Dissecting the mechanism of STIM coupling to Orai

Abstract

Store-operated Ca2+ entry (SOCE) triggered by the depletion of endoplasmic reticulum (ER) luminal Ca2+ stores is a major Ca2+ entry pathway in non-excitable cells and is essential in physiological Ca2+ signaling and homeostasis. STIM proteins are sensors of ER luminal Ca2+, which translocate to ER-plasma membrane (PM) junctional regions to activate the family of Orai channels mediating Ca2+ entry. This study is focused on dissecting the mechanism of STIM interacting with Orai. A powerful modifier of SOCE, 2-aminoethoxydiphenyl borate (2-APB) is utilized. First, the action of 2-APB on the mammalian Orai homologues are characterized using the DT40 STIM knockout cells. 50 ìM 2-APB directly activates Orai3 but not Orai1 or Orai2. Second, while it stimulates the STIM2-mediated constitutive Ca2+ entry through Orai, 2-APB also induces the cytosolic STIM C-terminus fragments to translocate to the PM and activate Orai1. These data reveal 50 ìM 2-APB enhances STIM-Orai coupling. Further, this enhanced binding of STIM and Orai leads to a conformational change within the STIM-Orai complex, which is possibly the underlying mechanism for the 50 ìM 2-APB inhibitory effect on SOCE. Finally, six residues (344-349) at the N-terminus of the STIM-Orai activation region (SOAR) prove to be critical for this inhibitory action. These same six amino acid region also constitutes an ancillary Orai binding site within SOAR, in addition to the main polybasic region. The deletion of this ancillary site abolishes the ability of SOAR to bind to and activate Orai1, but can be compensated for by the STIM-Orai binding enhancing effect of 50 ìM 2-APB. The majority of STIM1 is located on the ER membrane, while a small proportion of STIM1 is on the PM. Using an extracellularly applied STIM1 antibody, the PM STIM1 can be aggregated to exert an influence on the ER STIM1. Although the PM STIM1 is not obligatory for STIM1-mediated Orai activation, it nevertheless may have a functional presence in the PM. Lastly, a regulatory link between voltage-gated Ca2+ channels (Cav channels) and the STIM proteins is established. After activation by store depletion, STIM strongly suppresses the Cav1.2 channels. There is a biochemical interaction between STIM1 and the Cav1.2 pore subunit á1C. This inhibitory effect is independent of Orai1 activation. Hence, STIM1 interacts with and reciprocally controls two major Ca2+ channels.