CSPα co-expression alters BK channel whole cell current density in transiently transfected CAD cells.

<p>(A) Representative recordings of single CAD cells voltage clamped as depicted in the protocol using the whole-cell configuration of the patch clamp technique. The left-hand column displays macroscopic BK channel currents recorded from CAD cells transiently-transfected with BKα subunits alone. The middle and right-hand columns display BK currents recorded from cells co-transfected either a CSPα mutant (i.e. CSPα_HPD-AAA) or wild-type CSPα, respectively. Recordings were done 40–48 hours post-transfection. In all three conditions, whole cell currents were largely inhibited following addition of penitrem-A (100 nM). Penitrem-A sensitive currents, obtained by digital subtraction, are displayed in the bottom row of traces. The vertical and horizontal scale bars shown apply to all current tracings. The current-voltage plot shown in panel B quantifies the average penitrem A-sensitive peak currents observed under each transfection condition and highlights the significant differences in current density magnitude found between CAD cells transiently-transfected with BKα subunits alone, and those co-transfected with either WT or a mutant form of CSPα (Dunnett post-hoc ANOVA, one-way Vs BK channel alone; *p<0.05, **p<0.01). Inset shows BK channel expression in CAD cells transiently co-transfected with 0.75 µg cDNA encoding either CSPα or CSPα_HPD-AAA. For detection of surface expressed BK channel, transfected CAD cells were labeled for 30 min at 4°C with bath applied Biotin, as described in the methods. Following lysis, 1 mg of soluble protein lysate was subjected to overnight streptavidin pull-down at 4°C. Proteins isolated by streptavidin pull down (top panel) and 30 µg of soluble cellular lysate (bottom panel) were analyzed by Western Blot with an anti-BKα subunit antibody. Data is representative of 3 similar experiments.