Expression profile and protein translation of TMEM16A in murine smooth muscle
Expression profile and protein translation of TMEM16A in murine smooth muscle
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TMEM16A 在小鼠平滑肌中的表达谱和蛋白质翻译
DOI：
10.1152/ajpcell.00018.2010
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发表时间：
2010-11-01
影响因子：
5.5
通讯作者：
Leblanc, Normand
中科院分区：
文献类型：
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作者：
Davis, Alison J.;Forrest, Abigail S.;Leblanc, Normand
Davis AJ，Forrest AS，Jepps TA，Valencik ML，Wiwchar M，Singer CA，Sones WR，Greenwood IA，Leblanc N. TMEM 16 A在小鼠平滑肌中的表达谱和蛋白翻译。美国生理学杂志细胞生理学299：C948-C959，2010年。首次发表于2010年8月4日; doi：10.1152/ajpcell.00018.2010。最近，基因TMEM 16 A和TMEM 16 B的过表达已被证明在血管平滑肌中产生与天然Ca 2+激活的Cl-电流（I-ClCa）定性相似的电流。然而，在血管平滑肌中没有关于这个新基因家族的信息，其中Cl-通道是主要的去极化机制。在BALB/c小鼠门静脉、胸主动脉和颈动脉分离的平滑肌细胞中，用含有500 nM Ca 2+的移液器溶液诱发定性相似的Cl-电流。使用SYBR绿色化学和跨膜蛋白（TMEM）16 A或密切相关的TMEM 16 B特异性引物的定量PCR显示TMEM 16 A表达如下：门静脉>胸主动脉>颈动脉>脑。此外，还检测到TMEM 16 A的几种可变剪接变体转录本。相反，TMEM 16 B在平滑肌中的表达非常低。用针对TMEM 16 A的不同抗体进行的Western印迹分析显示，除了门静脉外，许多产物具有类似于120 kDa的一致条带，其中80-kDa条带占主导地位。在门静脉、胸主动脉和颈动脉的4 μ m厚切片的平滑肌层中鉴定了TMEM 16 A蛋白。在分离的肌细胞中，在整个细胞质以及膜附近弥漫地检测到对TMEM 16 A抗体特异性的荧光。在来自血管组织的裂解物的Western印迹分析中使用的相同抗体也识别在HEK 293细胞中表达的类似于147-kDa小鼠TMEM 16 A-绿色荧光蛋白（GFP）融合蛋白，其与GFP抗体检测到的类似条带相关。膜片钳实验显示，通过在HEK 293细胞中转染TMEM 16 A-GFP产生的I-ClCa显示出与血管肌细胞中记录的I-ClCa的显著相似性，包括缓慢的动力学、陡峭的向外整流以及与药理学试剂尼氟灭酸相似的反应。这项研究表明，TMEM 16 A的表达是强大的鼠血管平滑肌细胞，巩固的观点，该基因是一个可行的候选人的天然钙激活的Cl-通道在这种细胞类型。
Davis AJ, Forrest AS, Jepps TA, Valencik ML, Wiwchar M, Singer CA, Sones WR, Greenwood IA, Leblanc N. Expression profile and protein translation of TMEM16A in murine smooth muscle. Am J Physiol Cell Physiol 299: C948-C959, 2010. First published August 4, 2010; doi: 10.1152/ajpcell.00018.2010.-Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca2+-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 Ca2+ 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 similar to 120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-mu m-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 similar to 147-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 Ca2+-activated Cl- channel in this cell type.