Steady-State Modulation of Voltage-Gated K+ Channels in Rat Arterial Smooth Muscle by Cyclic AMP-Dependent Protein Kinase and Protein Phosphatase 2B

Voltage-gated potassium channels (K_v) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that K_v channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that K_v channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on K_v and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of K_v currents. Tonic PKA-mediated activation of K_v appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance K_v currents. We also show that this modulation of K_v by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of K_v by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of K_v requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the K_v current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on K_v channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.