NDLI: Expression profile and protein translation of TMEM16A in murine smooth muscle

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Davis, Alison J. Forrest, Abigail S. Jepps, Thomas A. Valencik, Maria L. Wiwchar, Michael Singer, Cherie A. Sones, William R. Greenwood, Iain A. Leblanc, Normand
Description	Country affiliation: United kingdom Author Affiliation: Davis AJ ( Division of Biomedical Sciences, St. George's, University of London, London, United Kingdom.)
Abstract	Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca(2+)-activated Cl(-) currents (I(ClCa)) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl(-) channels are a major depolarizing mechanism. Qualitatively similar Cl(-) currents were evoked by a pipette solution containing 500 nM Ca(2+) in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle. Western blot analysis with different antibodies directed against TMEM16A revealed a number of products with a consistent band at â¼120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-µm-thick slices of portal vein, thoracic aorta, and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an â¼147-kDa mouse TMEM16A-green fluorescent protein (GFP) fusion protein expressed in HEK 293 cells, which correlated to a similar band detected by a GFP antibody. Patch-clamp experiments revealed that I(ClCa) generated by transfection of TMEM16A-GFP in HEK 293 cells displayed remarkable similarities to I(ClCa) recorded in vascular myocytes, including slow kinetics, steep outward rectification, and a response similar to the pharmacological agent niflumic acid. This study shows that TMEM16A expression is robust in murine vascular smooth muscle cells, consolidating the view that this gene is a viable candidate for the native Ca(2+)-activated Cl(-) channel in this cell type.
File Format	HTM / HTML
ISSN	03636143
e-ISSN	15221563
DOI	10.1152/ajpcell.00018.2010
Journal	American Journal of Physiology - Cell Physiology
Issue Number	5
Volume Number	299
Language	English
Publisher	American Physiological Society
Publisher Date	2010-11-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Cell Biology Chloride Channels Metabolism Myocytes, Smooth Muscle Physiology Protein Biosynthesis Protein Isoforms Alternative Splicing Animals Cell Line Genetics Gene Expression Profiling Mice Mice, Inbred Balb C Molecular Sequence Data Cytology Patch-clamp Techniques Recombinant Fusion Proteins Tissue Distribution Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Cell Biology Physiology

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