Fluorescence correlation spectroscopy detects galanin receptor diversity on insulinoma cells

Fluorescence correlation spectroscopy (FCS) allows the study of interaction s of fluorescently labeled ligand with receptors in living cells at single- molecule detection sensitivity. From the autocorrelation functions of fluor escence intensity fluctuations, the diffusion time of molecules through the confocal volume is analyzed, and from that, the molecular weights of free and bound molecules can be calculated. We have applied FCS to study the rec eptor diversity for the neuropeptide galanin (GAL) in cultured cells. FCS m easurement of the fluorophore rhodamine-labeled GAL (Rh-GAL) has been perfo rmed in 0.2-fL-confocal volume elements of the laser beam. The analysis of autocorrelation functions of Rh-GAL in solution above cells and at cell mem branes demonstrates that the diffusion time of unbound Rh-GAL is 0.16 ms, w hereas diffusion times of membrane-bound Rh-GAL are 22 and 700 ms. Because both of the diffusion times (22 and 700 ms) are much longer as compared to that of unbound Rh-GAL, they correspond to slow-diffusing complexes when Rh -GAL is bound to the cell membranes. Addition of excess nonlabeled GAL is a ccompanied by competitive displacement. Full saturation of the GAL binding is obtained at nanomolar concentrations. Scatchard analysis of binding data reveal one binding process, assuming one binding site per Rh-GAL (n=1). On the other hand, the appearance of two diffusion times, 22 and 700 ms, sugg ests the existence of two subpopulations of GAL receptor complexes or two s ubtypes of GAL receptor not detected before. This makes an important point that FCS permits the identification of receptors, which were not possible t o detect before by conventional binding techniques. The inhibitory effect o f pertussis toxin on the GAL binding considers a G-protein-involved alloste ric system, important for the clarification of essential steps in the G-pro tein-related signal transduction. This study is of pharmaceutical significa nce, since it will provide insights into how FCS can be used as a rapid tec hnique for studying ligand-receptor interactions in living cells, which is one step forward for large-scale drug screening in cell cultures.