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. 2016 Dec 8;7:13725. doi: 10.1038/ncomms13725

Figure 3. The Orai1-ANSGA mutant has modified STIM1 binding but is pharmacologically identical to the STIM1-activated Orai1-WT.

Figure 3

(a) E-FRET changes in HEK-STIM1-YFP cells transiently expressing CFP-Orai1-WT (black) or CFP-Orai1-ANSGA (red) following ER Ca2+ store depletion with ionomycin (2.5 μM). (b) Summary of E-FRET values for the Orai1 mutants-WT and mutant before and after store depletion. Calculated FRET image from CFP-Orai1-WT (c) and CFP-Orai1-ANSGA (d) 3 min after ionomycin. Scale bar, 5μm. (e,f) Using internal BAPTA-dialysed HEK-STIM1-YFP cells expressing CFP-Orai1-WT, ICRAC was observed on 20 mM external Ca2+ addition. A unit of 50 μM 2-APB induced rapid transient ICRAC activation (I–V curves shown were 8 s before 2-APB, and at the peak following 2-APB addition). (g,h) In store-replete HEK cells expressing CFP-Orai1-ANSGA, constitutive ICRAC was measured on external Ca2+ addition, and 50 μM 2-APB added as in e giving identical, rapid transient activation of ICRAC, as revealed from I–V curves 8 s before 2-APB, and at the peak following 2-APB addition. (i) Summary of 2-APB induced increase in ICRAC from individual cells. All values are means±s.e.m.