Rationale: Postsynaptic density-95 (PSD95) is a scaffolding protein that associates with voltage-gated, Shaker-type K(+) (KV1) channels and promotes the expression of KV1 channels in vascular smooth muscle cells of the cerebral (cVSMCs) circulation. However, the physiological role of PSD95 in mediating molecular signaling in cVSMCs is unknown.
Objective: We explored whether a specific interaction between PSD95 and KV1 channels enables protein kinase A phosphorylation of KV1 channels in cVSMCs to promote vasodilation.
Methods and results: Rat cerebral arteries were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of KV1.2α was designed as a dominant-negative peptide to disrupt the association of KV1 channels with PSD95. Application of KV1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and KV1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of KV1-C peptide. Patch-clamp recordings confirmed that KV1-C peptide attenuates KV1 channel blocker (5-(4-phenylalkoxypsoralen))-sensitive current in cVSMCs. Western blots using a phospho-protein kinase A substrate antibody revealed that cerebral arteries exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction.
Conclusions: These findings provide initial evidence that protein kinase A phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in cerebral arteries and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.
Keywords: PDZ domains; cerebral arteries; muscle, smooth, vascular; potassium channels.