title: The multiple expression of Ca²⁺-activated Cl⁻ channels via homo- and hetero-dimer formation of TMEM16A splicing variants in murine portal vein.
abstract: Ca(2+)-activated Cl(-) channel (CaCC) often plays substantial roles in the regulation of membrane excitability in smooth muscle cells (SMCs). TMEM16A, a member of the TMEM16 family, has been suggested as the molecular entity responsible for CaCC in several types of SMCs. In this study, the expression of TMEM16A splicing variants and their contribution to CaCC activity were examined in murine portal vein SMCs (mPVSMCs). Four transcripts of TMEM16A splicing variants, which include four alternatively spliced segments ("a" and "b" in N-terminus and "c" and "d" in the first intracellular loop), were identified; the expression ratio of four transcripts of "abc", "acd", "abcd" and "ac" was 64.5, 25.8, 4.8 and 4.8%, respectively. The immunostaining of isolated mPVSMCs with anti-TMEM16A antibody indicates the abundant expression of TMEM16A on the cell membrane. CaCC currents recorded in mPVSMCs were markedly reduced by T16A(inh)-A01, a specific TMEM16A inhibitor. When the two major TMEM16A splicing variants, abc and acd isoforms, were expressed separately in HEK293 cells, the CaCC currents, which possess similar electrophysiological characteristics to those in mPVSMCs were observed. The single-molecule photobleaching analyses using total internal reflection fluorescence (TIRF) microscope indicated that the distribution of stepwise photobleaching events was fit well with a binomial distribution for homodimer. Additionally, the heterodimer formation was suggested by fluorescence resonance energy transfer (FRET) analyses in HEK293 cells co-expressing CFP- or YFP-tagged variants. In conclusion, alternatively spliced variants of TMEM16A abc and acd in mPVSMCs are two major molecular entities of CaCC and may form hetero-/homo-dimers to be functional as CaCC in the regulation of membrane excitability and contractility in mPVSMCs.
authors: Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors: Junya
authors: J
authors: Ohshiro
authors: Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors: Hisao
authors: H
authors: Yamamura
authors: Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors: Takanori
authors: T
authors: Saeki
authors: Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
authors: Yoshiaki
authors: Y
authors: Suzuki
authors: Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan. Electronic address: yimaizum@phar.nagoya-cu.ac.jp.
authors: Yuji
authors: Y
authors: Imaizumi
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D019281:Dimerization; D005786:Gene Expression Regulation / Q000502:physiology; D008297:Male; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D011169:Portal Vein / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism*
chemical_list: C531181:ANO1 protein, mouse; D000075369:Anoctamin-1; D018118:Chloride Channels; D020033:Protein Isoforms
keywords: Ca(2+)-activated Cl(−) channel; Single molecular imaging; Splicing variants; TMEM16A; Vascular smooth muscle