Simon Bulley

TMEM16A/ANO1 Channels Contribute to the Myogenic Response in Cerebral Arteries

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 nonselective 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), also termed Anoctamin 1, chloride (Cl−) channels in arterial myocytes. Objective: To 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, nonselective 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-induced (−40 mm Hg) 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-induced (60 mmol/L K+) 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 nonselective cation channels stimulates TMEM16A channels to induce myogenic constriction.

TMEM16A channels generate Ca2+-activated Cl− currents in cerebral artery smooth muscle cells

Transmembrane protein (TMEM)16A channels are recently discovered membrane proteins that display electrophysiological properties similar to classic Ca(2+)-activated Cl(-) (Cl(Ca)) channels in native cells. The molecular identity of proteins that generate Cl(Ca) currents in smooth muscle cells (SMCs) of resistance-size arteries is unclear. Similarly, whether cerebral artery SMCs generate Cl(Ca) currents is controversial. Here, using molecular biology and patch-clamp electrophysiology, we examined TMEM16A channel expression and characterized Cl(-) currents in arterial SMCs of resistance-size rat cerebral arteries. RT-PCR amplified transcripts for TMEM16A but not TMEM16B-TMEM16H, TMEM16J, or TMEM16K family members in isolated pure cerebral artery SMCs. Western blot analysis using an antibody that recognized recombinant (r)TMEM16A channels detected TMEM16A protein in cerebral artery lysates. Arterial surface biotinylation and immunofluorescence indicated that TMEM16A channels are located primarily within the arterial SMC plasma membrane. Whole cell Cl(Ca) currents in arterial SMCs displayed properties similar to those generated by rTMEM16A channels, including Ca(2+) dependence, current-voltage relationship linearization by an elevation in intracellular Ca(2+) concentration, a Nerstian shift in reversal potential induced by reducing the extracellular Cl(-) concentration, and a negative reversal potential shift when substituting extracellular I(-) for Cl(-). A pore-targeting TMEM16A antibody similarly inhibited both arterial SMC Cl(Ca) and rTMEM16A currents. TMEM16A knockdown using small interfering RNA also inhibited arterial SMC Cl(Ca) currents. In summary, these data indicate that TMEM16A channels are expressed, insert into the plasma membrane, and generate Cl(Ca) currents in cerebral artery SMCs.

Smooth muscle cell transient receptor potential polycystin-2 (TRPP2) channels contribute to the myogenic response in cerebral arteries

•  Intravascular pressure is reported to activate several mechanosensitive ion channels, leading to smooth muscle cell (SMC) depolarization, voltage‐dependent Ca2+ channel activation and vasoconstriction; a process known as the ‘myogenic response’. •  Polycystin‐1 and ‐2 (TRPP1 and ‐2) have been shown to differentially regulate the mesenteric artery myogenic response, with TRPP2 expression attenuating vasoconstriction. •  We show that TRPP2 is the major TRPP isoform expressed and that TRPP2 is located primarily in the plasma membrane in cerebral artery SMCs. •  Selective TRPP2 knockdown reduced swelling‐induced non‐selective cation currents (ICat) in SMCs and myogenic tone in cerebral arteries. •  These data indicate that TRPP2 activation contributes to the cerebral artery myogenic response and suggest that TRPP2 performs differential functions in different vascular beds.

Cl− channels in smooth muscle cells

In smooth muscle cells (SMCs), the intracellular chloride ion (Cl−) concentration is high due to accumulation by Cl−/HCO3− exchange and Na+–K+–Cl− cotransportation. The equilibrium potential for Cl− (ECl) is more positive than physiological membrane potentials (Em), with Cl− efflux inducing membrane depolarization. Early studies used electrophysiology and nonspecific antagonists to study the physiological relevance of Cl− channels in SMCs. More recent reports have incorporated molecular biological approaches to identify and determine the functional significance of several different Cl− channels. Both “classic” and cGMP-dependent calcium (Ca2+)-activated (ClCa) channels and volume-sensitive Cl− channels are present, with TMEM16A/ANO1, bestrophins, and ClC-3, respectively, proposed as molecular candidates for these channels. The cystic fibrosis transmembrane conductance regulator (CFTR) has also been described in SMCs. This review will focus on discussing recent progress made in identifying each of these Cl− channels in SMCs, their physiological functions, and contribution to diseases that modify contraction, apoptosis, and cell proliferation.

