STIMULATION OF DROSOPHILA TRPL BY CAPACITATIVE CA2+ ENTRY
STIMULATION OF DROSOPHILA TRPL BY CAPACITATIVE CA2+ ENTRY
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电容性 CA2 输入对果蝇 TRPL 的刺激
DOI：
10.1042/bj3410041
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发表时间：
1999
影响因子：
4.1
通讯作者：
W. Schilling
中科院分区：
文献类型：
--
作者：
M. Estacion;W. Sinkins;W. Schilling
果蝇的Trp样蛋白（Trp-like protein，TrpL）是一种非选择性的阳离子通道，被认为是典型的受体激活通道。我们以前的研究表明，TrpL通道在Sf9细胞中表达时，不被内部Ca 2+储存耗尽激活。用Fura-2测定TrpL的阳离子内流，用细胞贴壁记录直接监测TrpL单通道活性，发现在胞外二价阳离子存在下，毒胡萝卜素诱导TrpL活性增加，Ca~（2+）> Sr~（2+）>> Ba~（2+）。La~（3+）可完全抑制内源性容性Ca~（2+）内流（CCE），但对TrpL无影响，表明TrpL通过CCE内流对Ca~（2+）具有反式刺激作用。TrpL具有两个推定的钙调素（CaM）结合结构域，命名为CBS-1和CBS-2。为了确定胞质游离Ca2+浓度（[Ca2 +] i）刺激TrpL可能需要哪个位点，创建了嵌合构建体，其中含有CBS-2的TrpL的C-末端结构域连接至人TrpC1，TrpC1是Trp的短同源物，其不被内部Ca2+储存的耗尽或[Ca2 +] i的升高激活。这种功能获得性突变体，命名为TrpC1-TrpL，表现出通过CCE的Ca 2+内流的反式刺激。在凝胶覆盖实验中对CaM结合的检查表明，TrpL和TrpC1-TrpL嵌合体结合CaM，但TrpC1或缺乏CBS-2的TrpL的截短版本不结合。这些结果表明，只有CBS-2结合天然TrpL中的CaM，并且含有该位点的C-末端结构域对于CCE对TrpL的反式刺激是重要的。
Trp-like protein (TrpL, where Trp is transient receptor-potential protein) of Drosophila, a non-selective cation channel activated in photoreceptor cells by a phospholipase C-dependent mechanism, is thought to be a prototypical receptor-activated channel. Our previous studies showed that TrpL channels are not activated by depletion of internal Ca2+ stores when expressed in Sf9 cells. Using fura-2 to measure cation influx via TrpL, and cell-attached patch recordings to monitor TrpL single-channel activity directly, we have found a thapsigargin-induced increase in TrpL activity in the presence of extracellular bivalent cations, with Ca2+>Sr2+>> Ba2+. The increase in TrpL channel activity was blocked by concentrations of La3+ that completely inhibited endogenous capacitative Ca2+ entry (CCE), but have no effect on TrpL, suggesting that TrpL exhibits trans-stimulation by cation entry via CCE. TrpL has two putative calmodulin (CaM)-binding domains, designated CBS-1 and CBS-2. To determine which site may be required for stimulation of TrpL by the cytosolic free Ca2+ concentration ([Ca2+]i), a chimaeric construct was created in which the C-terminal domain of TrpL containing CBS-2 was attached to human TrpC1, a short homologue of Trp that is not activated by depletion of internal Ca2+ stores or by a rise in [Ca2+]i. This gain-of-function mutant, designated TrpC1-TrpL, exhibited trans-stimulation by Ca2+ entry via CCE. Examination of CaM binding in gel-overlay experiments showed that TrpL and the TrpC1-TrpL chimaera bound CaM, but TrpC1 or a truncated version of TrpL lacking CBS-2 did not. These results suggest that only CBS-2 binds CaM in native TrpL and that the C-terminal domain containing this site is important for trans-stimulation of TrpL by CCE.