Regulation and Gating of Orai channels

In T-lymphocytes and mast cells, Ca2+ release activated Ca2+ (CRAC) channels represent the primary mechanism underlying long lasting elevations in cytosolic Ca2+ that is initated by depletion of endoplasmic reticulum (ER) Ca2+ stores. The main molecular components include the ER located STIM1 that senses luminal Ca2+ levels and Orai1 the pore-forming subunit of CRAC. Upon store-depletion STIM1 multimerises and redistributes to discrete punctae close to plasma membrane. Coupling of STIM1 to Orai1 initiates robust increase in CRAC currents. In this project, I aim to characterize molecular key domains critical for Orai1 gating and regulation. Therefore I will focus on a highly conserved hydrophobic, polybasic cluster in Orai1 that resembles a possible mulitple interaction site for either PIP 2 and PIP 3 , Calmodulin as well as cholesterol recognition and is conserved among the Orai family. I will use the standard expression system HEK293 for a combined approach of several techniques including molecular biology, biochemical techniques, electrophysiology, confocal FRET microscopy and Ca2+ imaging to examine the function of this domain in Orai1 regulation. On the other hand these studies will be complemented by high-throughput analysis of Orai1 mutants generated by random mutagenesis PCR enabling to identify additional unbiased regulatory domains in the STIM1/Orai1 signalling cascade. Furthermore, I will extend my studies to Orai2 and Orai3 to compare their regulatory domains as well as gating properties with those of Orai1. Hence, the results from my studies will provide deeper insight into the molecular determinants contributing to CRAC channel gating/regulation and are the pursiut for the search of therapeutical targets in a long term.