Experiments in HEK293T Cells with Flash Photolysis.

HEK293T cells were cultured in DMEM (PAN Biotech) supplemented with 10% FCS (Biochrom AG), l-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C and 7% CO₂.

For FRET experiments, cells were seeded onto poly-d-lysine–coated UV-transparent quartz coverslips (Ted Pella, Inc.) in six-well plates 12–16 h before transfection. Cells were transfected using Effectene according to the manufacturer’s instructions. cDNA amounts were 150 ng of each protomer of the WT and mutant E sensors and 150 ng of the WT and mutant A sensors per coverslip. To minimize glutamate contact with receptors, cell culture medium was exchanged for DMEM-GlutaMAX (Gibco) 24 h after transfection. Approximately 60 h after transfection, cells were washed twice with HBSS (150 mM NaCl, 2.5 mM KCl, 2 mM MgCl₂, 4 mM CaCl₂, 10 mM Hepes, 10 mM glucose, pH 7.4) and incubated in HBSS buffer supplemented with 1.75 U/mL glutamate-pyruvate transaminase (Roche), 4 mM sodium pyruvate, and 0.1% BSA for 1 h.

For FRET experiments, coverslips mounted in an experimental chamber were placed on a custom-built inverted microscope (Axio Observer D1; Zeiss GmbH) kept at room temperature that was equipped with an oil-immersion objective (Fluar 100×/1.30 Oil UV; Zeiss GmbH) and a light-emitting diodes system (pE-4000; CoolLED Ltd.) as an excitation light source. On excitation with 435 ± 9 nm, fluorescence emission was simultaneously recorded at 483 ± 16 nm (CFP) and 535 ± 15 nm (YFP) before and after the addition of MNI-caged l-glutamate (1 mM final concentration). Fluorescence signals were detected by photometry systems, each of which contained a gated photomultiplier (ET Enterprises Ltd.) and photometer amplifier unit (Myotronic). Photocurrents were digitized at 10-kHz sampling frequency using an analog–digital converter Axon Digidata 1550 (Molecular Devices) and recorded with the pClamp software (Molecular Devices).

Photouncaging of MNI-caged l-glutamate was achieved by a short 375-nm laser flash generated by a trigger-controlled UV laser source (DL 375; Rapp OptoElectronic GmbH). The laser source was coupled to the microscope via a quartz fiber optic light guide and collimated to the objective. To estimate the size of UV laser focal spot size, we used caged fluorescein. A thin layer of 5-carboxymethoxy-2-nitrobenzyl fluorescein was laid on a quartz coverslip and allowed to dry to minimize lateral diffusion of the dye. The size of the fluorescent spot was measured through the obtained image from the CMOS camera (DCC3240N; Thorlabs). Intensity image was plotted in a 3D domain, and assuming that the laser beam has an ideal Gaussian intensity profile, we approximated it with a 2D Gaussian function:

where F₀ is the offset; A is the amplitude relative to the baseline; x_c and y_c are coordinates of the peak; w₁ and w₂ are x and y spreads of the blob, respectively; and θ is the orientation of the blob.

FWHM values were further calculated as follows:

Fluorescence emissions of both donor and acceptor were corrected for background, fluorophores quenching, and bleed through of donor light into the acceptor channel essentially as described previously (38). In detail, background was measured in each channel for every experiment as a fluorescence intensity of neighboring nontransfected cells. Fluorophore quenching was corrected by subtracting the corresponding exponential curves for CFP and YFP. Bleed through of CFP emission into the YFP channel was estimated as 36%. FRET ratios were further corrected for the transient inner filter effect of the nitrosoindole by-product of MNI–l-glutamate uncaging (39). Corrected photocurrents were analyzed with OriginPro software (OriginLab).

FRET values at time point t were determined as follows:

where R(t) is the observed YFP/CFP ratio, R_p is a plateau ratio value at the peak of transient signal, and R_b is a baseline ratio value before uncaging.