Figure 5: Evidence for a unimolecular interaction between the SOAR dimer and Orai1.
From: STIM1 dimers undergo unimolecular coupling to activate Orai1 channels
Two possible models for SOAR–Orai1 interactions: (a) bimolecular binding of a homomeric SOAR dimer (red) to two adjacent Orai1 subunits is required to open the hexameric Orai1 channel (purple). (b) Unimolecular binding of a single monomer within the SOAR dimer (red) to a single Orai1 subunit is sufficient to open the hexameric Orai1 channel (purple). Changes in the association of heterodimers of SOAR comprising one WT and one F394H monomer (red/blue) occurring in the bimolecular (a, right) and unimolecular (b, right) models. (c) Near-PM values of E-FRET between Orai1-CFP in HEK-Orai1-CFP stable cells, and transiently expressed YFP-S-S (black; n=141), YFP-SH-S (green, n=243), YFP-S-SH (red, n=213) or YFP-SH-SH (blue, n=206). (d) YFP/CFP ratios of the near-PM-expressed Orai1-CFP and YFP dimers in the cells used for E-FRET in c. (e) Cartoon of Orai1 protein (left) and PM-CFP-Orai1CT construct (right) comprising C terminus of Orai1 (267–301) attached to CFP and a single PM transmembrane-spanning helix. (f) Near-PM E-FRET between PM-CFP-Orai1CT transiently expressed in HEK-WT cells and transiently expressed SOAR concatemer–dimers: YFP-S-S (black; n=51); YFP-SH-S (green; n=55); YFP-S-SH (red; n=39); and YFP-SH-SH (blue; n=85). (g) YFP/CFP ratios of near-PM-expressed Orai1-CFP and YFP dimers in the cells used (f). (h) Near-PM E-FRET between PM-CFP-Orai1CT stably expressed HEK cells and transiently expressed SOAR concatemer–dimers: YFP-S-S (black; n=141); YFP-SH-S (green; n=137); YFP-S-SH (red; n=389); YFP-SH-SH (blue; n=202); and YFP alone (yellow; n=202). (i) YFP/CFP ratios of near-PM-expressed Orai1-CFP and YFP dimers in the cells used in h. In all cases (c–i), results are means±s.e.m. of three independent experiments. (j) Interpretation of interactions between CFP-tagged Orai1CT (purple) and each of the four YFP-tagged SOAR dimer–concatemers (or YFP alone) used in the E-FRET experiments shown in h. Top row shows input reactants for each condition; bottom row shows resulting interactions. Each WT SOAR monomer (red) of the SOAR dimers is shown to independently bind a single Orai1CT molecule. F394H SOAR monomers (blue) do not bind Orai1CT.
