果蝇TRP离子通道C端一个新钙调蛋白结合位点的鉴定和分析

瞬时受体电位(TRP)通道是一类钙离子透过性的阳离子通道蛋白家族，参与了视觉、味觉、温度感受等重要的生物学过程。之前的研究表明，钙离子既能够正反馈也能够负反馈地调节瞬时受体电位通道的活性，而这种调节可能是通过钙调蛋白（calmodulin，CaM）与TRP通道的相互作用来进行的。为了阐明这一调控机制，我们首先需要对钙调蛋白与瞬时受体电位通道之间的相互作用进行详细的生化研究。在此项研究中，通过大肠杆菌表达系统，表达和纯化了果蝇瞬时受体电位通道羧基末端不同长短的蛋白片段，并发现了一个新的钙调蛋白结合位点。通过快速蛋白液相色谱、静态光散射以及等温量热滴定技术，鉴定了这一钙调蛋白结合位点与果蝇瞬时受体电位通道之间的相互作用，发现它们在钙离子依赖的条件下，可以形成亲和力非常强的稳定的蛋白复合物（解离常数在01～1微摩尔范围）。此外，通过合成多肽的方法，鉴定了果蝇瞬时受体电位通道913～939片段为该钙调蛋白结合位点的核心区域。最后，通过突变实验，进一步明确了果蝇瞬时受体电位通道922位的酪氨酸以及923位的缬氨酸为其钙调蛋白结合位点的关键氨基酸。总而言之，本研究发现和鉴定了果蝇瞬时受体电位通道上一个新的钙依赖的钙调蛋白结合位点，这一发现将为研究瞬时受体电位通道的体内功能提供生化基础，为阐明钙离子通过钙调蛋白调节瞬时受体电位通道的分子机制做出贡献。

Identification and Characterization of a New Calmodulin Binding Site at the C-terminus of Drosophila TRP Channel

The transient receptor potential (TRP) channels are a family of Ca2+ permeable cation channels, which are involved in many important biological processes such as vision, taste, temperature sensation. Previous studies showed that Ca2+ can bidirectionally regulate the activities of the TRP channel, which may be mediated by the binding of calmodulin (CaM) to the TRP channel. To unravel this regulation mechanism, the interaction between the CaM and the TRP channel should be first biochemically investigated in detail. In this study, by using bacterial expression system, we expressed and purified different fragments of the Drosophila TRP C terminus and identified a novel strong Ca2+ dependent CaM binding site (CBS). The interaction between this novel CBS and CaM were characterized by the fast protein liquid chromatography (FPLC), size exclusion chromatography coupled with multi angle laser light scattering (SEC MALS) and isothermal titration calorimetry (ITC), which showed they formed a stable protein complex in a Ca2+ dependant manner and the binding affinity was very strong (Kd in sub micromolar range). In addition, by using the synthetic peptide, the TRP913～939 fragment was further identified as the core region of the CBS. Finally, by mutagenesis studies, we defined the Tyr922 and Val923 as the critical residues involved in the Ca2+- CaM binding. In summary, our studies discover and characterize a new Ca2+ dependent CBS in the Drosophila TRP channel, which will provide the biochemical basis for the in vivo functional studies of TRP channel and the understanding of the Ca2+ mediated regulation mechanism of the TRP channel through CaM.