Calcium

STIM1 and calmodulin interact with Orai1 to induce Ca2 -dependent inactivation of CRAC channels
Franklin M. Mullinsa,b,1, Chan Young Parkc,1, Ricardo E. Dolmetschc,2, and Richard S. Lewisb,2
Departments of aPathology, bMolecular and Cellular Physiology, and cNeurobiology, Stanford University School of Medicine, Stanford, CA 94305 Edited by Richard W. Aldrich, University of Texas, Austin, TX,and approved July 22, 2009 (received for review June 17, 2009)

Ca2 -dependent inactivation (CDI) is a key regulator and hallmark of the Ca2 release-activated Ca2 (CRAC) channel, a prototypic store-operated Ca2 channel. Although the roles of the endoplasmic reticulum Ca2 sensor STIM1 and the channel subunit Orai1 in CRAC channel activation are becoming well understood, the molecular basis of CDIremains unclear. Recently, we defined a minimal CRAC activation domain (CAD; residues 342– 448) that binds directly to Orai1 to activate the channel. Surprisingly, CADinduced CRAC currents lack fast inactivation, revealing a critical role for STIM1 in this gating process. Through truncations of full-length STIM1, we identified a short domain (residues 470 – 491) C-terminal to CAD that is required forCDI. This domain contains a cluster of 7 acidic amino acids between residues 475 and 483. Neutralization of aspartate or glutamate pairs in this region either reduced or enhanced CDI, whereas the combined neutralization of six acidic residues eliminated inactivation entirely. Based on bioinformatics predictions of a calmodulin (CaM) binding site on Orai1, we also investigated a role for CaM inCDI. We identified a membrane-proximal N-terminal domain of Orai1 (residues 68 –91) that binds CaM in a Ca2 -dependent manner and mutations that eliminate CaM binding abrogate CDI. These studies identify novel structural elements of STIM1 and Orai1 that are required for CDI and support a model in which CaM acts in concert with STIM1 and the N terminus of Orai1 to evoke rapid CRAC channelinactivation.
calcium ion channel gating store-operated calcium entry patch-clamp calcium-binding proteins

are activated by a minimal soluble fragment of STIM1 [amino acids 342–448, known as the CRAC activation domain (CAD)], revealing a critical role for STIM1 in CDI (13). Consistent with such a role, a recent report showing that the extent of inactivation correlates with the STIM1/Orai1 transfectionratio further suggests that the number of STIM1 proteins bound to a CRAC channel influences its ability to inactivate (20). Another recent study identified a STIM1 domain (amino acids 475–485) that reduces CRAC channel activity, but its effect on CDI was not examined (21). In this study, we address the roles of STIM1 and CaM in the CDI of CRAC channels. Using a combination of serial truncations andmutagenesis, we identify a negatively charged region of STIM1 that is required for inactivation. We also identify a region in the N terminus of Orai1 that binds CaM in a Ca2 -dependent manner and demonstrate that CaM binding is required for CDI to occur. These results reveal the structural underpinnings of CRAC channel inactivation, identify Orai1 as a CaM-binding protein, and extend the knownroles of STIM1 from those of Ca2 sensor and activating ligand to that of a CRAC channel subunit that controls inactivation gating in response to local Ca2 accumulation. Results
CDI of Heterologously Expressed CRAC Channels. In HEK293 cells

tore-operated Ca2 channels provide a major route for receptor-stimulated Ca2 entry in nonexcitable cells (1). The Ca2 -release activated Ca2 (CRAC) channel, thebestcharacterized store-operated channel, is essential for generating Ca2 signals that drive the activation of T lymphocytes, mast cells, and platelets (2–4). CRAC channel activity is shaped by the combination of store-dependent activation and Ca2 -dependent inactivation (CDI) processes. The mechanisms linking depletion of Ca2 from the endoplasmic reticulum (ER) to activation of the CRAC...