Optical single-channel recording of CRAC channels with HaloTag and a Ca²⁺-sensitive ligand

Reviewer #1 (Public review):

Summary:

Dhillon and Lewis present an optical approach to record single CRAC channel activity, overcoming the long-standing barrier imposed by the channel's extremely small unitary conductance. By fusing HaloTag to Orai1, labeling with JF646-BAPTA, and combining TIRF microscopy with whole-cell voltage clamp (Patch-TIRF), the authors achieve genuine single-channel resolution. A central contribution is the recognition that JF646-BAPTA undergoes reversible photophysical blinking that can be readily mistaken for gating events. The authors exploit the multi-dye labeling of hexameric Orai1, combined with voltage-clamped definition of open and closed fluorescence levels, to distinguish true gating transitions from blinks. The result is the first kinetic characterization of single CRAC channel openings activated by STIM1, reporting multiple open and closed states with durations from about 0.1 s to tens of seconds, predominantly high open probabilities ({greater than or equal to} 0.7), and an unexpected population of "silent" channels that co-localize with STIM1 but show no detectable activity over the observation window.

Strengths:

The work is technically rigorous, and the controls are appropriate. The integration of patch-clamp voltage control with TIRF imaging is a thoughtful methodological choice that defines the open- and closed-channel fluorescence reference levels with precision, providing a quantitative framework that the field has lacked. The use of the non-conducting Orai1-E106A mutant as a specificity control (Figure 4C) is exactly the right experiment, and the demonstration that JF646-BAPTA signals require Ca²⁺ flux through Orai1 itself anchors the entire approach. The identification and characterization of JF646-BAPTA blinking (Figures 2 and 3) is a significant contribution in its own right. The authors show clearly that the dye exhibits long-lived dark states and that transitions to zero fluorescence, rather than to a finite calcium-free baseline, are diagnostic of blinking rather than channel closure. This caveat has immediate implications for the interpretation of recent work using the same dye on other calcium-permeable channels, and will recalibrate the broader field of HaloTag-based single-channel optical recording. The kinetic analysis itself reveals something that was previously inaccessible: seconds-long open times, multi-state gating behavior, and a population of channels that co-localize with STIM1 yet remain electrically silent. These findings are physiologically meaningful and would not have been detectable by macroscopic electrophysiology. Overall, an outstanding study.

Weaknesses:

The manuscript would benefit from a small number of additional analyses of the existing data and modest refinements to the presentation. The discrete-channel interpretation of the intensity histogram in Figure 6C, the open probability distribution in Figure 8C, and the assignment of the "silent" channel population are all interesting and likely correct, but each rests on assumptions that the authors are well positioned to test directly using data already in hand. Brief additional discussion of the dynamic range of JF646-BAPTA in situ and of how the temporal resolution of the recordings shapes the inferred kinetic model would also help readers calibrate the findings.

None of these points challenges the central claims of the paper, and none requires new experiments.

Reviewer #2 (Public review):

Summary:

Dhillon and Lewis use the enhanced brightness of the new calcium indicator dye JF646-BAPTA attached to Orai1-bound HaloTag to identify single CRAC channel events detected as [Ca2+]i fluctuations rather than currents. This enables them to detect Orai1single channel kinetics of permeation, overcoming the currently unmeasurable single channel CRAC conductances (~ 20-40 fS). TIRF microscopy narrows the z-section and improves calcium event localization.

JF646-BAPTA reversibly blinks between fluorescent and non-fluorescent states, complicating single-channel detection. Blinking occurs both in permeabilized cells with saturating Ca2+ and in intact cells at physiological [Ca2+]i. Using voltage clamp and TIRF imaging, CRAC gating events were distinguished from blinking by analyzing fluorescence responses to voltage changes.

Hyperpolarization (-100 mV) increases fluorescence, indicating channel opening. Responses blocked by La3+ confirm specificity for Orai1, while minimum fluorescence at +30 mV corresponds to closed channels. Dynamic range and response kinetics help differentiate genuine gating from blinking artifacts. Long channel openings (seconds to tens of seconds) are observed, with most open times around 1.2 seconds. Longer openings (tens of seconds) are present but difficult to sample. Silent channels constitute 11% of puncta.

The paper carefully examines a new method to sample CRAC kinetics, which should enable further mechanistic studies of STIM control of ORAI and modulation by other signaling components such as calcineurin. Development of bright nonblinking dyes or dyes whose blink rates are directly correlated with a calcium-binding site will enhance this route of investigation.

Comments:

This is an excellent methodological study, rigorous and thorough. I wondered whether La3+ alone could alter JF646-BAPTA blinking, but the authors show that JF646-BAPTA exhibits reversible transitions to a non-fluorescent state (blinking) under both Ca2+-saturated and physiological conditions, independent of channel activity or the presence of La3+.

Strengths:

A novel method providing additional tools to study store-depletion induced Ca currents mediated by Stim-Orai family members.

