Mean inward currents at −40 mV and −80 mV activated by exogenous addition of 8 μm arachidonic acid recorded in control STIM1-stable cells or the same cells transfected with either the E106QOrai1 monomer, or the corresponding E81QOrai3 monomer. Values are means ±s.e.m., values of n shown in parentheses.

A, mean ±s.e.m. inward arachidonic acid activated currents in cells transfected with the indicated trimeric construct alone, or co-expressed with either the wild-type Orai1–Orai3 dimer or the corresponding dimer containing the dominant-negative E81QOrai3 subunit. Currents were recorded at −40 mV following activation with 8 μm arachidonic acid. Values of n shown in parentheses. B, representative current–voltage relationships of the arachidonic acid activated currents seen on co-expression of the indicated trimers with the Orai1–Orai3 dimer.

A, arachidonic acid activated currents measured in STIM1-stable cells transfected with either a concatenated Orai3–Orai1–Orai1 trimer (311), or an Orai3–Orai1–Orai3 trimer (313). Also shown are the same currents following co-expression of these trimers with either a dominant-negative E106QOrai1 monomer, or the corresponding E81QOrai3 monomer. Values are means ±s.e.m. of inward currents at −40 mV, (values of n shown in parentheses). B, representative current–voltage relationships of arachidonic acid activated currents in STIM1-stable cells transfected with either the concatenated 311 or 313 trimer.

Mean ±s.e.m. inward currents measured at −40 mV activated either by arachidonic acid (open bars), or following store depletion (filled bars), in cells transfected with the indicated pentameric constructs. Values of n shown in parentheses. Data for the arachidonic acid activated currents in cells expressing the 31113 and 31311 pentamers (indicated by *) were taken from Fig. 7, and are shown here simply for comparison.

A, mean ±s.e.m. inward arachidonic acid activated currents at −40 mV in STIM1-stable cells transfected with the respective Orai1–Orai3 tetrameric constructs either alone, or co-transfected with either wild-type or dominant-negative Orai monomers as indicated. Values of n were 7–14 for the 3111 data, 8–15 for the 3113 data, 6–13 for the 3131 data, and 7–12 for the 3331 data. B, representative current–voltage relationships for the large arachidonic acid activated currents recorded on co-expression of the Orai3 monomer with the 3111 tetramer, and the Orai1 monomer with either the 3113 tetramer or the 3131 tetramer, as indicated.

A, mean ±s.e.m. inward currents measured at −40 mV activated by different concentrations of exogenous arachidonic acid in cells transfected with the 31311 pentamer. Values of n shown in parentheses. B, representative current–voltage relationships of the current activated by different concentrations of exogenous arachidonic acid recorded in cells expressing the 31311 pentamer. C and D, representative current–voltage relationships of the arachidonic acid activated current recorded in STIM1-stable cells expressing either the 31311 pentamer in the presence and absence of 5 μm gadolinium (C), or the 31113 pentamer in the presence or absence of 100 μm lanthanum (D).

A, mean inward currents at −40 mV (±s.e.m.) in STIM1-stable cells transfected with concatenated constructs of each of the Orai1–Orai3 tetrameric assemblies as indicated. Shown are the respective currents activated by exogenous addition of 8 μm arachidonic acid (black columns), and currents activated following store-depletion (grey columns). Values of n shown in parentheses. Also shown are representative current–voltage relationships for the arachidonic acid activated currents (B), or the store-operated currents (C) obtained in cells expressing the various tetrameric constructs as follows: •, 3111; ○, 3113; ▴, 3131; and ▵, 3331.

A, representative trace showing the activation of inward currents in STIM1-stable cells expressing the Orai1–Orai3 dimer following exogenous addition of arachidonic acid (8 μm, added at arrow). Currents were measured at −40 mV. B, mean (±s.e.m.) arachidonic acid activated currents measured in STIM1-stable cells, and in the same cells transfected with the Orai1–Orai3 dimer, or an Orai1–Orai3 dimer in which the Orai3 subunit bore the dominant-negative E81Q mutation. Also shown are the same currents following co-transfection of the Orai1–Orai3 dimer with either a dominant-negative E81QOrai3 mutant monomer, or the corresponding E106QOrai1 monomer. Values are inward currents measured at −40 mV (values of n shown in parentheses) following activation by addition of 8 μm exogenous arachidonic acid. C, representative current–voltage relationships of conductances activated by exogenous addition of arachidonic acid (8 μm) in STIM1-stable cells, and in the same cells transfected with a concatenated Orai1–Orai3 dimer (+ 1–3 dimer).

A, representative Western blot showing STIM1 protein levels in the various siRNA-transfected cells. Shown are STIM1 protein in control cells (C), in cells transfected with the STIM1 siRNA (S), and in siRNA-transfected cells expressing either the siRNA-resistant wild-type STIM1 (S+WT), or the corresponding siRNA-resistant N-glycosylation mutant STIM1 (S+M). Gels were stripped and reprobed with β-actin as a loading control. Note that the glycosylation mutant runs at a slightly lower molecular mass, as previously reported (Mignen et al. 2007). B, the effects of the N-glycosylation mutant STIM1 on arachidonic acid activated currents induced by expression of the 31311 and 31113 pentamers (0.2 μg DNA – see text for details). Mean ±s.e.m. inward current density at −40 mV in siRNA-transfected cells expressing the indicated pentamer construct together with either an siRNA-resistant wild-type STIM1 (black), or an siRNA-resistant N-glycosylation mutant STIM1 (grey). Also shown are the endogenous ARC channel currents in the same siRNA-transfected cells expressing the siRNA-resistant wild-type STIM1 alone (white). Values of n shown in parentheses.

A, representative Western blot showing the expression of the concatenated pentameric constructs in STIM1-stable cells. The gel shows lanes obtained from STIM1-stable cells transfected with the empty vector as a control (lane C), and from the same cells transfected with either the FLAG-tagged 31113 pentamer construct (lane 1), or the FLAG-tagged 31311 pentamer construct (lane 2). All lysates were treated identically and probed with anti-FLAG antibody. Gels were stripped and reprobed with β-actin as a loading control, as indicated. The prominent band running at approximately 200 kDa (indicated by the arrow) in the lanes transfected with the concatenated pentamers corresponds to the predicted molecular mass of the Orai1/Orai3 pentamer. Importantly, there was no obvious indication of breakdown of the pentameric constructs to smaller forms. B, mean ±s.e.m. inward arachidonic acid activated currents measured at −40 mV in cells transfected with the indicated pentameric construct alone, or the equivalent construct containing a single dominant-negative E81QOrai3 subunit. Also shown for the 31311 pentamer are the corresponding currents on expression of a pentamer containing a single dominant-negative E106QOrai1 subunit, or co-expression of the normal pentamer with either the E81QOrai3 or E106Q dominant-negative monomers. Values of n shown in parentheses. C, representative traces showing the activation of inward currents in STIM1-stable cells expressing either the 31311 pentamer (open circles) or the 31113 pentamer (filled circles) following exogenous addition of arachidonic acid (8 μm, added at arrow). Currents were measured at −40 mV. D, representative current–voltage relationships (between −100 mV and +100 mV) of the arachidonic acid activated currents recorded on expression of either the 31113 pentamer, or the 31311 pentamer.