PKA-Phosphorylated KV1 Channels in PSD95 Signaling Complex Contribute to the Resting Membrane Potential and Diameter of Cerebral Arteries
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 (cVSMCs) of the cerebral 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 PKA phosphorylation of KV1 channels in cVSMCs to promote vasodilation.
Methods and Results: Rat cerebral arteries (CA) were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the PDZ 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 CA rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced co-immunoprecipitation 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 (Psora4)-sensitive current in cVSMCs. Western blots employing a phospho-PKA substrate antibody revealed CA exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and PKA inhibitor peptide-mediated vasoconstriction.
Conclusions: These findings provide initial evidence that PKA phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in CA, and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.
- Received January 9, 2014.
- Revision received February 26, 2014.
- Accepted February 28, 2014.