Trebak Mohamed, Bird Gary St J, McKay Richard R, Putney James W
Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 2002 Jun 14;277(24):21617-23. doi: 10.1074/jbc.M202549200. Epub 2002 Apr 9.
Capacitative calcium entry or store-operated calcium entry in nonexcitable cells is a process whereby the activation of calcium influx across the plasma membrane is signaled by depletion of intracellular calcium stores. Transient receptor potential (TRP) proteins have been proposed as candidates for store-operated calcium channels. Human TRPC3 (hTRPC3), an extensively studied member of the TRP family, is activated through a phospholipase C-dependent mechanism, not by store depletion, when expressed in HEK293 cells. However, store depletion by thapsigargin is sufficient to activate hTRPC3 channels when expressed in DT40 avian B-lymphocytes. To gain further insights into the differences between hTRPC3 channels generated in these two expression systems and further understand the role of hTRPC3 in capacitative calcium entry, we examined the effect of two well characterized inhibitors of capacitative calcium entry, Gd3+ and 2-aminoethoxydiphenyl borane (2APB). We confirmed that in both DT40 cells and HEK293 cells, 1 microm Gd3+ or 30 microm 2APB completely blocked calcium entry due to receptor activation or store depletion. In HEK293 cells, 1 microm Gd3+ did not block receptor-activated hTRPC3-mediated cation entry, whereas 2APB had a partial (approximately 60%) inhibitory effect. Interestingly, store-operated hTRPC3-mediated cation entry in DT40 cells was also partially inhibited by 2APB, whereas 1 microm Gd3+ completely blocked store-operated hTRPC3 activity in these cells. Furthermore, the sensitivity of store-operated hTRPC3 channels to Gd3+ in DT40 cells was similar to the endogenous store-operated channels, with essentially 100% block of activity at concentrations as low as 0.1 microm. Finally, Gd3+ has a rapid inhibitory effect when added to fully developed hTRPC3-mediated calcium entry, suggesting a direct action of Gd3+ on hTRPC3 channels. The distinct action of these inhibitors on hTRPC3-mediated cation entry in these two cell types may result from their different modes of activation and may also reflect differences in basic channel structure.
非兴奋性细胞中的容量性钙内流或储存-操纵性钙内流是一个过程，即通过细胞内钙储存的耗竭来发出信号，激活跨质膜的钙内流。瞬时受体电位（TRP）蛋白已被提出作为储存-操纵性钙通道的候选者。人TRPC3（hTRPC3）是TRP家族中一个被广泛研究的成员，当在HEK293细胞中表达时，它是通过磷脂酶C依赖性机制激活的，而不是通过储存耗竭激活。然而，当在DT40禽类B淋巴细胞中表达时，毒胡萝卜素引起的储存耗竭足以激活hTRPC3通道。为了进一步深入了解在这两种表达系统中产生的hTRPC3通道之间的差异，并进一步了解hTRPC3在容量性钙内流中的作用，我们研究了两种特征明确的容量性钙内流抑制剂钆离子（Gd3+）和2-氨基乙氧基二苯硼烷（2APB）的作用。我们证实，在DT40细胞和HEK293细胞中，1微摩尔的Gd3+或30微摩尔的2APB都能完全阻断因受体激活或储存耗竭引起的钙内流。在HEK293细胞中，1微摩尔的Gd3+不能阻断受体激活的hTRPC3介导的阳离子内流，而2APB有部分（约60%）抑制作用。有趣的是，在DT40细胞中，储存-操纵性hTRPC3介导的阳离子内流也被2APB部分抑制，而1微摩尔的Gd3+完全阻断了这些细胞中储存-操纵性hTRPC3的活性。此外，DT40细胞中储存-操纵性hTRPC3通道对Gd3+的敏感性与内源性储存-操纵性通道相似，在低至0.1微摩尔的浓度下，基本上100%阻断活性。最后，当添加到完全发育的hTRPC3介导的钙内流中时，Gd3+具有快速抑制作用，表明Gd3+对hTRPC3通道有直接作用。这些抑制剂对这两种细胞类型中hTRPC3介导的阳离子内流的不同作用可能是由于它们不同的激活方式，也可能反映了基本通道结构的差异。
