| Store-operated Ca2+ entry is initiated by depletion of Ca2+ from the endoplasmic reticulum (ER). Upon depletion, the ER Ca2+ sensor STIM1 oligomerizes, promoting its accumulation at junctions between the ER and plasma membrane (PM), where it recruits and activates the Ca2+ release-activated Ca2+ (CRAC) channel subunit, Orai1. This dissertation focuses on the mechanism of STIM1 oligomerization and the steady-state dynamics of STIM1/Orai1 complexes at ER-PM junctions. The introductory chapter presents a brief review of the CRAC channel activation mechanism. The second chapter defines the roles of the cytoplasmic domains in STIM1 oligomerization using biochemical and fluorescence microscopy techniques. While the membrane-proximal coiled coil is sufficient for store-independent self-association of STIM1, the downstream CRAC activation domain (CAD), which is responsible for binding and activating Orai1, is required for store-dependent STIM1 oligomerization. Point mutations in the putative coiled coil within the CAD can promote constitutive oligomerization and clustering of STIM1, with or without activation of channels. The third chapter explores the steady-state dynamics of STIM1 and Orai1 during sustained Ca2+ store depletion. Photobleaching and photoactivation experiments showed that STIM1 and Orai1 at ER-PM junctions continuously and rapidly exchange with the surrounding diffuse protein pool. The relatively low affinity of STIM1 and Orai1 for these sites has important implications for the regulation of steady-state channel activity and the speed of deactivation of Ca 2+ influx upon store refilling. |
