Wang Xiaoxia, Pluznick Jennifer L, Wei Peilin, Padanilam Babu J, Sansom Steven C
Department of Physiology and Biophysics, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850, USA.
Am J Physiol Cell Physiol. 2004 Aug;287(2):C357-64. doi: 10.1152/ajpcell.00068.2004. Epub 2004 Mar 24.
Studies were performed to identify the molecular component responsible for store-operated Ca(2+) entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise Ca(2+)-selective and -nonselective cation channels in a variety of cells, we screened TRPC1-TRPC7 with the use of molecular methods and the fura 2 method to determine their participation as components of the mesangial store-operated Ca(2+) (SOC) channel. Using TRPC-specific primers and RT-PCR, we found that cultured MMC contained mRNA for TRPC1 and TRPC4 but not for TRPC2, TRPC3, TRPC5, TRPC6, and TRPC7. Immunocytochemical staining of MMC revealed predominantly cytoplasmic expression of TRPC1 and plasmalemmal expression of TRPC4. The role of TRPC4 in SOC was determined with TRPC4 antisense and fura 2 ratiometric measurements of intracellular Ca(2+) concentration (Ca(2+)). SOC was measured as the increase in Ca(2+) after extracellular Ca(2+) was increased from <10 nM to 1 mM in the continued presence of thapsigargin. We found that TRPC4 antisense, which reduced plasmalemmal expression of TRPC4, inhibited SOC by 83%. Incubation with scrambled TRPC4 oligonucleotides did not affect SOC. Immunohistochemical staining identified expressed TRPC4 in the glomeruli of mouse renal sections. The results of RT-PCR performed to distinguish between TRPC4-alpha and TRPC4-beta were consistent with expression of both isoforms in brain but with only TRPC4-alpha expression in MMC. These studies show that TRPC4-alpha may form the homotetrameric SOC in mouse mesangial cells.
开展了多项研究以鉴定负责小鼠系膜细胞（MMC）中储存性钙（Ca2+）内流的分子成分。由于之前已表明典型瞬时受体电位（TRPC）蛋白家族在多种细胞中包含Ca2+选择性和非选择性阳离子通道，因此我们运用分子方法和fura 2法筛选了TRPC1 - TRPC7，以确定它们作为系膜储存性钙（SOC）通道成分的参与情况。使用TRPC特异性引物和RT - PCR，我们发现培养的MMC含有TRPC1和TRPC4的mRNA，但不含有TRPC2、TRPC3、TRPC5、TRPC6和TRPC7的mRNA。MMC的免疫细胞化学染色显示TRPC1主要在细胞质中表达，而TRPC4主要在质膜上表达。通过TRPC4反义核酸以及fura 2比率法测量细胞内钙浓度（[Ca2+]i）来确定TRPC4在SOC中的作用。在持续存在毒胡萝卜素的情况下，当细胞外Ca2+从<10 nM增加到1 mM后，将[Ca2+]i的增加作为SOC进行测量。我们发现，降低TRPC4质膜表达的TRPC4反义核酸可使SOC受到83%的抑制。用乱序的TRPC4寡核苷酸孵育不影响SOC。免疫组织化学染色鉴定出小鼠肾切片肾小球中有TRPC4表达。为区分TRPC4 - α和TRPC4 - β而进行的RT - PCR结果表明，在脑中两种亚型均有表达，但在MMC中仅表达TRPC4 - α。这些研究表明，TRPC4 - α可能在小鼠系膜细胞中形成同四聚体的SOC。
