, 10 HEPES, pH 7.4 with NaOH. Patch pipettes (2–4 MΩ) were pulled from soda-lime glass (VWR, Lutterworth, Leicestershire, UK), and filled with the pipette solution, which contained (mM) 20 CsCl, 110 Cs aspartate, 2 MgCl
2, 10 glucose, 10 HEPES, 10 EGTA, 7.2 CaCl
2 (free Ca
2+ activity = 500 nM) and pH adjusted to 7.2 with CsOH. Membrane potential (
V m) was stepped for 450 ms from −120 to +80 mV, in +20 mV increments, from a holding
V m of −50 mV. Cl
− channel activities for wild-type and mutant bestrophins were measured as the chord conductances between
V m = 60 and 80 mV. Data are presented as mean ± SE, and differences between means were assessed by ANOVA or Student's
t-test for unpaired data where appropriate. The mean whole cell capacitance was 16.0 ± 0.5 pF (
n = 258). There were no significant differences (
P > 0.1 by ANOVA) between the mean capacitances of the cells in any of the different experimental groups.