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 specifi c shRNA to knockdown gene expression. By RT-PCR and immunocytochemistry, we identifi ed 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 μM) inhibited TRPM7 in a concentration-dependent manner. The molecular suppression of TRPM7 signifi cantly decreased MSC proliferation and viability as determined by MTT assay. In addition, TRPM7 gene expression was up-regulated during osteogenesis. These fi ndings demonstrate that TRPM7 is required for MSC survival and perhaps involved in the differentiation process. Introduction Stem cell therapy offers a promising approach to providing an advanced and reliable therapeutic strategy for tissue repair. Mesenchymal stem cells (MSCs) from bone marrow are a source of stem cells capable of differentiation into specialized tissue, including bone, fat, and muscle [ 1 ]. The process of stem cell growth and differentiation is controlled by a network of intracellular signaling molecules that are activated by hormone, growth factor, and/or cytokine stimulation. These signals control gene transcription and the differentiation process [ 2 , 3 ]. Despite a number of studies demonstrating the capability of MSCs to differentiate, the precise mechanism controlling the fate of stem cells is not well understood. Among second messengers, Ca 2+ ions are one of the most important and commonly used molecules for cellular function [ 4 ]. Calcium is available to the cell from 2 sources: fi rst, the extracellular environment, where the Ca 2+ concentration is in the low millimolar range. This Ca 2+ is available to the cytosol through plasma membrane ion channels. Second, there are fi nite intracellular Ca 2+ stores, typically membrane-delineated compartments, such as endoplasmic reticulum (ER), Golgi apparatus, mitochondria, and nucleus. This Ca 2+ is available to the cytosol to differing degrees and exits the storage compartment through intracellular Ca 2+ "release" channels. Increases in intracellular Ca 2+ concentration are a phenomenon commonly observed during stem cell differentiation and linked to the activation of transcriptional programs [ 5 , 6 ]. In MSCs, Ca 2+ oscillations are dependent on both Ca 2+ release from the ER and entry from the extracellular space [ 7 , 8 ]. These signals can be initiated by autocrine or paracrine m...

Shaker-type voltage-gated K + (K V 1) channels composed of K V 1.2 and K V 1.5 α-subunits are expressed in cerebral vascular smooth muscle cells (cVSMCs), where they contribute to the resting diameter and vasodilation of cerebral arteries (CAs).1,2 K V 1 channels are multiprotein structures composed of 4 K V α pore-forming subunits coassembled with intracellular K V β subunits, which may affect channel trafficking and kinetics. [3][4][5][6][7][8] In addition, post-translational modifications, such as glycosylation and protein kinase A (PKA)-mediated phosphorylation of the K V α subunits, may increase protein expression and activity of K V 1 channels. 9-15Recently, we reported the expression of a scaffolding protein, postsynaptic density protein-95 (PSD95), in rat CA. 16 Previously, PSD95 was studied primarily in neurons, where it provides an assembly platform at the plasma membrane for macromolecular signaling complexes including ion channels.17-22 However, we reported that PSD95 serves as a molecular scaffold for K V 1 channels in cVSMCs, and this interaction is required for the proper expression of K V 1 channels that exerts a tonic vasodilator influence. 16 Accordingly, antisense-mediated knockdown of PSD95 in rat CA resulted in a loss of K V 1 channel expression and caused vasoconstriction, inferring that PSD95 promotes the expression of K V 1 channels in cVSMCs. 16 Notably, the C terminus of the K V 1.2α subunit contains a structural motif that permits the channel to interact with Objective: We explored whether a specific interaction between PSD95 and K V 1 channels enables protein kinase A phosphorylation of K V 1 channels in cVSMCs to promote vasodilation. Methods and Results:Rat cerebral arteries were used for analyses. A membrane-permeable peptide (K V 1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of K V 1.2α was designed as a dominant-negative peptide to disrupt the association of K V 1 channels with PSD95. Application of K V 1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and K V 1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of K V 1-C peptide. Patch-clamp recordings confirmed that K V 1-C peptide attenuates K V 1 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 K V 1-C peptide showed markedly less phosphorylation of K V 1.2α subunits. Finally, phosphatase inhibitors blunted both K V 1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction. PSD95. [16][17][18][19][20] Collectively, the interactions of signaling proteins with PSD95 are enabled by 3 postsynaptic density-95, discs large, zonula occludens-1 (PDZ...

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. β1-Adrenergic receptors (β1ARs) also have a binding motif for PSD95. Functional association of β1AR with KV1 channels through PSD95 may represent a novel vasodilator complex in cerebral arteries (CA). We explored whether a β1AR-PSD95-KV1 complex is a determinant of rat CA dilation. RT-PCR and western blots revealed expression of β1AR in CA. Isoproterenol induced a concentration-dependent dilation of isolated, pressurized rat CA that was blocked by the β1AR blocker CGP20712. Cranial window imaging of middle cerebral arterioles in situ showed isoproterenol- and norepinephrine-induced dilation that was blunted by β1AR blockade. Isoproterenol-induced hyperpolarization of cVSMCs in pressurized CA was blocked by CGP20712. Confocal images of cVSMCs immunostained with antibodies against β1AR and PSD95 indicated strong colocalization, and PSD95 co-immunoprecipitated with β1AR in CA lysate. Blockade of KV1 channels, β1AR 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 β1AR and KV1 channels in cVSMCs. This complex may be critical for proper vasodilation in rat CA.