FIGURE 3.
PKC activation decreases KATP channel surface density and whole cell K+ conductance. A, PKC activation with PMA decreases surface density. Cells stably expressing KATP-HA were incubated with anti-HA antibodies for 1 h in the presence of vehicle (DMSO 0.1%), PMA (100 nm), 4α-PDD (100 nm), or chelerythrine (10 μm). The cells were then fixed, permeabilized, and stained with Cy3-conjugated anti-rat secondary antibodies. B, quantitative assay of surface density determined by chemiluminescence (see “Experimental Procedures”) following 1 h of treatment with the indicated drugs as for A (n = 3). C, distribution of KATP channels following treatment with chelerythrine before (panel i) and after (panel ii) removal of surface-bound antibody by acid strip. D, PKC inhibition blocks the effects of PMA. The cells were pretreated with chelerythrine (10 μm) or bisindolylmaleimide I (BIM I) (4 μm) for 30 min prior to and throughout 1 h of PMA application and incubation with anti-HA antibody. E, PKC activation with PMA decreases channel current density; current families from HEK293 cells stably expressing HA-tagged KATP channels evoked by 300-ms voltage steps between −100 and +50 mV following treatment of cells with vehicle (0.1% DMSO) or PMA (100 nm) for 1 h. The currents were recorded first in the absence and then in the presence of tolbutamide (Tolb., 200 μm). F, example I-V relationships showing KATP current at initial membrane rupture (▵), following ATP dialysis (●), and after tolbutamide application (■) under control conditions. G, mean tolbutamide-sensitive conductance measured from slopes of I-V curves recorded form cells treated with vehicle or PMA. The data points represent the means ± S.E. (n = 9 in each group); significance is indicated as * (p < 0.05) or ** (p < 0.005) assessed by Student's t test.