Rationale Pressure-induced arterial depolarization and constriction (the myogenic response), is a smooth muscle cell (myocyte)-specific mechanism that controls regional organ blood flow and systemic blood pressure. Several different non-selective cation channels contribute to pressure-induced depolarization, but signaling mechanisms involved are unclear. Similarly uncertain is the contribution of anion channels to the myogenic response and physiological functions and mechanisms of regulation of recently discovered transmembrane 16A (TMEM16A) chloride (Cl−) channels in arterial myocytes. Objective Investigate the hypothesis that myocyte TMEM16A channels control membrane potential and contractility and contribute to the myogenic response in cerebral arteries. Methods and Results Cell swelling induced by hyposmotic bath solution stimulated Cl− currents in arterial myocytes that were blocked by TMEM16A channel inhibitory antibodies, RNAi-mediated selective TMEM16A channel knockdown, removal of extracellular calcium (Ca2+), replacement of intracellular EGTA with BAPTA, a fast Ca2+ chelator, and Gd3+ and SKF-96365, non-selective cation channel blockers. In contrast, nimodipine, a voltage-dependent Ca2+ channel inhibitor, or thapsigargin, which depletes intracellular Ca2+ stores, did not alter swelling-activated TMEM16A currents. Pressure (−40 mmHg)-induced membrane stretch activated ion channels in arterial myocyte cell-attached patches that were inhibited by TMEM16A antibodies and were of similar amplitude to recombinant TMEM16A channels. TMEM16A knockdown reduced intravascular pressure-induced depolarization and vasoconstriction, but did not alter depolarization (60 mmol/L K+)-induced vasoconstriction. Conclusions Membrane stretch activates arterial myocyte TMEM16A channels, leading to membrane depolarization and vasoconstriction. Data also provide a mechanism by which a local Ca2+ signal generated by non-selective cation channels stimulates TMEM16A channels to induce myogenic constriction.

Thomas-Gatewood C, Neeb ZP, Bulley S, Adebiyi A, Bannister JP, Leo MD, Jaggar JH. TMEM16A channels generate Ca 2ϩ -activated Cl Ϫ currents in cerebral artery smooth muscle cells.

A ctivation of plasma membrane phospholipase (PL)Ccoupled receptors by vasoconstrictor agonists leads to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis and the generation of inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol. 1 In vascular myocytes, diacylglycerol (DAG) activates protein kinase (PK)C, leading to the phosphorylation of a wide variety of proteins, including ion channels. 2 IP 3 binds to sarcoplasmic reticulum (SR) IP 3 receptors (IP 3 Rs), resulting in SR Ca 2ϩ release, an elevation in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), and vasoconstriction. 3 Recent evidence also indicates that IP 3 -induced vasoconstriction can occur independently of SR Ca 2ϩ release and via the activation of type 1 IP 3 receptors (IP 3 R1) and type 3 canonical transient receptor potential (TRPC) channels. 4,5 However, the functional signaling mechanisms by which IP 3 Rs and TRPC channels communicate in arterial myocytes are unclear.The mammalian TRP channel superfamily is encoded by at least 28 different genes that are subdivided into 7 families. 6 These families encode ion channels with diverse ion selectivity, modes of regulation, and physiological functions. 6 Vascular myocytes express at least 4 TRP families, including TRPC, TRPM, TRPV, and TRPP. [7][8][9][10] These channels regulate arterial myocyte membrane potential, [Ca 2ϩ ] i , contractility, and proliferation, and are implicated in the etiology of vascular diseases. 4,8 -12 Given the diversity of vascular myocyte TRP channels, it has become important to identify signaling pathways that Original received July 15, 2009; resubmission received January 8, 2010; revision received March 22, 2010; accepted March 29, 2010. From the Department of Physiology, University of Tennessee Health Science Center, Memphis. Correspondence to Jonathan H. Jaggar, Department of Physiology, University of Tennessee Health Science Center, 894 Union Ave, Nash Building, Memphis, TN 38139. E-mail jjaggar@uthsc.edu © 2010 American Heart Association, Inc. Thus, TRPC3 and TRPC6 channels perform distinct physiological functions, but signaling pathways that mediate this differential regulation are unclear.Here, we studied mechanisms by which IP 3 R1, the principal molecular and functional arterial myocyte IP 3 R isoform, 5 stimulates TRPC currents in resistance-size cerebral arteries. Data suggest that IP 3 R1 is in close spatial proximity to, and associates with, TRPC3, but not TRPC6 or TRPM4 channels. Endothelin (ET)-1, a PLC-coupled receptor agonist, and IP 3 alter the interaction between the IP 3 R N terminus and the TRPC3 channel C terminus, leading to channel activation and vasoconstriction. Data indicate that IP 3 R1 selectively couples to TRPC3 channels because of the close spatial proximity of these proteins and that this mechanism is essential for mediating ET-1 and IP 3 -induced vasoconstriction. Methods Tissue PreparationAnimal protocols used were reviewed and approved by the Animal Care and Use Committee at the University of Tennessee Health Science ...

A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca2+ (CaV1.2) currents that induces pathological vasoconstriction. CaV1.2 channels are heteromeric complexes comprising a pore forming CaV1.2α1 with auxiliary α2δ and β subunits. Molecular mechanisms that elevate CaV1.2 currents during hypertension and the potential contribution of CaV1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α2δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats (SHR) was associated with an unequal elevation in α2δ-1 and CaV1.2α1 mRNA and protein in cerebral artery myocytes, with α2δ-1 increasing more than CaV1.2α1. Other α2δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive SHR displayed higher surface-localized α2δ-1 and CaV1.2α1 proteins, surface α2δ-1 to CaV1.2α1 ratio (α2δ-1:CaV1.2α1), CaV1.2 current-density and non-inactivating current, and pressure- and - depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α2δ-1 ligand, did not alter α2δ-1 or CaV1.2α1 total protein, but normalized α2δ-1 and CaV1.2α1 surface expression, surface α2δ-1:CaV1.2α1, CaV1.2 current-density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α2δ-1 expression that promotes surface trafficking of CaV1.2 channels in cerebral artery myocytes. This leads to an increase in CaV1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α2δ-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP3) that activates sarcoplasmic reticulum (SR) IP3 receptors (IP3Rs). In cerebral artery myocytes, IP3Rs release SR Ca2+ and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current (ICat) activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP3Rs control vascular contractility in systemic arteries and IP3R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ~2.7- and 2-fold higher in mesenteric arteries of hypertensive spontaneously hypertensive rats (SHR) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP3R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-hypertensive SHR and WKY rats. Control, IP3- and endothelin-1 (ET-1)-induced FRET between IP3R1 and TRPC3 was higher in hypertensive SHR than WKY myocytes. IP3-induced ICat was ~3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and CIRBP-TAT, an IP3R-TRP physical coupling inhibitor, reduced IP3-induced ICat and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a SR Ca2+-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP3R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP3R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension.