Fast Ca2+ Dependent Inactivation Of CRAC Channels Requires A Cytosolic Region Of STIM1

Abstract

The distinguishing biophysical features of mammalian Ca2+-release activated Ca2+ (CRAC) channels include high Ca2+ selectivity, small unitary conductance, and fast Ca2+-dependent inactivation on a millisecond time scale. Our previous studies of fast inactivation in Jurkat T cells suggested that Ca2+ binds to sites several nanometers from the intracellular mouth of the CRAC channel pore, possibly on the channel itself. The identification of STIM1 as the ER Ca2+ sensor and Orai1 as the pore-forming subunit of the CRAC channel has enabled studies of the molecular basis of activation and inactivation. We have recently identified a 107-residue cytosolic STIM1 fragment corresponding to the minimal STIM1 domain required for activation of the CRAC channel. The CRAC activation domain, or CAD, binds directly to Orai1 to activate CRAC current to the same mean level as wild-type STIM1, but while bypassing store depletion. CRAC currents were measured by whole-cell patch-clamp electrophysiology in HEK 293 cells coexpressing human Orai1 with a range of constructs derived from the cytosolic region of human STIM1. CAD-induced CRAC currents retain high Ca2+ selectivity, but surprisingly lack fast Ca2+-dependent inactivation, revealing a critical role for STIM1 in the inactivation gating process. Truncating STIM1 at the C-terminal end of CAD also yielded currents without fast inactivation in store-depleted cells. Extending CAD in the C-terminal direction partially reconstituted fast inactivation, but full reconstitution required both C- and N-terminal extensions of CAD. We conclude that a domain of STIM1 C-terminal to CAD is absolutely required for fast Ca2+-dependent inactivation of the CRAC channel. Elements of STIM1 N-terminal to CAD may enhance fast inactivation, possibly by increasing the local density of CRAC channels and Ca2+ influx, or by concentrating critical STIM1 domains near the inner pore mouth.