Abstract：
Physiological activation by light of the Drosophila TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP2) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. In this study, we re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells. Unlike previous studies, we added OAG into the cytosol via a patch-clamp pipette and observed robust activation of the expressed TRPL channels. However, TRPL channel activation was much slower than the physiologically activated TRPL by light. Therefore, we used a picosecond-fast optically activated DAG analogue, OptoDArG. Inactive OptoDArG was added into the intracellular solution with the patch-clamp pipette, and it slowly accumulated on the surface membrane of the recorded HEK cell in the dark. A fast application of intense UV light to the recorded cell resulted in a robust and relatively fast TRPL-dependent current that was greatly accelerated by the constitutively active TRPLF557I pore-region mutation. However, this current of the mutant channel was still considerably slower than the native light-induced TRPL current, suggesting that DAG alone is not sufficient for TRPL channel activation under physiological conditions.
摘要：
果蝇TRP和TRP样（TRPL）通道的光的生理激活需要磷脂酶Cβ（PLC）的激活。磷脂酰肌醇4,5,二磷酸酯(PIP2)的PLC水解是目前尚不清楚的光活化的关键步骤,而PLC产生的二酰甘油（DAG）似乎参与。在这项研究中,我们重新检查了DAG类似物1-油酰基-2-乙酰基-sn-甘油（OAG）激活HEK细胞中表达的TRPL通道的能力。与以往的研究不同,我们通过膜片钳移液管将OAG添加到细胞溶质中,并观察到表达的TRPL通道的强烈激活.然而,TRPL通道激活比生理激活的TRPL慢得多。因此,我们使用了皮秒快的光激活DAG类似物，OptoDArG.用膜片钳移液管将非活性OptoDArG加入细胞内溶液中，它在黑暗中缓慢积累在记录的HEK细胞的表面膜上。向记录的细胞快速施加强UV光导致稳健且相对快速的TRPL依赖性电流,其通过组成型活性TRPLF557I孔区域突变而大大加速。然而,突变通道的这种电流仍然比天然光诱导的TRPL电流慢得多，提示在生理条件下单独DAG不足以激活TRPL通道。
