Molecular Determinants of CRAC Channel Gating

Abstract

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels regulate numerous cellular functions including transcription, motility, and proliferation in many cell types. The discoveries of Orai1, the CRAC channel pore subunit, and STIM1, the ER Ca2+ sensor, have produced rapid progress in our understanding of the molecular features of the CRAC channel pore and the cellular events involved in channel activation. It is now known that following depletion of intracellular Ca2+ stores, STIM1 activates CRAC channels by interacting with the cytoplasmic N- and C-terminal domains of Orai1. Orai1 and Orai3 CRAC channels can additionally be activated in a store-independent fashion by the compound 2-APB. However, the molecular and structural mechanisms of STIM1- and 2-APB-mediated gating remain poorly understood. Using a combination of site-directed mutagenesis and cysteine accessibility analysis, we are probing the structural alterations in the pore that occur during STIM1- or 2-APB-mediated activation of Orai1 and Orai3 channels. Our data indicate that the pore of the CRAC channel changes significantly as it transitions from the closed to the open state. Specifically, STIM1 binding causes large modification of the pore architecture, giving rise to features classically associated with CRAC channels such as its narrow pore and high Ca2+ selectivity. Thus, in addition to serving as the ER Ca2+ sensor and activator of the CRAC channel, STIM1 appears to function as a channel subunit, modifying the structural features of the pore to confer its unique permeation profile in the active state.