Protein Kinase C ( PKC ) Activity Regulates Functional Effects of K β 1 . 3 Subunit on K 1 . 5 Channels

K 1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (I ). The regulatory K β1.3 subunit converts K 1.5 channels from delayed rectifiers with a modest degree of slow inactivation to channels with both fast and slow inactivation components. Previous studies have shown that inhibition of PKC with calphostin C abolishes the fast inactivation induced by K β1.3. In this study, we investigated the mechanisms underlying this phenomenon using electrophysiological, biochemical, and confocal microscopy approaches. To achieve this, we used HEK293 cells (which lack K β subunits) transiently cotransfected with K 1.5+K β1.3 and also rat ventricular and atrial tissue to study native α-β subunit interactions. Immunocytochemistry assays demonstrated that these channel subunits colocalize in control conditions and after calphostin C treatment. Moreover, coimmunoprecipitation studies showed that K 1.5 and K β1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, K β1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed K β1.3-induced inactivation, indicating that PKC inhibition causes a dramatic positive shift of the inactivation curve. Our results demonstrate that calphostin C-mediated abolishment of fast inactivation is not due to the dissociation of K 1.5 and K β1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between K 1.5, K β1.3, the receptor for activated C kinase (RACK1), PKCβI, PKCβII, and PKCθ in HEK293 cells. A very similar K 1.5 channelosome was found in rat ventricular tissue but not in atrial tissue.