Stoichiometric Requirements for Trapping and Activation of CRAC Channels by STIM1 At ER-Plasma Membrane Junctions

Abstract

Store-operated Ca2+ entry is regulated by physical interactions of the ER Ca2+ sensor STIM1 and the CRAC channel protein Orai1. Recent studies support a diffusion-trap mechanism in which Ca2+ depletion from the ER causes STIM1 to accumulate at ER-plasma membrane (ER-PM) junctions, where its CRAC activation domain (CAD) binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. The ability of the CAD to cluster CRAC channels suggests that each channel contains multiple STIM1 binding sites, but the number of sites that must be occupied to trap and activate CRAC channels is unknown. By competing for a limited amount of STIM1, increasing levels of Orai1 are expected to reduce the number of STIM1s bound per channel until the minimum stoichiometry sufficient for channel trapping is reached. In HEK293 cells expressing a fixed amount of mCherry-STIM1 and increasing levels of GFP-Orai1, the junctional STIM1:Orai1 ratio reached a minimum of ∼0.3; thus, binding of 1-2 STIM1 appears sufficient to immobilize tetrameric CRAC channels at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current (ICRAC) was a highly nonlinear bell-shaped function of Orai1 expression, and the minimum STIM1:Orai1 stoichiometry for channel trapping failed to evoke significant activation. A simple cooperative gating model fitted to the data suggests that while STIM1 binding to a single site is sufficient to trap CRAC channels, four sites must be occupied to cause significant activation. This highly nonlinear behavior supports conclusions based on current fluctuation analysis (Prakriya and Lewis, J. Gen. Physiol. 128:373-86, 2006) that the slow development of whole-cell ICRAC after store depletion reflects the stepwise recruitment of individual channels from a silent to a high open-probability state as they enter ER-PM junctional sites.