Rab25 influences functional Cav1.2 channel surface expression in arterial smooth muscle cells
Rab25 influences functional Cav1.2 channel surface expression in arterial smooth muscle cells
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DOI：
10.1152/ajpcell.00345.2015
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发表时间：
2016-06-01
影响因子：
5.5
通讯作者：
Jaggar, Jonathan H.
中科院分区：
文献类型：
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作者：
Bannister, John P.;Bulley, Simon;Jaggar, Jonathan H.
质膜Ca（V）1.2通道是动脉平滑肌细胞（肌细胞）中主要的钙（Ca（V）1.2）内流途径。Ca（V）1.2通道调节多种细胞功能，包括收缩性和基因表达，但控制这些蛋白质表面表达的运输途径尚不清楚。同样，表达和生理功能的小Rab GTP酶，控制囊泡运输动脉肌细胞的蛋白质，知之甚少。在这里，我们研究了Rab蛋白，控制脑动脉肌细胞中Ca（V）1.2通道的功能表面丰度。蛋白质印迹表明Rab 25，一个以前与顶端再循环内体相关的GTdR，在脑动脉肌细胞中表达。免疫荧光Forster共振能量转移（immunofRET）显微镜显示Rab 25在心肌细胞中与Ca（V）1.2通道在空间上非常接近。如使用动脉生物素化测定的，使用siRNA敲低Rab 25降低动脉中Ca（V）1.2表面和细胞内丰度。Ca（V）1.2蛋白不位于Rab 11 A或Rab 4附近，且Rab 11 A或Rab 4A基因敲除后Ca（V）1.2蛋白未发生改变。Rab 25敲低导致Ca（V）1.2通过涉及溶酶体和蛋白酶体途径的机制降解，并降低全细胞Ca（V）1.2电流密度，但不改变分离的肌细胞中电流激活或失活的电压依赖性。Rab 25敲低还抑制脑动脉中的去极化（20-60 mM K+）和压力诱导的血管收缩（肌源性张力）。这些数据表明Rab 25在动脉肌细胞中表达，其中Rab 25促进Ca（V）1.2通道的表面表达以控制压力和去极化诱导的血管收缩。
Plasma membrane-localized Ca(V)1.2 channels are the primary calcium (Ca(V)1.2 ) influx pathway in arterial smooth muscle cells (myocytes). Ca(V)1.2 channels regulate several cellular functions, including contractility and gene expression, but the trafficking pathways that control the surface expression of these proteins are unclear. Similarly, expression and physiological functions of small Rab GTPases, proteins that control vesicular trafficking in arterial myocytes, are poorly understood. Here, we investigated Rab proteins that control functional surface abundance of Ca(V)1.2 channels in cerebral artery myocytes. Western blotting indicated that Rab25, a GTPase previously associated with apical recycling endosomes, is expressed in cerebral artery myocytes. Immunofluorescence Forster resonance energy transfer (immunoFRET) microscopy demonstrated that Rab25 locates in close spatial proximity to Ca(V)1.2 channels in myocytes. Rab25 knockdown using siRNA reduced Ca(V)1.2 surface and intracellular abundance in arteries, as determined using arterial biotinylation. In contrast, Ca(V)1.2 was not located nearby Rab11A or Rab4 and Ca(V)1.2 protein was unaltered by Rab11A or Rab4A knockdown. Rab25 knockdown resulted in Ca(V)1.2 degradation by a mechanism involving both lysosomal and proteasomal pathways and reduced whole cell Ca(V)1.2 current density but did not alter voltage dependence of current activation or inactivation in isolated myocytes. Rab25 knockdown also inhibited depolarization (20-60 mM K+) and pressure-induced vasoconstriction (myogenic tone) in cerebral arteries. These data indicate that Rab25 is expressed in arterial myocytes where it promotes surface expression of Ca(V)1.2 channels to control pressure- and depolarization-induced vasoconstriction.