(A) APP+ is a fluorescent analogue of MPP+. Excitation and emission spectra of APP+ occur at wavelengths ranging from 410 to 450 nm and 510 to 550 nm, respectively, which depends on solvent properties. (B) Left, schematics of the setup used in this study. The setup included (1) a dichroic glass that specifically reflects light of 440 nm but allows transmission of those with a wavelength of 550 nm, (2) an inverted microscope with a x100 objective (for single-cell fluorescence recordings), (3) an electrode and patch-clamp amplifier that allows for voltage control, (4) a photomultiplier tube (PMT) that converts emitted fluorescence into electrical signal, and (5) and an acquisition system that allows for filtering, digitizing, and two-channel recordings of APP+-induced fluorescence and currents. Right, a theoretical trace for a two-channel recording that displays simultaneous APP+-induced currents (red trace) and fluorescence (orange trace) in real time. (C) A simplified scheme of a secondary active transporter that employs Na+ and Cl- as co-substrates. Substrate-induced currents comprise two components: peak current (reactions highlighted in red and b in (B), right) that represents movement of charged substrate/co-substrates through the membrane electric field and steady-state current (reactions highlighted in blue and a in (B), right) that represents transporters functioning in the physiological forward mode. (D) Representative traces of the APP+-induced currents and fluorescence in empty HEK293 cells or in HEK293 cells expressing dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT) patched under normal physiological ionic conditions. In all traces, the rapid rise and decline in fluorescence on applying and washing off APP+, respectively (see 'Results'), are shown. All three transporters showed a linear increase in fluorescence as a function of time, indicative of APP+ accumulation in cells expressing DAT, SERT, and NET. APP+ induced robust DAT-mediated currents that comprise both peak and steady-state currents. Only peak currents (represented as magnified inset) were seen on APP+ application to cells expressing NET and SERT. AU – arbitrary units.