Weaknesses:

Limited by blinking dyes, the only ones currently sensitive enough to measure the calcium fluxes through single channels.

Reviewer #3 (Public review):

Summary:

Previous work from the Cahalan lab used fluorescent Genetically Encoded Ca2+ Indicators (GECI), like GCaMP6f, tethered to the N- or C- terminus of Orai1 to monitor CRAC channel optical signals (Dynes et al., PNAS 2016 PMID: 26712003; J Gen Physiol 2020 PMID: 32589186; PNAS 2023 PMID: 37729200). In this study from the Lewis lab, the HaloTag system enables C-terminal labeling of Orai1 with a reactive JF646-BAPTA loaded into cells. The article raises two key issues with the Ca2+ indicator probe that may limit potential applications: probe loading conditions and blinking.

Making Sense of Probe Probe-lems:

This is a three-component system: the hexameric Orai1 channel, the Halo tag, and the Ca2+ indicator (four components if you count the GFP- or mCherry-tagged STIM1 in the endoplasmic reticulum membrane that activates the plasma membrane Orai1 channel). The Orai1 channel, tagged with the Halo protein, appears to function normally, judging from the characteristic inwardly rectifying Ca2+ current first observed in T lymphocytes (Lewis and Cahalan, Cell Regulation 1989 PMID: 2519622). One problem is to find a condition for indicator dye loading that results in complete and uniform labeling with the covalently linked JF646 indicator. JF646-BAPTA is a far-red fluorescent indicator related to BAPTA, with a Kd of ~150 nM. The esterified form can be loaded into cells, as is routinely done for Ca2+ indicators like fura-2 or fluo-4. Ideally, to monitor local Ca2+ in the cytosolic nanodomain of the Orai1 channel, the indicator should react with each and every Halo tag of the hexameric channel. The authors assessed published methods by varying the exposure time to the JF646-BAPTA-esterified probe. The authors then used green JF552 labeling following red JF646-BAPTA loading to assess the completeness of labeling. Even overnight incubation of Halo-tagged cells was not sufficient. The addition of Pluronic treatment for 1 hr improved labeling, and a standard condition was adopted. Under this condition, no additional labeling with the green JF552 was seen, implying complete labeling with JF646-BAPTA. However, even with complete labeling, several additional effects might reduce the effective signal-to-noise, which is lower in these studies than expected from in vitro measurements - for example, if the JF646-BAPTA molecules are incompletely de-esterified, or if there is quenching between the closely spaced probes attached to the channel hexamer.

A second, more serious problem analyzed by this article is that the JF646-BAPTA probe blinks on and off spontaneously, making it problematic to monitor true single-channel events in which the channel open state is assessed by the fluorescent probe. The authors distinguish blinking from channel-gating events by carefully noting the residual level of fluorescence in the absence of Ca2+ influx. Blinking events occur in bursts that reduce fluorescence transiently to zero, whereas the closed channel labeled with JF646-BAPTA retains a low level of fluorescence (~20%). To circumvent the blinking issue, the authors use whole-cell patch recording, in conjunction with optical recording (Patch-TIRF). This allows channel-gating events to be identified by step-wise changes in fluorescence due to Ca2+ entry upon hyperpolarization to -100 mV, above a baseline level of fluorescence at +30 mV, which the authors presume represents the closed channel level of fluorescence. Irreversible photobleaching is an additional issue, limiting the recording times to less than 1 minute.

Visualizing Orai1 Single-Channels:

With the blinking problem circumvented, at least in part, the authors uncovered a wide variety of single-channel events. Cells with low expression levels of Orai1 revealed 0-3 active Orai1 channels per STIM1 puncta. The range of gating behavior at the single-channel level is one of the revelations in this study. A substantial fraction (11%) of puncta contained "silent" channels that did not open (detected by the non-zero level of baseline fluorescence for closed channels). At the other extreme, some channels remained open for tens of seconds. On average, channels that opened and closed stochastically exhibited a bi-exponential distribution of bright states (open channels), with a major component of fast events (92 ms) and a minor component of slower ones (1190 ms), as well a single-exponential distribution of dark states (closed channels), and open probabilities >0.7. Channel open/closed times and the high open probability of active Orai1 channels seen here reinforce previous work based on analysis of CRAC current fluctuations in whole-cell recording, and optical single-channel recording using a different genetically encoded Ca2+ indicator, G-GECO1, tethered to Orai1 (Prakriya and Lewis, J Gen Physiol 2006 PMID: 16940559; Dynes et al., PNAS 2016 PMID: 26712003).

