Gutorov Rita, Katz Ben, Peters Maximilian, Minke Baruch
Faculty of Medicine, Institute for Medical Research Israel-Canada (IMRIC), Edmond and Lily Safra Center for Brain Sciences (ELSC), The Hebrew University, Jerusalem, Israel.
Faculty of Medicine, Institute for Medical Research Israel-Canada (IMRIC), Edmond and Lily Safra Center for Brain Sciences (ELSC), The Hebrew University, Jerusalem, Israel.
J Biol Chem. 2024 Jan;300(1):105484. doi: 10.1016/j.jbc.2023.105484. Epub 2023 Nov 21.
Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-β-cyclodextrin (MβCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein-coupled phospholipase Cβ (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings. Here, we studied the effects of MβCD on Drosophila phototransduction using electrophysiology while fluorescently monitoring PIP2 hydrolysis, aiming to examine the effects of sterol modulation on PIP2 hydrolysis and the ensuing light-response in the native system. Incubation of photoreceptor cells with MβCD dramatically reduced the amplitude and kinetics of the TRP/TRPL-mediated light response. MβCD also suppressed PLC-dependent TRP/TRPL constitutive channel activity in the dark induced by mitochondrial uncouplers, but PLC-independent activation of the channels by linoleic acid was not affected. Furthermore, MβCD suppressed a constitutively active TRP mutant-channel, trpP365, suggesting that TRP channel activity is a target of MβCD action. Importantly, whole-cell voltage-clamp measurements from photoreceptors and simultaneously monitored PIP2-hydrolysis by translocation of fluorescently tagged Tubby protein domain, from the plasma membrane to the cytosol, revealed that MβCD virtually abolished the light response when having little effect on the light-activated PLC. Together, MβCD uncoupled TRP/TRPL channel gating from light-activated PLC and PIP2-hydrolysis suggesting the involvement of distinct nanoscopic lipid domains such as lipid rafts and PIP2 clusters in TRP/TRPL channel gating.
甾醇是疏水分子，已知其能使信号膜蛋白聚集在脂筏中，而甲基-β-环糊精（MβCD）一直是调节膜甾醇含量以研究其对膜蛋白（包括瞬时受体电位（TRP）通道）影响的主要工具。果蝇的光敏感TRP通道在G蛋白偶联磷脂酶Cβ（PLC）级联反应的下游被激活。在光转导过程中，PLC是一种水解磷脂酰肌醇4,5-二磷酸（PIP2）生成二酰基甘油、肌醇三磷酸和质子的酶，导致TRP和TRP样（TRPL）通道开放。在此，我们利用电生理学方法研究了MβCD对果蝇光转导的影响，同时通过荧光监测PIP2水解，旨在研究甾醇调节对PIP2水解以及天然系统中后续光反应的影响。用MβCD孵育光感受器细胞会显著降低TRP/TRPL介导的光反应的幅度和动力学。MβCD还抑制了线粒体解偶联剂在黑暗中诱导的PLC依赖性TRP/TRPL组成型通道活性，但亚油酸对通道的PLC非依赖性激活不受影响。此外，MβCD抑制了组成型活性TRP突变通道trpP365，表明TRP通道活性是MβCD作用的靶点。重要的是，通过对光感受器进行全细胞电压钳测量，并同时通过荧光标记的Tubby蛋白结构域从质膜向细胞质的转位来监测PIP2水解，结果显示MβCD在对光激活的PLC影响很小的情况下几乎消除了光反应。总之，MβCD使TRP/TRPL通道门控与光激活的PLC和PIP2水解解偶联，这表明不同的纳米级脂质结构域（如脂筏和PIP2簇）参与了TRP/TRPL通道门控。