Transcriptional Upregulation of α2δ-1 Elevates Arterial Smooth Muscle Cell Voltage-Dependent Ca2+ Channel Surface Expression and Cerebrovascular Constriction in Genetic Hypertension

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 composed of a pore-forming CaV1.2&agr;1 with auxiliary &agr;2&dgr; and &bgr; 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 &agr;2&dgr; subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in &agr;2&dgr;-1 and CaV1.2&agr;1 mRNA and protein in cerebral artery myocytes, with &agr;2&dgr;-1 increasing more than CaV1.2&agr;1. Other &agr;2&dgr; isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized &agr;2&dgr;-1 and CaV1.2&agr;1 proteins, surface &agr;2&dgr;-1:CaV1.2&agr;1 ratio, CaV1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an &agr;2&dgr;-1 ligand, did not alter &agr;2&dgr;-1 or CaV1.2&agr;1 total protein but normalized &agr;2&dgr;-1 and CaV1.2&agr;1 surface expression, surface &agr;2&dgr;-1:CaV1.2&agr;1, 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 &agr;2&dgr;-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 &agr;2&dgr;-1 targeting is a novel strategy that may be used to reverse pathological CaV1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

9-Phenanthrol inhibits recombinant and arterial myocyte TMEM16A channels

In arterial smooth muscle cells (myocytes), intravascular pressure stimulates membrane depolarization and vasoconstriction (the myogenic response). Ion channels proposed to mediate pressure‐induced depolarization include several transient receptor potential (TRP) channels, including TRPM4, and transmembrane protein 16A (TMEM16A), a Ca2+‐activated Cl− channel (CaCC). 9‐Phenanthrol, a putative selective TRPM4 channel inhibitor, abolishes myogenic tone in cerebral arteries, suggesting that either TRPM4 is essential for pressure‐induced depolarization, upstream of activation of other ion channels or that 9‐phenanthrol is non‐selective. Here, we tested the hypothesis that 9‐phenanthrol is also a TMEM16A channel blocker, an ion channel for which few inhibitors have been identified.

Angiotensin II stimulates internalization and degradation of arterial myocyte plasma membrane BK channels to induce vasoconstriction.

Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated K(+) (BK) channel α and auxiliary β1 subunits that modulate arterial contractility. In arterial myocytes, β1 subunits are stored within highly mobile rab11A-positive recycling endosomes. In contrast, BKα subunits are primarily plasma membrane-localized. Trafficking pathways for BKα and whether physiological stimuli that regulate arterial contractility alter BKα localization in arterial myocytes are unclear. Here, using biotinylation, immunofluorescence resonance energy transfer (immunoFRET) microscopy, and RNAi-mediated knockdown, we demonstrate that rab4A-positive early endosomes traffic BKα to the plasma membrane in myocytes of resistance-size cerebral arteries. Angiotensin II (ANG II), a vasoconstrictor, reduced both surface and total BKα, an effect blocked by bisindolylmaleimide-II, concanavalin A, and dynasore, protein kinase C (PKC), internalization, and endocytosis inhibitors, respectively. In contrast, ANG II did not reduce BKα mRNA, and sodium nitroprusside, a nitric oxide donor, did not alter surface BKα protein over the same time course. MG132 and bafilomycin A, proteasomal and lysosomal inhibitors, respectively, also inhibited the ANG II-induced reduction in surface and total BKα, resulting in intracellular BKα accumulation. ANG II-mediated BK channel degradation reduced BK currents in isolated myocytes and functional responses to iberiotoxin, a BK channel blocker, and NS1619, a BK activator, in pressurized (60 mmHg) cerebral arteries. These data indicate that rab4A-positive early endosomes traffic BKα to the plasma membrane in arterial myocytes. We also show that ANG II stimulates PKC-dependent BKα internalization and degradation. These data describe a unique mechanism by which ANG II inhibits arterial myocyte BK currents, by reducing surface channel number, to induce vasoconstriction.

