Store operated Ca2+ entry (SOCE) is a ubiquitous process in nonexcitable cells important for receptor signaling in hematopoietic and other cell types. The ERtransmembrane Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel Orai1 are the two essential protein components of SOCE. To further understand the mechanism by which STIM1 and Orai1 facilitate SOCE, I have characterized bimolecular and supramolecular electrostatic interactions involving these proteins, using an imagingbased fluorescence resonance energy transfer (FRET) assay in RBL mast cells. Using this assay I initially identified positively-charged small molecule inhibitors of the STIM1-Orai1 interaction. Based on this information I hypothesized that these small molecules could be binding to and disrupting the C-terminal acidic coiled-coil of Orai1. Mutation of these acidic residues in Orai1 reduced its association with STIM1 and caused constitutive clustering of Orai1 at the plasma membrane. I then identified a short polybasic sequence in the Ca2+ activating domain (CAD) of STIM1 that binds to this acidic region of Orai1. Mutation of this three amino acid basic sequence prevented association with wild type Orai1, but not with the Orai1 coiled-coil mutant. Despite the residual association between the Orai1 and STIM1 mutants, they cannot initiate SOCE, suggesting that the polybasic sequence in STIM1 and the acidic coiled-coil of Orai1 are important for Ca2+ gating. Using multiple isoforms of type I phosphoinositide-5-kinase (PI5KI) and lipid domain targeted inositol-5-phosphatases, I found that the STIM1-Orai1 interaction has a dual dependence on two pools of of phosphoinositide-(4,5)-bisphosphate (PIP2) in the plasma membrane that are distinguishable by their association with detergent resistant membranes and detergent solubilized membranes. These correspond to distinctive ordered lipid subregions and disordered lipid subregions in the membrane, respectively. Deletion of an N-terminal polyarginine sequence on Orai1 or a Cterminal polylysine sequence on STIM1 interferes with these selective interactions with PIP2 pools localized to these subregions. Based on these findings, I propose a model in which Orai1 must translocate between functionally distinct membrane domains in a PIP2 dependent fashion, to engage STIM1 associated with PIP2 in ordered lipid subregions of the membrane.