Molecular basis of TRPC4 and TRPC5 channel activation

TRPC 4和TRPC 5通道是瞬时受体电位经典（TRPC）通道家族的非选择性钠、钙离子通道。它们的激活是受体操纵的和磷脂酶C依赖的。最近，我们发现TRPC 4和TRPC 5通道对第二信使甘油二酯（DAG）敏感，类似于所有其他功能性TRPC通道。这一发现导致DAG敏感和不敏感亚家族中TRPC通道分类的范式变化。我们发现TRPC 4/5通道的DAG敏感性受到接头蛋白NHERF 1和NHERF 2的严格调节。此外，我们得到了C-末端构象变化的第一个证据，这与TRPC 5通道活性的动力学和幅度有关。预期研究项目的目的是阐明TRPC 4和TRPC 5通道激活与TRPC 6通道激活相比的分子基础。为此，我们将首先使用分子内动态Förster共振能量转移（FRET）的技术，以广泛和部分的C-末端内的C-末端和相对于两个细胞内的环的构象变化的特点。此外，进行定点氨基酸交换，我们的目标是定位蛋白质结构域，这是必需的DAG敏感性。此外，蛋白激酶C磷酸化对TRPC 5和TRPC 6通道激活和失活的影响将使用光开关DAG衍生物进行详细分析，该衍生物不受洗出和洗入效应的影响，从而允许测定快速激活和失活动力学。另一个目的是鉴定有效和选择性TRPC 4/5通道激活剂（-）-Englerin A的结合位点。利用基于结构的药物设计产生的新型（-）-Englerin A衍生物，我们计划通过进行特异性氨基酸交换来表征（-）-Englerin A的潜在结合位点，这将有助于阐明结合位点处的空腔和特异性相互作用。这些发现有助于理解TRPC 4/5和TRPC 6通道的不同生理和病理生理功能。

TRPC4 and TRPC5 channels are unselective sodium and calcium permeable cation channels, which belong to the transient receptor potential classical (TRPC) channel family. Their activation is receptor-operated and phospholipase C-dependent. Recently, we identified that TRPC4 and TRPC5 channels are sensitive to the second messenger diacylglycerol (DAG) similar to all other functional TRPC channels. This finding leads to a paradigm change in the classification of TRPC channels in DAG sensitive and insensitive subfamilies. We found that the DAG sensitivity of TRPC4/5 channels is tightly regulated by the adapter proteins NHERF1 and NHERF2. Moreover, we got first evidence for conformational changes of the C-terminus, which correlate to the TRPC5 channel activity with regard to dynamics and magnitude. The aim of the intended research project is the elucidation of the molecular basis of the TRPC4 and TRPC5 channel activation compared to the TRPC6 channel activation. For this, we will first use the technique of intramolecular dynamic Förster resonance energy transfer (FRET) to extensively and sectionally characterize C-terminal conformational changes within the C-terminus and relative to the two intracellular loops. In addition, performing site directed amino acid exchanges we aim to localize protein domains, which are essential for the DAG sensitivity. Moreover, the influence of protein kinase C phosphorylation on TRPC5 and TRPC6 channel activation and inactivation will be analyzed in detail using photoswitchable DAG derivates, which are free from wash out and wash in effects and thus allow for the determination of fast activation and inactivation kinetics. Another aim is the identification of the binding site for the potent and selective TRPC4/5 channel activator (-)-Englerin A. Using novel (-)-Englerin A derivates, which are generated by structure-based drug design, we plan to characterize the potential binding site for (-)-Englerin A by performing specific amino acid exchanges, which will help to elucidate the cavitary and the specific interactions at the binding site. These findings might help to understand the different physiological and pathophysiological functions of TRPC4/5 and TRPC6 channels.