Angiotensin II-activated protein kinase C targets caveolae to inhibit aortic ATP-sensitive potassium channels

Abstract

Objective: The vasoconstrictor angiotensin II (Ang II) acts at G q/11 -coupled receptors to suppress ATP-sensitive potassium (K ATP ) channel activity via activation of protein kinase C (PKC). The aim of this study was to determine the PKC isoforms involved in the Ang II-induced inhibition of aortic K ATP channel activity and to investigate potential mechanisms by which these isoforms specifically target these ion channels.Methods and results: We show that the inhibitory effect of Ang II on pinacidil-evoked whole-cell rat aortic K ATP currents persists in the presence of Gö6976, an inhibitor of the conventional PKC isoforms, but is abolished by intracellular dialysis of a selective PKCε translocation inhibitor peptide. This suggests that PKC-dependent inhibition of aortic K ATP channels by Ang II arises exclusively from the activation and translocation of PKCε. Using discontinuous sucrose density gradients and Western blot analysis, we show that Ang II induces the translocation of PKCε to cholesterol-enriched rat aortic smooth muscle membrane fractions containing both caveolin, a protein found exclusively in caveolae, and Kir6.1, the pore-forming subunit of the vascular K ATP channel. Immunogold electron microscopy of rat aortic smooth muscle plasma membrane sheets confirms both the presence of Kir6.1 in morphologically identifiable regions of the membrane rich in caveolin and Ang II-evoked migration of PKCε to these membrane compartments. Conclusions: Ang II induces the recruitment of the novel PKC isoform, PKCε, to arterial smooth muscle caveolae. This translocation allows PKCε access to K ATP channels compartmentalized within these specialized membrane microdomains and highlights a potential role for caveolae in targeting PKC isozymes to an ion channel effector.

α1‐Adrenoceptor activation of PKC‐ε causes heterologous desensitization of thromboxane receptors in the aorta of spontaneously hypertensive rats

“…The variation in relative intensity of these two molecular weight bands in 36 weeks WKY and SHR implies the presence of mixtures of isoforms in their aortic smooth muscle. Indeed, several of the PKC isoforms have similar molecular weights (Ohanian et al, 1996;Shin et al, 2000;Sampson et al, 2007). The present results thus support the interpretation that variations in PKC isozymes underlie the desensitization of TP receptors observed in the SHR aorta.…”

Protein Kinase C-ε Induces Caveolin-Dependent Internalization of Vascular Adenosine 5′-Triphosphate–Sensitive K + Channels

“…15,37 However, the mechanism by which PKC inhibits K ATP channels is unknown. Both PKC-and Kir6.1 are localized in the caveolae of arterial smooth muscles, 16,17 which are specialized microdomains in the plasma membrane and serve to compartmentalize and integrate numerous signaling events. 38 Caveolin-1 is a major integral membrane component of caveolar membranes in vascular smooth muscles.…”

“…22,39 Interestingly, both Kir6.1 and PKC-have been reported to coimmunoprecipitate with caveolin-1. 17 This caveolin-1 association could serve to cluster PKC and signaling molecules with K ATP channels, allowing for specific modulation while preventing crosstalk with other pathways. In the present study, we found that caveolae disruption with M␤CD or caveolin-1-specific siRNA prevented not only an inhibitory effect of PKC on K ATP currents but also PKC-induced internalization of K ATP channels, suggesting the functional importance of this compartmentalization.…”

“…16,17 We evaluated the effect of methyl-␤-cyclodextrin (M␤CD) on PMA-induced inhibition of K ATP currents in HEK293 cells transfected with Kir6.1/SUR2B. M␤CD is a drug that disrupts caveolae and lipid rafts by depleting the cell membrane of cholesterol.…”

“…16 Angiotensin II, a potent physiological vasoconstrictor, was reported to recruit a novel PKC isoform, PKC-, to arterial smooth muscle caveolae, indicating a potential role for caveolae in the regulation of K ATP channel function. 17 However, the molecular mechanism by which PKC inhibits vascular K ATP channels remains unknown.…”