For patch-clamp recordings under single turnover conditions, the coding sequence of CsR was cloned into the pEGFP-N1 vector. Patch pipettes were prepared from borosilicate glass capillaries (G150F-3, Warner Instruments) using a P-1000 micropipette puller (Sutter Instruments) and fire polished for a final pipette resistance of 1.5 to 3.0 megohm.

Single fluorescent cells were identified using an Axiovert 100 inverted microscope (Carl Zeiss). Monochromatic light for continuous illumination was provided by a Polychrome V monochromator (TILL Photonics) and temporally controlled by a VS25 and VCM-D1 shutter system (Vincent Associates). For single turnover experiments, nanosecond laser pulses were generated by an Opolette Nd:YAG laser/OPO system (OPOTEK) and selected using a LS6ZM2 shutter system (Vincent Associates). Both light sources were coupled into the microscope and delivered to the sample using a 90/10 beamsplitter (Chroma). Light intensities were measured after passing through all of the optics using either a P-9710 optometer (Gigahertz-Optik) or a calibrated S470C thermal power sensor and a PM100D power and energy meter (Thorlabs) for continuous illumination or single laser pulses, respectively. Average light intensity at continuous illumination was 2.47 mW × mm−2, and average laser pulse energy adjusted by the built-in motorized variable attenuator was 11.3 ± 1.4 μJ.

During whole-cell patch-clamp recordings, membrane resistance was generally higher than 1 gigohm, and access resistance was below 10 megohm. Pipette capacity, series resistance, and cell capacity compensation were applied. Recorded signals were filtered at 2 or 100 kHz using an Axopatch 200B amplifier (Molecular Devices) and digitized using a Digidata 1440A digitizer (Molecular Devices) at a sampling rate of 250 kHz. A 140 mM NaCl agar bridge was used for the reference bath electrode. Extracellular bath solutions contained 110 mM NaCl, 1 mM KCl, 1 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, and 10 mM Hepes at pHe 7.2 (with glucose added up to 310 mOsm). The intracellular pipette solution contained 110 mM NaCl, 1 mM KCl, 1 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM EGTA, and 10 mM Hepes at pHi 7.2 (glucose added up to 290 mOsm). All electrical recordings were controlled by pCLAMP software (Molecular Devices).

The recordings were baseline-corrected and time-shifted after measurements to align the rising edges of the Q-switch signals of the activating laser pulses using Clampfit 10.4 software (Molecular Devices). The recordings were then binned to 50 logarithmically spaced data points per temporal decade, normalized to peak photocurrents at +30 mV, and averaged for up to 10 individual repetitions for each cell and voltage condition with a custom MATLAB script (MathWorks). Kinetic analysis was performed in Origin 9.1 (OriginLab). To determine statistical significance, each measurement was repeated multiple times with different biological replicates in at least two independent experiments. The number of biological replicates for each measurement is provided in the figure legends.

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