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. 2015 Jun 8;209(5):653–670. doi: 10.1083/jcb.201412060

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© 2015 Rana et al.

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Figure 8. — STIM2β inhibits Orai1 channels through a sequence-specific interaction. (A) Chimeric system for detecting inhibitory interactions. A dimer of S domains is covalently attached to constitutively active Orai1(V102C). Inhibitory interactions between the S domains and Orai are detected by a reduced rate of constitutive Ca²⁺ influx. (B) Constitutive Ca²⁺ influx in HEK293 cells expressing Orai1(V102C) or Orai1(V102C)-SS chimeras, measured upon addition of 2 mM Ca²⁺ (n > 45 cells for each curve). (C) The relative Ca²⁺ influx rates from the experiments in B, quantified by the initial slopes of the fura-2 ratio. Fusion of wild-type (P < 0.0001), but not L2R mutant (P = 0.67), STIM2β S domains to Orai1(V102C) strongly inhibited constitutive Ca²⁺ entry (Mann–Whitney test). (D) The cumulative frequency distribution of surface HA intensities for the cells analyzed in B and C shows that surface expression for all three channel constructs was similar. a.u., arbitrary unit. Error bars show means ± SEM.