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 (Kv) 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 Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv 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 Kv 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 Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone

Mechanotransduction and Vascular Resistance

International audienceMechanotransduction is the process by which any cell transduces (converts) a mechanical signal into chemical cues. The vessel wall is permanently sheared by the moving blood particles as well as stretched and compressed by the pressure applied by the blood. Multiple types of mechanical stress fields are associated with flow patterns and unsteadiness.Mechanosensing occurs locally at the plasma membrane. It relies on detection of local changes in protein conformation that lead to ion channel opening, protein unfolding, modified enzyme kinetics, and variations in molecular interactions following exposure of buried binding site or, conversely, hiding them.Mechanotransduction initiates several signaling pathways. Multiple mediators include: At the cell surface, G-protein-coupled and protein tyrosine kinase receptors, ion channels, enzymes, adhesion molecules, and specialized plasmalemmal nanodomains At the cell cortex, the cortical actin network that regulates the cell-surface mechanics and signaling adaptors and effectors (e.g., small monomeric guanosine triphosphatases and heterotrimeric guanine nucleotide-binding proteins, kinases, phosphatases, and ubiquitins, among others) In the cytosol, enzymes, scaffolds, carriers such as endosomes, calcium concentration, and transcription factors In the nucleus, nuclear pore carriers, enzymes, and the transcriptional and translational machineryMechanotransduction by vascular cells regulate the contraction–relaxation state of vascular smooth myocytes, thereby regulating locally and quickly the size of the vascular lumen, that is, the local vascular resistance to blood flow. Once experiencing an unusual mechanical stress, vascular smooth myocytes react by contracting or relaxing according to the magnitude of the mechanical stress, the value of which rises above or falls below the range in which it fluctuates in normal conditions. Moreover, they receive chemical and electrochemical signals from endotheliocytes, themselves sensing the wall shear stress at their wetted (luminal) surface.Mechanotransduction thus regulates locally blood flow more rapidly than the endocrine regulation by remote tissues and even than that of the nervous system, which transmits signals very rapidly via afferent nerves and, after processing in the centers of the spinal cord and brain, efferent nerves