HaloTag labeling of Orai1 does not perturb Orai1 function.

(A) TIRF images of TG-treated (store-depleted) HEK293 cells expressing Orai1-HaloTag and mCh-STIM1 after loading with JF646-BAPTA. The merged image shows colocalization of mCh-STIM1 (green) and Orai1-HaloTag (red). (B) Line scans of mCh-STIM1 (green) and Orai1-HaloTag (red) from selected puncta in the merged image from A. (C) Induction of ICRAC in a HEK293 cell expressing mCh-STIM1 and Orai1-HaloTag after break-in to the whole-cell recording configuration with EGTA in the recording pipette. Each point is the current during a step to -100 mV delivered from a holding potential of +30 mV. (D) The I-V relation measured with a 100-ms voltage ramp from - 100 to +100 mV.

Orai1-HaloTag labeled with JF646-BAPTA exhibits blinking in saturating Ca2+.

COS-7 cells expressing Orai1-HaloTag and mCh-STIM1 were treated with TG to induce puncta formation before exposure to 20 µM digitonin and 10 mM Ca2+ to saturate JF646-BAPTA. (A) Left, after stepwise bleaching of one channel to a single active JF646-BAPTA dye, rapid fluctuations appear. Right, spontaneous recovery of fluorescence in a second channel after a prolonged dark state period. The gray bars mark sections expanded below to show transitions to the zero-fluorescence level. 33 ms exposure with 3-frame boxcar averaging, 15 mW laser power. (B) Dwell time histograms of the bright and dark events like those shown in A. A biexponential fit to the bright state distribution (τ1=89 ms, A1=0.62; τ2=609 ms, A2=0.38) and biexponential fit to the dark state distribution (τ1=72 ms, A1=0.94; τ2=1166 ms, A2=0.06) are shown. Data compiled from 99 channels from 2 cells. In this and all subsequent figures, c.u. corresponds to camera output units, and unless otherwise noted, all values have been corrected for local background fluorescence (see Methods).

Blinking behavior of Orai1-HaloTag labeled with JF646-BAPTA in intact cells after ER Ca2+ depletion.

COS-7 cells expressing mCh-STIM1 and Orai1-HaloTag labeled with JF646-BAPTA were treated with 2 µM CPA in the presence of 20 mM extracellular Ca2+. (A) Representative single-channel traces show fluctuations to the zero-fluorescence level alternating with long-lived bright or dark events. 20 ms exposure, 3 mW laser power, 5 frame boxcar averaging. (B) Transitions to and from the zero-fluorescence level are complete within the single-frame exposure time of 10 ms. The section marked by the gray bar is expanded to the right. 9 mW laser power. (C) Dwell time histograms of bright and dark events. Biexponential fits to the bright state distribution (τ1=217 ms, A1=0.86; τ2=1030 ms, A2=0.14) and dark state distribution (τ1=156 ms, A1=0.86; τ2=650 ms, A2=0.14) are shown. Data compiled from 128 channels from 4 cells.

JF646-BAPTA selectively reports Ca2+ entering through Orai1 channels during whole-cell recording.

(A) Images of HEK293 cells expressing mCh-STIM1 and either WT Orai1-HaloTag (top row) or Orai1-E106A-HaloTag (bottom row). The fluorescence of JF646-BAPTA increases at - 100 mV for WT Orai1 but not Orai1-E106A, despite being co-clustered with STIM1. Bar=10 µm. (B) Fluorescence of WT Orai1-HaloTag across the cell footprint increases during 100-ms pulses to -100 mV from a holding potential of +30 mV (bars). The response is blocked by 100 µM La3+ as expected for influx through Orai1. (C) Fluorescence of Orai1-E106A-HaloTag across the cell footprint does not respond to hyperpolarizing pulses, indicating that the fluorescence increase of WT Orai1 derives directly from Ca2+ flux through Orai1. Orai1-E106A-HaloTag expression level in C was ∼3-fold higher than in B in order to optimize detection of even a small amount of Ca2+ from other sources. 10 ms exposure, 1 mW laser power.

In situ characterization of Orai1-HaloTag labelled with JF646-BAPTA.

