Wild type or mutant TACR3 in pIRES2-AcGFP1 (Clontech) were transfected into HEK293A cells (QBiogene) using polyethylenimine (Sigma) before seeding cells into poly-L-lysine-coated glass-bottomed dishes 16 hours later. At 24 hours cells were loaded with Fura2-AM for 30 min. Experiments were performed on an inverted fluorescence microscope (Eclipse TE2000, Nikon, UK) with a 40x oil-immersion objective. Fura2 was excited at 340, 360 and 380 nm, and GFP at 475 nm, using a 75W xenon arc lamp and a monochromator (Cairn Research, Faversham, UK) and MetaFluor software (Molecular Devices, UK). Emission, filtered at 510/80 and 535/25 for Fura2 and GFP respectively, was recorded with a QuantEM CCD camera (Photometrics, Roper Scientific, UK). NKB (Sigma) or custom made mutant NKB with or without C terminal amidation (New England Peptides) were perfused in bath solution at ~1 ml/min with a chamber volume of ~0.2 ml. 340/380 nm ratio was calculated from images taken at 100 ms excitation at each wavelength at 0.5 Hz. Free Ca2+-concentrations were calculated for individual cells after background subtraction using the equation of Grynkiewicz et al (1985) assuming a KD