Expression levels for single-channel optical recording must be low; accordingly, puncta contained only 0-3 active channels. However, under conditions of high STIM1 and Orai1 expression, conventionally used to investigate channel function, as in Figure 1, cells with large currents express many thousands of active channels. The number of active channels per cell can be calculated by dividing the peak current (~-100 pA) by the voltage (-100 mV); this corresponds to a whole-cell conductance (G) of ~1 nS (conductance is measured in Siemens). The single channel conductance (gamma, too low to detect electrically) is estimated by noise analysis to be 20-40 fS. Thus, the number of active channels is given by G / gamma corresponding to a range of > 25,000 - 50,000 open channels per cell. Under similar conditions of high STIM1/Orai1 co-expression in HEK cells, individual Orai1 channels were visualized at high density in puncta by freeze-fracture electron microscopy (Perni et al., PNAS 2015 PMID: 26351694), revealing puncta packed with Orai1 particles corresponding to hundreds to >1000 channels per punctum. Measuring the center-to-center distances between particles in puncta revealed two peaks in a distribution of inter-particle lengths: 9 nm (consistent with the approximate width of the Orai1 channel hexamer) and 15 nm (possibly due to two adjacent Orai1 channels held together by intervening STIM1 dimers).

Strengths:

The authors do an excellent job of analyzing and discussing probe artifacts that can confound measurements at the single-channel level. On the technical side, we thank the authors for including a photon 'budget' for their imaging experiments by including: the conversion factor from camera intensity units (c.u.) to photoelectrons, cell background fluorescence levels, and nominally Ca2+ free single channel fluorescence levels. One parameter missing from the list is the size of the region of interest used for channel recording. We expect the intensity measurements provided in the channel traces to correspond to mean ROI intensity levels. Upon knowing the ROI size in pixels, the magnitude of fluorescent signals could then be calculated in photons. Taken together, these values will aid comparisons to previous work and help guide subsequent researchers doing their own optical recording.

The most important finding of this study is the ability to analyze single-channel properties of active Orai1 channels using the HaloTag approach. By direct measurement, the authors confirm previous work that there are at least two open states and that the CRAC channel open probability is greater than 0.7.

Like any good study, this work suggests opportunities for further work. At the chemistry level, one focus should be the development of new probes that don't blink and have lower affinity for Ca2+ to circumvent unwanted responses to global Ca2+ signaling. Far-red probes like JF646-BAPTA have the advantage of reduced scattering for in vivo imaging applications. At the level of channel molecular function, the results pave the way for unraveling mechanisms of channel gating, such as the requirement for STIM1 binding to activate sub-states of Orai1, and how the channel undergoes Ca2+-dependent inactivation. At the cellular physiology level, localized Ca2+ probes should help to clarify mechanisms that couple to changes in gene expression and reveal Ca2+ signaling in subcellular structures, including dendritic spines. As a nice proof of principle, Halo-tagging enabled Ca2+ signals to be measured in primary cilia (Deo et al., J Am Chem Soc 2019 PMID: 31430138). Future users of HaloTag and GECI Ca2+ indicators will need to confront the issues (probe-lems) at the single-channel level that are carefully raised and analyzed in this article.

Weaknesses:

The major confounding issue identified here is probe blinking. The authors find a way to circumvent the issue, but not to prevent it. Is it triggered by high laser light intensity? Do the six JF646-BAPTA molecules tagging a single Orai1 channel exhibit quenching or correlated blinking?

Which type of probe is better for understanding more about the CRAC channel function? It is difficult to evaluate the pros and cons of the HaloTag and GECI approaches without a side-by-side comparison under identical conditions (except for the probe, obviously). With respect to Ca2+ affinities, higher Kd values (lower affinity) are probably better. JF646-BAPTA has a relatively low Kd value (150 nm) compared to Orai1-GCaMP6f (620 nM in situ), which may account for the saturation of optical signals at potentials more negative than -75 mV in this study. In contrast, saturation did not occur at negative potentials with Orai1-GCaMP6f in the study by Dynes et al., 2020. Lower affinity also makes the probe more resistant to unwanted signals from global increases in Ca2+. With respect to response kinetics, the finding that JF646-BAPTA has faster Ca2+ binding and unbinding kinetics than GECIs in Deo et al., 2019, occurred before publication of the jGCaMP8 series indicators in Y. Zhang et al., Nature 2023. Kinetic measurement of Orai1-jGCaMP8f fusions was reported in Dynes et al., PNAS 2023, and these measurements were performed using the same patch-TIRF approach as the present manuscript. While photoinactivation of jGCaMP8f fused to Orai1 interfered with kinetic measurements, Orai1-jGCaMP8f V203Y (a mutant with greatly reduced photoinactivation) exhibited a tauon of 10 ms and tauoff of 15 ms, roughly twice as fast as the values reported for Orai1-HaloTag-JF646-BAPTA in the present manuscript. The manuscript text comparing Halo-Tag kinetics with GECI should be revised accordingly.

The authors suggest that single-channel events reported previously for Piezo1 channels (Bertaccini et al., Nat Comm 2025 PMID: 40593468) may be due to probe blinking. However, that study included two critical controls that demonstrate that signals reflect bona fide channel activity rather than blinking artifacts. Notably: (1) treatment with channel activator Yoda1 increased bright-state occupancy (Figure 3C - 3G), and (2) increasing channel open probability by administering a mechanical stimulus increased bright-state occupancy (Supplementary Figure 13).