Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels

Abstract

Receptor-operated Ca(2+) entry (ROCE) and store-operated Ca(2+) entry (SOCE) into cells are functions performed by all higher eukaryotic cells, and their impairment is life-threatening. The main molecular components of this pathway appear to be known. However, the molecular make-up of channels mediating ROCE and SOCE is largely unknown. One hypothesis proposes SOCE channels to be formed solely by Orai proteins. Another proposes SOCE channels to be composed of both Orai and C-type transient receptor potential (TRPC) proteins. Both hypotheses propose that the channels are activated by STIM1, a sensor of the filling state of the Ca(2+) stores that activates Ca(2+) entry when stores are depleted. The role of Orai in SOCE has been proven. Here we show the TRPC-dependent reconstitution of Icrac, the electrophysiological correlate to SOCE, by expression of Orai1; we also show that R91W-Orai1 can inhibit SOCE and ROCE and that Orai1 and STIM1 expression leads to functional expression of Gd-resistant ROCE. Because channels that mediate ROCE are accepted to be formed with the participation of TRPCs, our data show functional interaction between ROCE and SOCE components. We propose that SOCE/Icrac channels are composed of heteromeric complexes that include TRPCs and Orai proteins.

Fig. 1.

Expression of Orai in TRPC-expressing cells reconstitutes Icrac. (A) Summary of Gd³⁺-sensitive Icrac at −100 mV developed 3–5 min after break-in in the various cell types transfected as shown. EV, empty vector. Shown below each group are the nanograms of WT or mutant or Orai1-carrying expression vector used in the standard transfection to which the analyzed cells had been subjected. (B) I–V relationships recorded from a TRPC6-expressing cell transfected with Orai1 after break-in (trace 1, black), at 180 sec before addition of 0.5 μM GdCl₃ (trace 2, red) and at 500 sec after Gd³⁺ block had set in (trace 3, blue). The green trace indicates net Icrac (trace 2 minus blue trace 3). (Inset) Time course of current at −100 mV during the course of the experiment. (C) (Left) Changes in Icrac recorded from a TRPC3-expressing cell transfected with Orai1 as seen by changing the ionic composition of the external solution from Ca²⁺ to divalent-free and Ba²⁺. (Right) I–V relations of the leak-subtracted currents at the times indicated in Left.

Fig. 2.

Inhibition of SOCE by mutant Orai. (A) Representative SOCE elicited in Fura2-loaded, TRPC3-expressing cells transfected with YFP and empty vector alone (YFP), with YFP plus Orai1, or YFP plus R91W-Orai1. In this and all other figures, SOCE was triggered by the addition of 2 μg/ml Tg after previous removal of Ca²⁺ from the medium. SOCE developed after 450 sec is seen by the rate of rise in [Ca²⁺]_i and the extent of increase in [Ca²⁺]_i. The time of addition of Ca²⁺ is indicted by the black bar. Numbers in parentheses denote numbers of cells analyzed. (B) Survey of TRPC expression in HEK293 cells by RT-PCR. Primer sequences are available upon request. (C) Expression of STIM1 protein in control HEK293 and TRPC-expressing cells is unaffected by expression of SOCE augmenting levels of Orai1 as seen by Western blot analysis of total cell lysates. (D) Western blot analysis shows that the exogenous myc-tagged WT and R91W-Orai1 are expressed at similar levels in transfected cells. N223A, WT myc-tagged Orai1 in which the site for posttranslational glycosylation has been changed from Asn to Ala. (E) Mutations cognate to R91W-Orai1 in Orai2 and Orai3 also inhibit SOCE. HEK293 cells were used.

Fig. 3.

Block of inhibition of SOCE and effects of WT and mutant Orai coexpressed with STIM1 as seen in HEK293 cells. The results shown are from a single representative experiment. (A) Block of the inhibitory effect of excess WT Orai1. (B) Block of the effect of low levels of R91W-Orai1.

Fig. 4.

Block of V1a receptor-triggered ROCE in TRPC3-expressing cells (A and C) and in control HEK cells (B). (B and C) Block of the inhibition by STIM1.

Fig. 5.

Development of resistance of ROCE, but not SOCE, to inhibition by Gd³⁺ upon coexpression of Orai1 and STIM1. (A) Gd³⁺ block of SOCE. (Ai) SOCE in HEK cells is blocked by 1 μM Gd³⁺. (Aii) SOCE induced by coexpression of Orai1 and STIM1 also is blocked by Gd³⁺. (B) Gd³⁺ block of ROCE and appearance of Gd³⁺-resistant ROCE in cells cotransfected with Orai1 plus STIM1. (Bi) ROCE is highly sensitive to inhibition by Gd³⁺. (Bii) Coexpression of Orai1 and STIM1 leads to the appearance of Gd³⁺-resistant ROCE (Biii) Gd³⁺-resistant ROCE requires both STIM1 and inhibitory levels of WT Orai1. The results are from a single experiment; each trace represents the averaged changes in the indicated number of cells.

Fig. 6.

Model of the interplay between TRPCs, Orai, and STIM. TRPCs are depicted as being stabilized in an inactive state by interacting with Orai. Receptor signaling is proposed to cause fast dissociation of Orai from TRPC, promoting its activation. Activated TRPC gating in the nonselective cation channel mode is then trapped by STIM–Orai complexes induced by depletion of Ca²⁺ from the store and dissociation of Ca²⁺ from STIM's luminal, Ca²⁺-sensing N terminus. Clustered aggregates of STIM–Orai associate with activated TRPCs and stabilize their conformation in a state in which it operates as an Icrac channel. Normal cells have excess of neither Orai nor STIM but do have more TRPC molecules than Orai molecules. For further discussion of the model, see the text.