Axopatch 200a

HEK293T cells were maintained in DMEM (Invitrogen) with 10% fetal bovine serum on poly-L-lysine-coated coverslips. Cells were patch-clamped in whole-cell configuration 16–24 h after transfection in high potassium solution containing 120 mM KCl, 25 mM NaCl, 10 mM HEPES, 2 mM CaCl₂, and 1 mM MgCl₂, pH 7.4. Glass pipettes with a resistance of 3–7 MΩ were filled with intracellular solution containing 140 mM KCl, 10 mM HEPES, 3 mM Na₂ATP, 0.2 mM Na₂GTP, 5 mM EGTA, and 3 mM MgCl₂, pH 7.4. Cells were voltage clamped to −60 or −80 mV using an Axopatch 200A (Molecular Devices) amplifier.
All pharmacological compounds were applied using a gravity-driven perfusion system. Illumination was applied to the entire field of view using a Polychrome V monochromator (TILL Photonics) through a 20× objective (4 mW/mm² at 460 nm or 0.5 mW/mm² at 360 nm). pClamp software was used for both data acquisition and control of illumination. For bistable switching, the shutter was manually turned on or off at specific time points. To conjugate MAP to WT and cysteines mutants of D1R or D2R, cells were incubated with 30 μM MAP for 60 min in the dark at 23–27 °C in standard extracellular cell buffers. These conditions are comparable to those used to label other engineered light-gated receptors with maleimide-azobenzene photoswitches. ^{48 (link),77 (link)}