Postsynaptic density‐95 scaffolding of Shaker‐type K⁺ channels in smooth muscle cells regulates the diameter of cerebral arteries

Abstract

Non-technical summary Shaker-type potassium channels are found on the smooth muscle cells of blood vessels in the brain and are important in keeping the blood vessels open or dilated. We show that a protein called PSD95, previously found in nerve cells, interacts with these potassium channels. PSD95 may act as a scaffold to ensure that the potassium channels are expressed in adequate numbers and in the right location on the smooth muscle cells. When we reduced the number of PSD95 proteins, we saw that the potassium channels were also reduced and the blood vessels were not as dilated compared to blood vessels with normal amounts of PSD95. This research may help us understand how abnormal constriction of blood vessels in the brain occurs in diseases such as high blood pressure and stroke.Abstract Postsynaptic density-95 (PSD95) is a 95 kDa scaffolding molecule in the brain that clusters postsynaptic proteins including ion channels, receptors, enzymes and other signalling partners required for normal cognition. The voltage-gated, Shaker-type K + (K V 1) channel is one key binding partner of PSD95 scaffolds in neurons. However, K V 1 channels composed of α1.2 and α1.5 pore-forming subunits also are expressed in the vascular smooth muscle cells (cVSMCs) of the cerebral circulation, although the identity of their molecular scaffolds is unknown. Since α1.2 contains a binding motif for PSD95, we explored the possibility that cVSMCs express PSD95 as a scaffold to promote K V 1 channel expression and cerebral vasodilatation. Cerebral arteries from Sprague-Dawley rats were isolated for analysis of PSD95 and K V 1 channel proteins. PSD95 was detected in cVSMCs and it co-immunoprecipitated and co-localized with the pore-forming α1.2 subunit of the K V 1 channel. Antisense-mediated knockdown of PSD95 profoundly reduced K V 1 channel expression and suppressed K V 1 current in patch-clamped cVSMCs. Loss of PSD95 also depolarized cVSMCs in pressurized cerebral arteries and induced a strong constriction associated with a loss of functional K V 1 channels. Our findings provide initial evidence that PSD95 is expressed in cVSMCs, and the K V 1 channel is one of its important binding partners. PSD95 appears to function as a critical 'dilator' scaffold in cerebral arteries by increasing the number of functional K V 1 channels at the plasma membrane. Abbreviations AKAP150, A-kinase anchoring protein 150; AS, antisense; CA, cerebral arteries; cVSMCs, cerebral vascular smooth muscle cells; E m , membrane potential; GK, guanylate kinase; IP, immunoprecipitate; MAGUK, membrane-associated guanylate kinase; NMDA, N -methyl-D-aspartate; NOS, nitric oxide synthase; PDZ, postsynaptic density-95, discs large, zonula occludens-1; PKA, protein kinase A; Psora, 5-(4-phenylalkoxypsoralen); PSD95, postsynaptic density protein-95; PSS, physiological salt solution; SAP97, synapse-associated protein 97; ScTx, stromatoxin; SH3, Src homology 3.

Beta1-Adrenergic Receptor-Mediated Dilation of Rat Cerebral Artery Requires Shaker-Type KV1 Channels on PSD95 Scaffold

“…31 In vascular smooth muscle, we have recently shown the presence and functional importance of PSD95 in rat cVSMCs where it associates with K V 1 channels and regulates the basal tone of CA. 11,12 This association of PSD95 with K V 1 channels in cVSMCs enables PKA-mediated phosphorylation and opening of K V 1 channels, which maintains the basal tone of CA. Disruption of PSD95-K V 1 interaction by a dominant-negative K V 1-C peptide resulted in constriction of CA ex vivo and in vivo.…”

“…We showed previously that CA preparations have minimal contamination from brain tissue. 11 Real-Time RT-PCR Real-time PCR was used to detect β1AR mRNA in rat CA as described before. 21 cDNA was generated from 500 ng of RNA by reverse transcription and amplified by real-time PCR with β1AR-specific primers (forward: 5′-AGACGCTCACCAACCTCTTCATCA-3′, reverse: 5′-ACATAGCACGTCTACCG AAGTC-3′, Integrated DNA Technologies, Coralville, IA, USA) The threshold cycles (Ct) for β1AR and α-actin were determined from the amplification curves.…”

“…The specificity of PSD95 antibody had been shown in cVSMCs by antisense-mediated knockdown of PSD95 in our earlier publication. 11 Coimmunoprecipitation Coimmunoprecipitation (coIP) was performed with Novex Dynabeads Protein G and a DynaMag-2 separation magnet (Life Technologies, Oslo, Norway). According to the manufacturer's instructions, 75 μL of bead slurry was prepared and 50 μL (10 μg) of β1AR antibody was added to the beads.…”

“…9,10 Previously, we reported that PSD95 associates with Shaker-type voltage-gated K + (K V 1) channels in rat cVSMCs to mediate vasodilation. 11 In that study, PSD95 and K V 1.2α subunits were shown to colocalize in cVSMCs and coimmunoprecipitate in rat CA lysate while antisense knockdown of PSD95 in rat CA resulted in a concomitant loss of K V 1 channel protein and reduced basal CA diameter. In addition, we recently reported that PSD95 scaffolding is required for proper protein kinase A (PKA) phosphorylation and vasodilatory function of K V 1 channels in cVSMCs.…”

Social Networking Among Voltage-Activated Potassium Channels

“…In rat cerebral artery myocytes, endogenous PSD-95 and Kv1.2 a subunits interact based on coimmunoprecipitation and colocalization experiments. 8 Interestingly, downregulation of PSD-95 by antisense oligonucleotides induced a significant reduction in both Kv1.2 protein level and Kv1-mediated currents recorded in isolated myocytes. Myocytes in pressurized arteries treated with PSD-95 antisense oligonucleotides exhibited a 20 mV positive shift in resting membrane potential and the arteries had a significantly reduced basal diameter, 8 indicating that the association of PSD-95 with Kv1.2 is essential for Kv channel function and the maintenance of more negative membrane voltages that oppose myocyte contraction and cerebral artery constriction.…”