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David M, Macías A, Moreno C, Prieto Á, Martínez-Mármol R, Vicente R, González T, Felipe A, Tamkun MM, Valenzuela C

Protein kinase C (PKC) activity regulates functional effects of Kvβ1.3 subunit on KV1.5 channels: identification of a cardiac Kv1.5 channelosome.

J. Biol. Chem.. 2012 Jun;287(25):21416-28, PMID: 22547057

K(v)1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (I(Kur)). The regulatory K(v)β1.3 subunit converts K(v)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(v)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(v)β subunits) transiently cotransfected with K(v)1.5+K(v)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(v)1.5 and K(v)β1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, K(v)β1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed K(v)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(v)1.5 and K(v)β1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between K(v)1.5, K(v)β1.3, the receptor for activated C kinase (RACK1), PKCβI, PKCβII, and PKCθ in HEK293 cells. A very similar K(v)1.5 channelosome was found in rat ventricular tissue but not in atrial tissue.


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