Molecular basis of TRPC4 and TRPC5 channel activation

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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.