HEK293 cells expressed mCh-STIM1 and Orai1-HaloTag labeled with JF646-BAPTA. All data are shown relative to local background fluorescence. (A) Responses of 3 puncta to voltage ramps from -100 to +100 mV. (B) Saturation of JF646-BAPTA in cells expressing Orai1-HaloTag. Mean fluorescence ± SEM was compiled from the responses of 22 puncta to 4 voltage ramps like those in A. Binding kinetics of Ca2+ for JF646-BAPTA reduces the response over the first several points of the ramp (- 100 to -99 mV). The response is minimal at potentials above ∼+20 mV, and saturates at potentials of -75 mV or below. (C) Kinetics of JF646-BAPTA fluorescence in response to a voltage step from +30 to -100 mV. Expanded sections indicated by colored bars show single-exponential fits to the off and on transitions (τoff=30 ms, τon=18 ms). Mean ± SEM of 6 puncta. In A-C, 10 ms exposure, 1 mW laser power. A had 15 frame boxcar averaging.

Identification of puncta containing a single Orai1-HaloTag channel.

(A) TIRF image of Orai1-HaloTag in a HEK293 cell coexpressing mCh-STIM1 during whole-cell recording at -100 mV. Note the range of JF646-BAPTA fluorescence intensities among different puncta. Arrowheads mark puncta plotted in B. Bar=10 µm. (B) Traces of single Orai1-HaloTag puncta from A upon hyperpolarization to -100 mV from a +30 mV holding potential, showing examples of 1-, 2-, and 3-channel puncta. (C) Distribution of fluorescence intensities at -100 mV relative to the baseline fluorescence at +30 mV, compiled from 289 puncta in 10 cells. Gaussian fits to the histogram indicate peaks at 5.1, 10.9, and 15.8 c.u., corresponding to 1-, 2-, and 3-channel puncta. In B and C, 33 ms exposure, 1 mW laser power, 2 frame boxcar averaging.

Single-channel behavior of Orai1-HaloTag during whole-cell recording.

HEK293 cells expressed mCh-STIM1 and Orai1-HaloTag labeled with JF646-BAPTA. All data are shown relative to local background fluorescence. (A) Examples of single-channel responses to hyperpolarization to -100 mV. In each case, fluorescence is ∼5 c.u. above the 0-current level (defined as the intensity at +30 mV, dashed line) and drops to the 0-current level during closing events. (B) Thresholding procedure for detecting gating transitions. Levels were set for the open and closed states from fluorescence intensities at -100 and +30 mV, respectively (top). A 50% threshold was applied to detect transitions between open and closed states (middle), to generate an idealized trace (bottom). (C) Open dwelltimes follow a biexponential distribution (τ1=0.09 s, A1=0.74; τ2=1.19 s, A2=0.26). (D) Closed dwelltimes were exponentially distributed (τ=0.14 s). Data compiled from 36 channels in 12 cells. In A-D, 33 ms exposure, 1 mW laser power, 3 frame boxcar averaging.

Open probability of Orai1-HaloTag channels during whole-cell recording.

HEK293 cells expressed mCh-STIM1 and Orai1-HaloTag labeled with JF646-BAPTA. (A) Example traces showing single channels with no apparent activity over a period of 40 s (‘silent channels’). (B) TIRF images of Orai1-HaloTag with arrowheads showing the channels displayed in A with merged images showing colocalization of mCh-STIM1 (green) and Orai1-HaloTag (red). Bar=1 µm. (C) The relative frequency of PO values from ‘silent’ channels (out of 559 channels at STIM1 puncta) and 28 active channels from Fig. 7C and D.

A method for labeling Orai1-HaloTag with the Ca2+-sensitive fluor JF646-BAPTA.

(A) Structure of JF646-BAPTA showing the AM esterified and de-esterified forms (Deo et al., 2019). (B) Testing for JF646-BAPTA labeling efficiency. HEK 293 cells were transfected with mCh-STIM1 and Orai1-HaloTag and treated with 1 µM JF646-BAPTA overnight (left) or for 1 h in the presence of 0.02% pluronic F-127 (right column), followed by exposure to 100 nM JF-552 for 10 min to label unoccupied HaloTag sites. TIRF images of the cell footprints after TG treatment show that pluronic F-127 is needed to achieve complete labeling with JF646-BAPTA. Bar=10 µm.

Single-channel optical recordings analyzed for the dwell time histograms.

36 recordings were selected for analysis based on the criteria outlined in Methods. Dashed lines indicate the intensity at +30 mV, as an indicator of the expected closed channel intensity. 33 ms sampling, 1 mW laser power, 3 frame boxcar averaging.