Graded activation of CRAC channel by binding of different numbers of STIM1 to Orai1 subunits

Abstract

The Ca 2+ release-activated Ca 2+ (CRAC) channel pore is formed by Orai1 and gated by STIM1 after intracellular Ca 2+ store depletion. To resolve how many STIM1 molecules are required to open a CRAC channel, we fused different numbers of Orai1 subunits with functional two-tandem cytoplasmic domains of STIM1 (residues 336-485, designated as S domain). Whole-cell patch clamp recordings of these chimeric molecules revealed that CRAC current reached maximum at a stoichiometry of four Orai1 and eight S domains. Further experiments indicate that two-tandem S domains specifically interact with the C-terminus of one Orai1 subunit, and CRAC current can be gradually increased as more Orai1 subunits can interact with S domains or STIM1 proteins. Our data suggest that maximal opening of one CRAC channel requires eight STIM1 molecules, and support a model that the CRAC channel activation is not in an "all-or-none" fashion but undergoes a graded process via binding of different numbers of STIM1.

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

“…The CRAC channel states defined in this way become more stable the more STIM1 dimers participate in forming the channel, which is quantified by the cooperativity factor ␤ (16). Each of these four states contributes in a specific, predefined way to the total CRAC channel current, I CRAC ϭ 0.001⅐OS 1 ϩ 0.025⅐OS 2 ϩ 0.275⅐OS 3 ϩ 1.0⅐OS 4 , modeling the experimentally observed graded channel currents (15,16). The parameters (reaction rates and diffusion constants) of our model are shown in Table 1.…”

“…The domains of clustered STIM1/Orai1 molecules are the sites of Ca 2ϩ influx (12)(13)(14). The minimal requirement for activation are two STIM1 molecules bound per Orai1 tetramer; however, this stoichiometry results in channels with very low open probability (P o ), whereas maximal P o is achieved with eight bound STIM1 proteins per Orai1 tetramer (15,16). Failure to activate this Ca 2ϩ entry pathway either by defective STIM1 molecules or by mutations in Orai1 proteins results in severe defects in immune cell function but also in defective skin, platelet, and neuronal functions, among others (5,17,18).…”

Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels

“…STIM2β S domain dimers were based on previously published STIM1 S domains (Li et al, 2011) and consisted of residues E427-P472 joined with a 24-residue linker (GGSGGSGGGILQSTGGSGGSGGSG; see primer sequences below; residue numbers based on reference sequence below). Orai1 or Orai1(V102C) was cloned between the NheI and XhoI sites of the pEYFP-N1 vector downstream of a cytomegalovirus promoter, and STIM2α or STIM2β S-domain dimers were then inserted between the Xho1 and BamH1 sites to produce YFP-tagged chimeric constructs with a 13-residue linker (LEGVSTATMGGSG).…”

Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel

“…Tetracycline was added to induce the expression of MSS (monomer ORAI1 covalently linked with two S 336-485 domains, abbreviated as MSS) [25] and the ORAI1 V102A mutant. The customized medium was used (no calcium) to decrease the toxicity effect of high calcium caused by the constitutively open MSS and V102A channel.…”

“…Electrophysiology and patch clamp experiments were performed at room temperature using the standard whole cell recording configuration [25] . Cells were plated on poly-L-lysinecoated coverslips 12-24 h prior to the experiments.…”