Figure 3.
Measurement of Orai1-GECI affinity and kinetics. HEK 293A cells were cotransfected with Orai1-GCaMP6f and mCherry-STIM1, or with Orai1-G-GECO 1.2 and mCherry-STIM1. ER Ca2+ stores were depleted by whole-cell break-in and dialysis with pipette solutions containing IP3. (A–D) Unroofed cells were sequentially perfused with buffered Ca2+ solutions in the order indicated and imaged by TIRF microscopy. (A and B) Orai1-GCaMP6f in situ binding affinity. (A) Normalized Orai1-GCaMP6f fluorescence from the cell footprint. Note the lack of photobleaching of Orai1-GCaMP6f. (B) Corresponding association curve, fitted by the Hill equation with Kd of 620 nM and an hc of 1.56 (gray line; R2 = 0.993). 95% CIs were 590–660 nM for the Kd and 1.44–1.68 for the hc. n = 7 cells acquired over 3 d. (C and D) Orai1-G-GECO 1.2 in situ binding affinity. Normalized Orai1-G-GECO 1.2 fluorescence from the cell footprint (C) and association curve (D), fitted by the Hill equation with Kd of 1,010 nM and hc of 1.88 (gray line; R2 = 0.994). 95% CIs were 950–1,070 nM for the Kd and 1.74–2.05 for the hc. n = 6 cells acquired over 2 d. (E–G) Orai1-GCaMP6f fluorescence rise and fall kinetics during whole-cell recording. n = 6 cells. (E) Rising phase of Orai1-GCaMP6f fluorescence in response to a 600-ms test pulse to −100 mV in 2 mM extracellular Ca2+. The gray line, representing the time course of Ca2+ association with the tethered GCaMP6f probe, indicates a fit to the sum of a single exponential association with a time constant of 71 ms and a linear rising function (R2 = 0.949). The red line indicates the linear component of the rising phase. Half-rise time was 49 ms (43–56 ms, 95% CI). (F) Mean rising phase Orai1-GCaMP6f fluorescence of HEK 293A cells in response to a 600-ms test pulse to −100 mV in 20 mM extracellular Ca2+. Gray line indicates the fit to a single exponential association function with a time constant of 33 ms (R2 = 0.961). (G) Mean falling phase of Orai1-GCaMP6f fluorescence of HEK 293A cells upon stepping back to +20 mV from −100 mV in 2 mM extracellular Ca2+. Gray line indicates a single exponential decay function with a time constant of 62 ms (R2 = 0.989). Half-fall time was 43 ms (41–45 ms, 95% CI). Error bars are ± SEM.

Measurement of Orai1-GECI affinity and kinetics. HEK 293A cells were cotransfected with Orai1-GCaMP6f and mCherry-STIM1, or with Orai1-G-GECO 1.2 and mCherry-STIM1. ER Ca2+ stores were depleted by whole-cell break-in and dialysis with pipette solutions containing IP3. (A–D) Unroofed cells were sequentially perfused with buffered Ca2+ solutions in the order indicated and imaged by TIRF microscopy. (A and B) Orai1-GCaMP6f in situ binding affinity. (A) Normalized Orai1-GCaMP6f fluorescence from the cell footprint. Note the lack of photobleaching of Orai1-GCaMP6f. (B) Corresponding association curve, fitted by the Hill equation with Kd of 620 nM and an hc of 1.56 (gray line; R2 = 0.993). 95% CIs were 590–660 nM for the Kd and 1.44–1.68 for the hc. n = 7 cells acquired over 3 d. (C and D) Orai1-G-GECO 1.2 in situ binding affinity. Normalized Orai1-G-GECO 1.2 fluorescence from the cell footprint (C) and association curve (D), fitted by the Hill equation with Kd of 1,010 nM and hc of 1.88 (gray line; R2 = 0.994). 95% CIs were 950–1,070 nM for the Kd and 1.74–2.05 for the hc. n = 6 cells acquired over 2 d. (E–G) Orai1-GCaMP6f fluorescence rise and fall kinetics during whole-cell recording. n = 6 cells. (E) Rising phase of Orai1-GCaMP6f fluorescence in response to a 600-ms test pulse to −100 mV in 2 mM extracellular Ca2+. The gray line, representing the time course of Ca2+ association with the tethered GCaMP6f probe, indicates a fit to the sum of a single exponential association with a time constant of 71 ms and a linear rising function (R2 = 0.949). The red line indicates the linear component of the rising phase. Half-rise time was 49 ms (43–56 ms, 95% CI). (F) Mean rising phase Orai1-GCaMP6f fluorescence of HEK 293A cells in response to a 600-ms test pulse to −100 mV in 20 mM extracellular Ca2+. Gray line indicates the fit to a single exponential association function with a time constant of 33 ms (R2 = 0.961). (G) Mean falling phase of Orai1-GCaMP6f fluorescence of HEK 293A cells upon stepping back to +20 mV from −100 mV in 2 mM extracellular Ca2+. Gray line indicates a single exponential decay function with a time constant of 62 ms (R2 = 0.989). Half-fall time was 43 ms (41–45 ms, 95% CI). Error bars are ± SEM.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal