Protein kinase A-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 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.

Transient receptor potential melastatin 4 channel controls calcium signals and dental follicle stem cell differentiation.

Elevations in the intracellular Ca(2+) concentration are a phenomena commonly observed during stem cell differentiation but cease after the process is complete. The transient receptor potential melastatin 4 (TRPM4) is an ion channel that controls Ca(2+) signals in excitable and nonexcitable cells. However, its role in stem cells remains unknown. The aim of this study was to characterize TRPM4 in rat dental follicle stem cells (DFSCs) and to determine its impact on Ca(2+) signaling and the differentiation process. We identified TRPM4 gene expression in DFSCs, but not TRPM5, a closely related channel with similar function. Perfusion of cells with increasing buffered Ca(2+) resulted in a concentration-dependent activation of currents typical for TRPM4, which were also voltage-dependent and had Na(+) conductivity. Molecular suppression with shRNA decreased channel activity and cell proliferation during osteogenesis but not adipogenesis. As a result, enhanced mineralization and phosphatase enzyme activity were observed during osteoblast formation, although DFSCs failed to differentiate into adipocytes. Furthermore, the normal agonist-induced first and secondary phases of Ca(2+) signals were transformed into a gradual and sustained increase which confirmed the channels' ability to control Ca(2+) signaling. Using whole genome microarray analysis, we identified several genes impacted by TRPM4 during DFSC differentiation. These findings suggest an inhibitory role for TRPM4 on osteogenesis while it appears to be required for adipogenesis. The data also provide a potential link between the Ca(2+) signaling pattern and gene expression during stem cell differentiation.

Beta1-adrenergic receptor-mediated dilation of rat cerebral artery requires Shaker-type KV1 channels on PSD95 scaffold.

Postsynaptic density-95 (PSD95) is a scaffolding protein in cerebral vascular smooth muscle cells (cVSMCs), which binds to Shaker-type K(+) (KV1) channels and facilitates channel opening through phosphorylation by protein kinase A. ß1-Adrenergic receptors (ß1ARs) also have a binding motif for PSD95. Functional association of ß1AR with KV1 channels through PSD95 may represent a novel vasodilator complex in cerebral arteries (CA). We explored whether a ß1AR-PSD95-KV1 complex is a determinant of rat CA dilation. RT-PCR and western blots revealed expression of ß1AR in CA. Isoproterenol induced a concentration-dependent dilation of isolated, pressurized rat CA that was blocked by the ß1AR blocker CGP20712. Cranial window imaging of middle cerebral arterioles in situ showed isoproterenol- and norepinephrine-induced dilation that was blunted by ß1AR blockade. Isoproterenol-induced hyperpolarization of cVSMCs in pressurized CA was blocked by CGP20712. Confocal images of cVSMCs immunostained with antibodies against ß1AR and PSD95 indicated strong colocalization, and PSD95 co-immunoprecipitated with ß1AR in CA lysate. Blockade of KV1 channels, ß1AR or disruption of PSD95-KV1 interaction produced similar blunting of isoproterenol-induced dilation in pressurized CA. These findings suggest that PSD95 mediates a vasodilator complex with ß1AR and KV1 channels in cVSMCs. This complex may be critical for proper vasodilation in rat CA.

Transient receptor proteins illuminated: current views on TRPs and disease.

The transient receptor potential proteins (TRPs) make up a very important family of ion channels responsible for a wide array of cellular functions. Originally identified in the visual system of Drosophila melanogaster, these channels are ubiquitously distributed throughout the mammalian system. The TRP family is divided into seven subfamilies in two groups: the first group comprises TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPN (Drosophila NOMPC) and TRPA (ankyrin). The second group contains TRPML (mucolipin) and TRPP (polycystin). The biophysical characterization of TRPs has revealed significantly different activation mechanisms and selectivity between channels. Functional studies have demonstrated that TRPs are necessary for a number of physiological processes, including sensation (such as taste, smell and temperature), hormone secretion and development. TRPs mediate these effects mainly by controlling the intracellular Ca(2+) concentration, which acts as a second messenger. Recent research has linked TRPs to different diseases. This review considers the impact of TRPs on cell physiology and the abnormalities observed with channel dysfunction.

Transient receptor potential melastatin type 7 channel is critical for the survival of bone marrow derived mesenchymal stem cells.

The transient receptor potential melastatin type 7 channel (TRPM7) is a member of the TRP family of ion channels that is essential for cell proliferation and viability. Mesenchymal stem cells (MSCs) from bone marrow are a potential source for tissue repair due to their ability to differentiate into specialized cells. However, the role of TRPM7 in stem cells is unknown. In this study, we characterized TRPM7 in mouse MSCs using molecular biology, immunocytochemistry, and patch clamp. We also investigated TRPM7 function using a lentiviral vector and specific shRNA to knockdown gene expression. By RT-PCR and immunocytochemistry, we identified TRPM7, but not TRPM6, a close family member with similar function. Electrophysiological recordings during depletion of intracellular Mg(2+) or Mg(2+)-ATP resulted in the development of currents typical for the channel. Furthermore, 2-aminoethoxydiphenyl borate (1 pM-100 microM) inhibited TRPM7 in a concentration-dependent manner. The molecular suppression of TRPM7 significantly decreased MSC proliferation and viability as determined by MTT assay. In addition, TRPM7 gene expression was up-regulated during osteogenesis. These findings demonstrate that TRPM7 is required for MSC survival and perhaps involved in the differentiation process.