Identification and localization of TRPC channels in the rat kidney
Identification and localization of TRPC channels in the rat kidney
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DOI：
10.1152/ajprenal.00376.2005
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发表时间：
2006-05-01
影响因子：
4.2
通讯作者：
Schilling, WP
中科院分区：
文献类型：
--
作者：
Goel, M;Sinkins, WG;Schilling, WP
已经确定瞬时受体电位（TRP）通道在与PLC连接的G蛋白偶联膜受体的刺激后被激活，但是它们在肾肾单位的各种细胞中的差异表达尚未被描述。在本研究中，免疫沉淀从大鼠肾脏裂解物，然后使用TRPC特异性，亲和纯化的抗体进行Western印迹分析，揭示了TRPC 1，-C3和-C6的存在。TRPC 4、-C5和-C7未检出。TRPC 1免疫荧光在肾小球和特定的肾小管细胞的皮质和外髓质。TRPC 1与水通道蛋白1共定位，水通道蛋白1是近端小管和细降支的标志物，但不与水通道蛋白2共定位，水通道蛋白2是连接小管和集合管细胞的标志物。TRPC 3和-C6免疫标记主要局限于肾小球和特定的肾小管细胞的皮质和外部和内部的髓质。TRPC 3和-C6与水通道蛋白2共定位，但不与Na（+）/Ca（2+）交换体或花生凝集素共定位。因此，TRPC 3和-C6蛋白在集合管的主要细胞中表达。在极化培养的M1和IMCD-3集合管细胞中，TRPC 3仅定位于顶端域，而TRPC 6被发现在基底侧和顶端膜。TRPC 3和TRPC 6也在原代足细胞培养物中检测到，而TRPC 1仅在系膜细胞培养物中表达。在肾切片或细胞系中未观察到TRPC 4、-C5或-C7的特异性免疫阳性标记。这些结果表明，TRPC 1，-C3，和-C6可能发挥PLC依赖的信号在肾单位的特定区域的功能作用。
It is well established that transient receptor potential (TRP) channels are activated following stimulation of G protein-coupled membrane receptors linked to PLC, but their differential expression in various cells of the renal nephron has not been described. In the present study, immunoprecipitations from rat kidney lysates followed by Western blot analysis using TRPC-specific, affinity-purified antibodies revealed the presence of TRPC1, -C3, and -C6. TRPC4, -C5, and -C7 were nondetectable. TRPC1 immunofluorescence was detected in glomeruli and specific tubular cells of the cortex and outer medulla. TRPC1 colocalized with aquaporin-1, a marker for proximal tubule and thin descending limb, but not with aquaporin-2, a marker for connecting tubule and collecting duct cells. TRPC3 and -C6 immunolabeling was predominantly confined to glomeruli and specific tubular cells of the cortex and both the outer and inner medulla. TRPC3 and -C6 colocalized with aquaporin-2, but not with the Na(+)/Ca(2+) exchanger or peanut lectin. Thus TRPC3 and -C6 proteins are expressed in principle cells of the collecting duct. In polarized cultures of M1 and IMCD-3 collecting duct cells, TRPC3 was localized exclusively to the apical domain, whereas TRPC6 was found in both the basolateral and apical membranes. TRPC3 and TRPC6 were also detected in primary podocyte cultures, whereas TRPC1 was exclusively expressed in mesangial cell cultures. Specific immunopositive labeling for TRPC4, -C5, or -C7 was not observed in kidney sections or cell lines. These results suggest that TRPC1, -C3, and -C6 may play a functional role in PLC-dependent signaling in specific regions of the nephron.