Ca2+ release-activated Ca2+ (CRAC) channels are plasma membrane Ca2+-selective channels that open in response to depletion of endoplasmic reticulum Ca2+ stores. CRAC channels generate Ca2+ signals in many cell types and regulate a wide variety of effector cell responses including gene expression, cell proliferation, exocytosis, and motility, which in turn impact physiological processes ranging from lymphocyte activation to neural stem cell proliferation. Prototypic CRAC channels are formed by Orai1, the pore-forming subunit, and STIM1, which functions as the endoplasmic reticulum Ca2+ sensor and CRAC channel activator. The crystal structure of Drosophila Orai in the closed state has revealed that the CRAC channel has a hexameric subunit stoichiometry, with the transmembrane helices (TMs) arranged in concentric rings around a central pore formed by TM1. Despite the availability of this static structure, it remains unclear how STIM1 binding at the cytosolic side of Orai1 leads to pore opening. The overall goal for my research is to understand the molecular mechanisms of how human Orai1 channels open in response to STIM1 binding. The dOrai structure indicates that the N-terminus forms a cytosolic helical extension continuous with the pore-lining TM1 segment, and previous structure-function studies have implicated this region in STIM1 gating, but the precise contribution of this putative inner pore to ion conduction and gating remains elusive. Further, growing evidence indicates that in addition to the N-terminus, the Orai1 C-terminus is also essential for STIM-mediated gating. However, little is known of how STIM1-binding at the Orai1 C-terminus is transduced through the non pore-lining transmembrane helices (TMs 2-4) into pore opening. I hypothesize that STIM1 binding at the C-terminus transmits a conformational wave through the transmembrane helices of human Orai1 to activate the channel through rotation of TM1 and the N-terminal helical extension. Aim 1 will use our lab's newly developed approach of recording CRAC currents in excised patches to examine the role of the N-terminal extension in ion permeation and gating. Aim 2 will take advantage of gain-of-function mutations identified in the laboratory to study how STIM1 binding at the C-terminus is transduced through the TMs into pore opening. Together, the proposed experiments will help further our understanding of the molecular mechanisms of CRAC channel gating and lay the foundation for future efforts in developing mechanism-based drugs targeting CRAC channels.