Unknown

Outside-out membrane patches from transfected HEK293 cells were held under voltage-clamp at a holding potential of _60 mV using an Axopatch 200B amplifier. During recordings, cells were perfused continuously with extracellular control solution containing 20 mM sucrose, 145 mM NaCl, 5.4 mM KCl, 5 mM HEPES, 1 mM MgCl2, 1.8 mM CaCl2, and 0.01 mg/ml phenol red, pH 7.3. Currents were evoked with test solutions containing 10 mM glutamate. Control and test solutions were perfused through quartz tubing. The patch pipette tip was positioned in the control solution stream near the interface between the control and glutamate-containing streams. When modulators were used, they were added to both test and control solutions in the following concentrations: 100 mkM CTZ, 100 mkM CX614, and 10 mkM CMPDA or CMPDB. Modulator stock solutions (10 mM) were dissolved in DMSO before dilution in extracellular solutions; final DMSO concentrations were 0.3 to 1%. When applying glutamate in the absence of modulator, after having perfused modulator through the tubing for a previous patch, we noticed that the steady-state current responses were not completely desensitized.