CRACM1 Multimers Form the Ion-Selective Pore of the CRAC Channel

Abstract

Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). 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(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). 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 Ca(2+) 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 Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.

Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 Regulate Ca2+ Influx by Altered Oligomerization of STIM1 and by Destabilization of the Ca2+ Channel Orai1

“…To separate effects on kinetics from effects on stoichiometry, as well as to address a possible mechanism for the STIM1 DQinduced reduction in Orai1 protein, we co-transfected STIM1 WT or STIM1 DQ with the pore mutant of Orai1 E106D (33). Orai1 E106D mutants show IP 3 -induced currents, but because of mutation of the selectivity filter, they lack Ca 2ϩ selectivity.…”

Voltage Gating at the Selectivity Filter of the Ca2+ Release-activated Ca2+ Channel Induced by Mutation of the Orai1 Protein

“…Several groups simultaneously identified Glu 106 (Glu 180 in Drosophila) as a residue that coordinates the Ca 2ϩ binding to the selectivity filter of the CRAC channel (24,25). 3 These experiments were repeated by another group using our Orai1 E106D construct (26). All these results provided unequivocal evidence that Orai1 is a poreforming subunit of the CRAC channel.…”

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

“…Overexpression of STIM1 with Orai1 results in very large CRAClike currents in Drosophila cell lines, Jurkat T-cells, RBL cells, and HEK293 cells (Mercer et al, 2006;Peinelt et al, 2006;Soboloff et al, 2006;Zhang et al, 2006). Recent studies have shown that Orai1 forms multimers and that targeted mutations in Orai1 alter the conductance properties of the CRAC channel Vig et al, 2006a;Yeromin et al, 2006;Gwack et al, 2007b). In order for ER-localized STIM1 to activate PM Orai1 channels, the two proteins must move to a common site and interact (Lewis, 2007;Putney, 2007b).…”

“…Recent work has shown that STIM1 and Orai1 are in membrane contact points between the ER and PM and that movement of a complex containing the two proteins requires at least 10 nm between the two membranes (Varnai et al, 2007). The current consensus is that STIM1 senses the depletion of Ca 2ϩ from ER stores and then moves to sites of ER-PM apposition where interactions between STIM1 and Orai1 cause the opening of CRAC channels composed of Orai1 protein subunits Vig et al, 2006a;Wu et al, 2006;Feske, 2007;Hogan and Rao, 2007;Lewis, 2007;Putney, 2007a;Varnai et al, 2007).We used confocal imaging techniques to visualize the dynamic movement of STIM1 and Orai1 after T-cell activation. We were particularly interested in demonstrating close interactions between fluorescently tagged versions of STIM1 and Orai1 using the technique of Fö rster resonance energy transfer (FRET).…”

“…In activated cells expressing both STIM1-CFP and Orai1-YFP, both proteins were present in puncta near the stimulatory surface ( Figure 1C). The merge panel shows extensive colocalization of the two proteins in these clusters.Although many studies have shown colocalization of STIM1 and Orai1 and the two proteins can be coimmunoprecipitated (Vig et al, 2006a;Yeromin et al, 2006;Gwack et al, 2007b), a direct interaction after store depletion assessed by FRET has only been reported recently in HEK293 cells (Muik et al, 2008). We used FRET techniques to determine whether the two proteins interact in the puncta induced by TCR stimulation.…”