Local coupling of TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies vasoconstriction in cerebral arteries

Abstract

[ANO1, also known as transmembrane protein 16A (TMEM16A)] is a Ca 2ϩ -activated Cl Ϫ channel expressed in arterial myocytes that regulates membrane potential and contractility. Signaling mechanisms that control ANO1 activity in arterial myocytes are poorly understood. In cerebral artery myocytes, ANO1 channels are activated by local Ca 2ϩ signals generated by plasma membrane nonselective cation channels, but the molecular identity of these proteins is unclear. Arterial myocytes express several different nonselective cation channels, including multiple members of the transient receptor potential receptor (TRP) family. The goal of this study was to identify localized ion channels that control ANO1 currents in cerebral artery myocytes. Coimmunoprecipitation and immunofluorescence resonance energy transfer microscopy experiments indicate that ANO1 and canonical TRP 6 (TRPC6) channels are present in the same macromolecular complex and localize in close spatial proximity in the myocyte plasma membrane. In contrast, ANO1 is not near TRPC3, TRP melastatin 4, or inositol trisphosphate receptor 1 channels. Hyp9, a selective TRPC6 channel activator, stimulated Cl Ϫ currents in myocytes that were blocked by T16Ainh-A01, an ANO1 inhibitor, ANO1 knockdown using siRNA, and equimolar replacement of intracellular EGTA with BAPTA, a fast Ca 2ϩ chelator that abolishes local Ca 2ϩ signaling. Hyp9 constricted pressurized cerebral arteries, and this response was attenuated by T16Ainh-A01. In contrast, T16Ainh-A01 did not alter depolarization-induced (60 mM K ϩ ) vasoconstriction. , and Cl Ϫ , as well as nonselective cation channels (4,12,14,19). An understanding of the signaling processes that control the activity of these ion channels provides a better understanding of mechanisms that regulate arterial contractility. Anoctamin-1 [ANO1, also known as transmembrane protein 16A (TMEM16A)] is a recently described Ca 2ϩ -activated Cl Ϫ (Cl Ca ) channel expressed in arterial myocytes that regulates membrane potential and contractility (4). Mechanisms that control ANO1 activity in arterial myocytes are poorly understood but important to determine given the functional significance of this protein in the vasculature. ANO1 channel message and protein have been described in the vasculature, including rat cerebral, pulmonary, and carotid arteries, murine portal vein, retinal and skeletal muscle arterioles, and cultured rat pulmonary artery myocytes (7,15,23,30 (6,23,30). ANO1 knockdown reduced Cl Ca current density in rat cerebral and mesenteric arteries and cultured pulmonary artery myocytes (23, 30). T16Ainh-A01, an ANO1 inhibitor, relaxed methoxamine-contracted murine and human blood vessels (8). Selective ANO1 knockdown attenuated intravascular pressure-induced cerebral artery depolarization and vasoconstriction (5). Cell-specific knockout of ANO1 reduced Cl Ca currents in aortic and cerebral arteriole myocytes, agonist-induced vasoconstriction in retinal and skeletal muscle arterioles, and systemic blood pressure and attenuated hypertensio...

TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis

“…TMEM16A, also known as ANO1, DOG1, and ORAOV2, is a calcium-activated chloride channel (CaCC) (3)(4)(5) with physiological functions in epithelial tissues, exocrine glands, dorsal root ganglion neurons, and smooth muscles (6)(7)(8). TMEM16A has also been detected in multiple cancers, including breast cancer, head and neck squamous cell carcinomas (HNSCC), gastrointestinal squamous tumors (GIST), lung cancer, pancreatic cancer, and ovarian cancer (9,10).…”

Drug Repurposing: The Anthelmintics Niclosamide and Nitazoxanide are Potent TMEM16A Antagonists that Fully Bronchodilate Airways

“…Channels can also associate with other cell surface molecules to form large macromolecular signaling complexes. Indeed, TMEM16A has been shown to interact with the ezrin-radixin-moesin network (Perez-Cornejo et al 2012) and with TrpV1 (Takayama et al 2015), TrpC6 (Wang et al 2016) and the IP3 receptor (Jin et al 2013) for coupled GPCR and ion channel signaling. Interestingly, the key signaling molecule -catenin involved in numerous cancers was also found recently to associate with the ion channels KCNQ1 (Rapetti-Mauss et al 2017) and BKCa (Bian et al 2011) to modulate Wnt signaling.…”

Recent advances in TMEM16A: Structure, function, and disease

“…TMEM16A downregulation in tail arteries was found to be associated with reduction in CACNA1C protein (vascular L ‐type Ca 2+ channel) expression, TMEM16A modulates arterial contractility may be indirectly via regulation of CACNA1C expression (Jensen et al, ). Similarly, Q. Wang et al () found that canonical transient receptor potential receptor (TRPC6) channels stimulate TMEM16A currents via local Ca 2+ signaling. TMEM16A and TRPC6 channels are coupled in arterial myocytes and this interaction stimulates vasoconstriction in cerebral arteries (Q. Wang et al, ).…”

“…Similarly, Q. Wang et al (2016) found that canonical transient receptor potential receptor (TRPC6) channels stimulate TMEM16A currents via local Ca 2+ signaling. TMEM16A and TRPC6 channels are coupled in arterial myocytes and this interaction stimulates vasoconstriction in cerebral arteries (Q.…”

“…These data indicated that TMEM16A may control cerebrovascular contraction and affect blood pressure. Furthermore, Q. Wang et al () proved that intracellular calcium signal generated by TRPC6 channel activates TMEM16A in nearby myocytes to stimulate vasoconstriction. This may be a mechanism by which TMEM16A regulates cerebral arterial vasoconstriction.…”