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Summary: STIM1 in the endoplasmic reticulum and CRACM1 in the plasma membrane are essential molecular components for controlling the store-operated CRAC current . CRACM1 proteins multimerize and bind STIM1 , and the combined overexpression of STIM1 and CRACM1 reconstitutes amplified CRAC currents . Mutations in CRACM1 determine the selectivity of CRAC currents, demonstrating that CRACM1 forms the CRAC channel''s ion-selective pore , but the CRACM1 homologs CRACM2 and CRACM3 are less well characterized . Here, we show that both CRACM2 and CRACM3, when overexpressed in HEK293 cells stably expressing STIM1, potentiate ICRAC to current amplitudes 15–20 times larger than native ICRAC. A nonconducting mutation of CRACM1 (E106Q) acts as a dominant negative for all three CRACM homologs, suggesting that they can form heteromultimeric channel complexes. All three CRACM homologs exhibit distinct properties in terms of selectivity for Ca2+ and Na+, differential pharmacological effects in response to 2-APB, and strikingly different feedback regulation by intracellular Ca2+. Each of the CRAC channel proteins'' specific functional features and the potential heteromerization provide for flexibility in shaping Ca2+ signals, and their characteristic biophysical and pharmacological properties will aid in identifying CRAC-channel species in native cells that express them. [Copyright &y& Elsevier]

Summary: Receptor-mediated Ca2+ release from the endoplasmic reticulum (ER) is often followed by Ca2+ entry through Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca2+ sensor and CRACM1 (Orai1; ) as the putative store-operated Ca2+ channel. Overexpression of both proteins is required to reconstitute CRAC currents (ICRAC; ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na+, Ba2+, and Sr2+ permeation relative to Ca2+. Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca2+ binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca2+ ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca2+-selective CRAC-channel pore. [Copyright &y& Elsevier]