FIGURE 4.
PKC activity regulates KATP channel recycling with no effect on channel endocytosis. A, effect of PKC on recycling. HEK293 cells stably expressing HA-tagged KATP channels were allowed to accumulate anti-HA antibodies for 1 h. Recycling was performed for 1 h in the presence of either vehicle (DMSO 0.1%), PMA (100 nm), or 4α-PDD (100 nm) and the HRP-conjugated secondary antibody; after washing, recycled channels were assayed by chemiluminescence. Normalized mean (±S.E.) data are shown (n = 3). ** denotes statistical significance at p < 0.005 assessed by analysis of variance with Tukey post-hoc means comparison test. B, effect of PKC on endocytosis. Stable cells were pretreated with either vehicle or PMA (100 nm). Internalization of anti-HA antibody-labeled channels was performed for 10 min in the continued presence of the drug. Surface density was estimated by chemiluminescence; normalized mean (±S.E.) data are presented (n = 3). C, endocytosis in PKC inhibited cells during the recycling block. Distribution of endocytic pool of KATP channels in stable cells after 1 h of continual endocytosis in the presence of chelerythrine (10 μm) and monensin (200 μm). D, channels allowed to internalize in the presence of chelerythrine (10 μm) are able to recycle back; the assay was performed as described in Fig. 2B. The scale bars equal 10 μm.