Reciprocal regulation between taurine and glutamate response via Ca2+- dependent pathways in retinal third-order neurons

Although taurine and glutamate are the most abundant amino acids conducting neural signals in the central nervous system, the communication between these two neurotransmitters is largely unknown. This study explores the interaction of taurine and glutamate in the retinal third-order neurons. Using specific antibodies, both taurine and taurine transporters were localized in photoreceptors and Off-bipolar cells, glutamatergic neurons in retinas. It is possible that Off-bipolar cells release juxtaposed glutamate and taurine to activate the third-order neurons in retina. The interaction of taurine and glutamate was studied in acutely dissociated third-order neurons in whole-cell patch-clamp recording and Ca2+ imaging. We find that taurine effectively reduces glutamate-induced Ca2+ influx via ionotropic glutamate receptors and voltage-dependent Ca2+ channels in the neurons, and the effect of taurine was selectively inhibited by strychnine and picrotoxin, but not GABA receptor antagonists, although GABA receptors are present in the neurons. A CaMKII inhibitor partially reversed the effect of taurine, suggesting that a Ca2+/calmodulin-dependent pathway is involved in taurine regulation. On the other hand, a rapid influx of Ca2+ through ionotropic glutamate receptors could inhibit the amplitude and kinetics of taurine-elicited currents in the third-order neurons, which could be controlled with intracellular application of BAPTA a fast Ca2+ chelator. This study indicates that taurine is a potential neuromodulator in glutamate transmission. The reciprocal inhibition between taurine and glutamate in the postsynaptic neurons contributes to computation of visual signals in the retinal neurons.

Taurine activates delayed rectifier KV channels via a metabotropic pathway in retinal neurons

•  Although taurine is one of the most abundant amino acids in human tissues, and serves a number of important functions ranging from cell development to cytoprotection, its precise mode of action is often obscure. •  Here we present evidence that, in the vertebrate retina, taurine regulates voltage‐gated potassium (KV) channels that are sensitive to the inhibitors of KV1, KV2 and KV4 subunits. •  Taurines effect was shown to be a metabotropic response, involving a G‐protein linked, PKC‐dependent intracellular pathway. •  Noteworthy was the finding that responses to taurine were blocked by a specific antagonist of 5‐HT2A receptors. Taurine activation of 5‐HT2A receptors was further confirmed in HEK cells that expressed recombinant 5‐HT2A receptors. •  Taurine has been shown to be beneficial in the management of a number of brain disorders. Its interaction with serotonergic pathways suggests that taurine may also play a role in various cognitive functions of the CNS.

Membrane depolarization activates BK channels through ROCK-mediated β1 subunit surface trafficking to limit vasoconstriction

Depolarization of arterial myocytes increases the surface abundance of the β1 subunit of BK channels to promote vasodilation. Trafficking patterns for vasodilation Constriction of the small arteries that regulate regional organ blood flow occurs due to membrane depolarization of arterial myocytes, which stimulates voltage-dependent Ca2+ channels that mediate the influx of Ca2+ ions. Dilation of these blood vessels from the constricted state can occur due to BK channels, which are activated by Ca2+, partially reversing the membrane depolarization of arterial myocytes. Leo et al. found that membrane depolarization triggered a signaling pathway that ensured the activation of BK channels. Ca2+ influx through voltage-dependent Ca2+ channels activated kinases that increased the trafficking of the β1 auxiliary subunit of the BK channel to the plasma membrane, where it bound to the pore-forming subunit to increase its sensitivity to Ca2+. Thus, BK channels are activated in depolarized arterial myocytes not only because of direct stimulation by Ca2+, but also because of the increased plasma membrane abundance of the subunit that determines their sensitivity to Ca2+. Membrane depolarization of smooth muscle cells (myocytes) in the small arteries that regulate regional organ blood flow leads to vasoconstriction. Membrane depolarization also activates large-conductance calcium (Ca2+)–activated potassium (BK) channels, which limits Ca2+ channel activity that promotes vasoconstriction, thus leading to vasodilation. We showed that in human and rat arterial myocytes, membrane depolarization rapidly increased the cell surface abundance of auxiliary BK β1 subunits but not that of the pore-forming BKα channels. Membrane depolarization stimulated voltage-dependent Ca2+ channels, leading to Ca2+ influx and the activation of Rho kinase (ROCK) 1 and 2. ROCK1/2-mediated activation of Rab11A promoted the delivery of β1 subunits to the plasma membrane by Rab11A-positive recycling endosomes. These additional β1 subunits associated with BKα channels already at the plasma membrane, leading to an increase in apparent Ca2+ sensitivity and activation of the channels in pressurized arterial myocytes and vasodilation. Thus, membrane depolarization activates BK channels through stimulation of ROCK- and Rab11A-dependent trafficking of β1 subunits to the surface of arterial myocytes.