The Study Of Auto-inhibitory Mechanism Of STIM1in CRAC Channel

Calcium (Ca2+) influx is required for the activation and function of all cells in the immune system. It is mediated mainly by store-operated Ca2+entry (SOCE) through Ca2+release-activated Ca2+(CRAC) channels located in the plasma membrane. CRAC channels are composed of ORAI proteins in the plasma membrane and are activated by stromal interaction molecules (STIM) located in the membrane of endoplasmic reticulum (ER). Orai molecules that form Ca2+-selective pore of CRAC channel have three subtypes which are Orai1, Orai2and Orai3, of which Orai1plays the most important role. STIM proteins that can sense the change of the intraluminal Ca2+concentration in the ER and active Orai molecules have two subtypes which are STIM1and STIM2, of which STIM1is the crucial one. The CRAC channel that is composed with Orail and STIM1is the major pathway that mediate Ca2+influx in immune and non-excitable cells.In the resting state, STIM1is uniformly distributed in the ER membrane and the calcium concentration of cytosol is at a low level (～100nM). A decrease of Ca2+concentration in the ER induces STIM1multimerization and translocation into puncta close to the plasma membrane where they bind to and activate Orai channel, leading to the vast Ca2+entry. In the process of immunity, the activation of STIM1is crucial for immune cells carrying out the immune effects.Under physiological conditions, the intra-molecular auto-inhibitions in STIM1-CT (STIM1-C terminus) and STIM1-NT (STIM1-N terminus) play essential roles in keeping STIM1in an inactive state. The mechanism of auto-inhibition in the STIM1-NT was so clear because the atomic crystal structure has been solved. However, the auto-inhibitory mechanism of STIM1-CT is still unclear. In this study, upon the different behaviors between CeSTIMl and hSTIMl in resting cells and the different amino acid sequence between the two proteins, we first predicted a short inhibitory domain (310-317) in human STIM1that might determine the different localizations of human and C. elegans STIM1in resting cells. Next, we confirmed the prediction and further identified an aromatic amino acid residue Y316that played a crucial role in maintaining STIM1in a closed conformation in quiescent cells. Full length STIM1-Y316A formed constitutive clusters near the plasma membrane and activated the CRAC channel in the resting state when co-expressed with Orai1. And the introduction of Y316A mutation caused the higher-order oligolization of in vitro purified STIM1fragment containing both auto-inhibitory domain and CAD domain. We proposed that the Y316residue may be involved in the auto-inhibitory mechanism of STIM1-CT in the quiescent state. This inhibition could be achieved either by interacting with the CAD domain using hydrogen and/or hydrophobic bonds or by intermolecular interaction using repulsive forces, which maintained a dimeric STIM1.