Orai1 channel dynamics critical for pore opening

Changes in cellular Ca2+ concentration play a universal role in a wide variety of cellular processes and human body functions. Abnormal Ca2+ levels in the cell can lead to diseases such as severe immune deficiency or muscle dysfunction. A main Ca2+ entry pathway into the cell is Ca2+ release-activated Ca2+ (CRAC) ion channel, which is crucial for maintenance of immune response for example. Since the discovery of the molecular determinants (STIM, Orai) of the CRAC channel 15 years ago, a deep understanding of their structure and function has been achieved. Nevertheless, several aspects, in particular, the dynamic resolution of the structure/function relationship of this Ca2+ ion channel has so far not been achieved, partly due to limitations of traditional techniques. The focus of this project is to characterize critical communication sites and the dynamics of individual transmembrane (TM) domains that control pore opening, especially at the periphery of the channel, which are currently still poorly understood. To investigate the interplay of decisive checkpoints in the CRAC channel complex, we will use a combined approach of conventional biophysical methods together with the currently emerging genetic code expansion technology to provide a deep understanding of protein dynamics depending on a single amino acid in the living cell. In particular, we will transfer light-sensitivity to individual building blocks of the CRAC channel protein to gain novel insights into the structure/function relationship at the atomic level in vivo. The main novelty of this proposal is that we are developing a series of light-sensitive Orai1 mutants to unravel unprecedented dynamics of the Orai1 pore opening mechanism. These tools will be valuable in the long term for resolving intra- and intermolecular binding interfaces with amino acid resolution and to control downstream signaling processes, which goes significantly beyond previous research. In the long term, our novel results will provide a new basis for the development of future target-specific therapeutic strategies.