Plan neofluar

VCF recordings were made on Xenopus oocytes provided by Ecocyte Bioscience (Germany), injected with 50 ng/μL of plasmid DNA encoding for the m5-HT3A receptor mutant S19’C and after 2 to 6 days of expression in Barth solution at 17°C. They were labeled with MTS-TAMRA (Toronto chemicals) 5 min at 17°C in ND96 buffer without CaCl2 + 10 μM 5-HT, then rinsed and stored at 17°C up to 4h before recording in ND96. Recording were made in a TEVC set up previously described¹³ and adapted for fluorescence recording. Briefly recordings were done with a VCF dedicated chamber with only a fraction of the oocyte perfused and illuminated by a LED (coolled PE-4000). Light was collected using a fluorescence microscope (Zeiss Axiovert135) equipped with a 40x objective (Plan neofluar), a TRITC filter set and a photomultiplier tube (Hamamatsu photonics). Recordings were made at -60mV clamp, 500 Hz sampling rate with a 550 nm excitation wavelength. Data were filtered, corrected for baseline and photobleaching when necessary and analyzed using pClamp and Axograph. Dose response curves were fitted to the Hill equation I/Imax = 1/(1 + (EC₅₀/[5-HT])^nH) using Prism. Serotonin dose-response relations were shifted to the left with half-responses for fluorescence and current at 40 and 240 nM respectively (the EC₅₀ of the wild-type receptor is 800 nM).