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

Following ER Ca²⁺ depletion, Ca²⁺ release-activated Ca²⁺ (CRAC) channels are activated by STIM1 at ER-plasma membrane junctions. The restricted localization and low conductance of the CRAC channel (<40 fS) precludes single-channel recordings, limiting studies of CRAC channel gating. Here we describe an optical approach to characterize the gating of HaloTag-fused Orai1 channels labeled with JF646-BAPTA, a Ca²⁺-sensitive fluorescent dye. While Ca²⁺ influx through single channels generates fluorescence fluctuations, identifying true gating events is complicated by stochastic transitions of JF646-BAPTA to a non-fluorescent state. To overcome this, we combine TIRF microscopy with whole-cell voltage clamp to control the driving force for Ca²⁺ entry. We show the open channel intensity at -100 mV reflects Ca²⁺ saturation of the dyes on each channel, while the closed-channel intensity is defined by the fluorescence at +30 mV, where influx is absent. True gating events can be identified from transitions between the open- and closed-channel levels, distinguishing them from transitions to a non-fluorescent state. We describe the gating behavior of CRAC channels activated by STIM1 after store depletion. Dwell time distributions indicate at least two open and closed states with durations of 0.1 to several seconds, with most channels having an open probability of ≥0.7. We also detect 'silent' channels that colocalize with STIM1 but show no activity over tens of seconds, a population that would be undetectable by whole-cell electrophysiology alone. This method offers an approach to explore CRAC channel gating mechanisms and may be applicable to other Ca²⁺-permeable channels not amenable to patch-clamp techniques.