Properties of single Drosophila Trpl channels expressed in Sf9 insect cells.
Properties of single Drosophila Trpl channels expressed in Sf9 insect cells.
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Sf9 昆虫细胞中表达的单个果蝇 Trpl 通道的特性。
DOI：
10.1152/ajpcell.1997.272.1.c27
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Schilling,WP
中科院分区：
文献类型：
--
作者：
Kunze,DL;Sinkins,WG;Vaca,L;Schilling,WP
瞬时受体电位(Trp)样蛋白(TRPL)基因被认为编码果蝇光感受器细胞信号转导的重要离子通道。与这一假设一致的是，TRPL编码蛋白(TRPL)的异源表达与外向整流、非选择性阳离子电流的出现有关。在本研究中，在多角体蛋白启动子的控制下，从含有重组杆状病毒的重组杆状病毒感染的Sf9昆虫细胞中，单通道被记录在细胞贴壁、由内向外和由外向外的膜片上。单通道电流-电压关系在-100~+80 mV范围内呈线性关系，斜率电导为89~110 ps。开路的概率是电压敏感的，在正电位下增加有助于整个电池电流的向外整流特性。单通道1)在感染B2型缓激肽受体基因的重组杆状病毒感染的Sf9细胞和未感染的Sf9细胞中从未观察到；2)在感染后与TRPL全细胞电流同时出现；3)对Na+、Ca+和Ba2+是非选择性的；4)被1-2 mM的La3+和Gd3+(但不是10微米)阻断；5)被4-8 mM的镁离子阻断。斑块形成后，单个TRPL通道的活性随时间自发增加，同时表达B2受体和TRPL蛋白的细胞应用缓激肽后，其活性进一步增强。这些结果表明，这种单通道活性反映了TRPL蛋白的表达，并提供了确凿的证据，证明TRPL编码的非选择性阳离子通道与其在果蝇光转导中的作用一致。
The transient receptor potential (trp)-like (trpl) gene is thought to encode an ion channel important for signal transduction in Drosophila photoreceptor cells. Consistent with this hypothesis, heterologous expression of the trpl-encoded protein (Trpl) is associated with the appearance of an outwardly rectifying, nonselective cation current. In the present study, single channels were recorded in cell-attached, inside-out, and outside-out membrane patches from Sf9 insect cells infected with recombinant baculovirus-containing trpl cDNA under control of the polyhedrin promoter. The single-channel current-voltage relationship was linear from -100 to +80 mV with a slope conductance of 89-110 pS. The probability of opening was voltage sensitive, increasing at positive potentials contributing to the outwardly rectifying properties of the whole cell currents. The single channels 1) were never observed in Sf9 cells infected with recombinant baculovirus containing the B2 bradykinin receptor cDNA or in noninfected Sf9 cells; 2) appear at the same time postinfection as the Trpl whole cell current; 3) were nonselective with respect to Na+, Ca2+, and Ba2+; 4) were blocked by 1-2 mM La3+ and Gd3+ (but not 10 microM); and 5) were blocked by 4-8 mM Mg2+. The single Trpl channel activity increased spontaneously with time after patch formation, and the activity was further increased by application of bradykinin to cells expressing both the B2 bradykinin receptor and the Trpl protein. These results suggest that this single-channel activity reflects expression of the Trpl protein and provides conclusive evidence that trpl encodes a nonselective cation channel consistent with its proposed role in Drosophila phototransduction.