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  • Book
    Paul J. Hoover.
    In excitable and non-excitable cells store-operated Ca2+ entry (SOCE) is a ubiquitous cellular mechanism that is critical for numerous processes such as gene expression, exocytosis, and metabolism. SOC channels are activated when stimulation of cell surface receptors initiates a signaling cascade leading to Ca2+ release from the endoplasmic reticulum (ER). ER Ca2+ release triggers activation of SOC channels in the plasma membrane (PM), the best characterized of which is the Ca2+ release-activated Ca2+ (CRAC) channel. An ER embedded protein, STIM1, acts as the ER Ca2+ sensor, and a tetramer of Orai1 subunits forms the pore of the active CRAC channel. After store depletion, STIM1 and Orai1 redistribute to sites of close apposition between the ER and the PM known as ER-PM junctions where they interact to produce Ca2+ entry. However, the fundamental question of how they interact to elicit Ca2+ entry was unknown. In our first line of inquiry, we show that the C-terminal polybasic domain of STIM1 targets STIM1 to ER-PM junctions independently of Orai1, that a cytosolic region of STIM1--CRAC activation domain (CAD)--binds directly to Orai1, that CAD is necessary and sufficient to cluster and activate CRAC channels, and that clustering and activation are functionally separable events. These results support a two-part diffusion trap mechanism for SOCE. After store depletion, STIM1 forms puncta independently of Orai1 underneath the PM where CAD is positioned to trap diffusing CRAC channels in the PM and induce a conformational change to activate channels. In the second line of inquiry we address the stoichiometric requirements for CRAC channel trapping and activation at ER-PM junctions. For this we developed a novel technique in single cells to control and measure the STIM1-Orai1 stoichiometry at ER-PM junctions while simultaneously recording channel activity. We find that binding of 1-2 STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions but this fails to evoke significant activation, while binding of 8 STIM1s causes maximal CRAC channel activity. We also find that the extent of fast Ca2+-dependent inactivation parallels STIM1-CRAC channel stoichiometry. The data are well described by a modified Monod-Wyman-Changeux model where STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity due to stabilization of the open state by STIM1. These results suggest a highly nonlinear dependence of CRAC channel activation on STIM:Orai stoichiometry.