The dynamics of agonist-receptor interactions within the complex environment of the cell membrane are still only partially understood. In particular, the microenvironment of the cell surface contains multiple compartments that are highly variable in both space and time. By employing a diffusion measurement technique (fluorescence correlation spectroscopy; FCS) and a high speed confocal optical imaging technique, we can directly observe and measure peptide binding to receptors on transfected cell lines and intact neurons.
In our experiments imaging was performed using a Leica SP5 laser scanning confocal microscope equipped with a high-speed (8kHz) resonant scanner and avalanche photo diode detectors. Photon counting data were acquired and analysed with LASAF and ISS Vista. Images were analysed using either the FCS software, or advanced functionality within ImageJ. Numerical data extracted from the images were analysed and modelled using SPSS.
We have demonstrated the application of this combination of techniques to G-protein coupled receptor (GPCR) agonist binding events involving a fluorescently labelled peptide hormone, angiotensin II (AngII) and the angiotensin II receptor (type1; AT1R). These interactions were studied in both live CHO cells transfected with AT1R (+/- enhanced green fluorescent protein; eGFP) and native sympathetic coeliac ganglion neurons from guinea-pig.
We found striking differences in the internalisation and desensitisation behaviour of AngII when comparing transfected CHO cells with native sympathetic neurons. Additionally we found the effective binding rates for AngII are significantly limited by reduced diffusion rates in the extra-cellular space. This toolkit of techniques has the potential to be applied to other GPCR-agonist combinations and other tissue preparations to better define the kinetics of diffusion and receptor binding.