Benzodiazepine Binding Studies on Living Cells: Application of Small Ligands for Fluorescence Correlation Spectroscopy

Abstract

We demonstrate the applicability of fluorescence correlation spectroscopy (FCS) for receptor binding studies using low molecular weight ligands on the membranes of living nerve cells. The binding of the benzodiazepine Ro 7-1986/602 (N-des-diethyl-fluorazepam), labeled with the fluorophore Alexa 532, to the benzodiazepine receptor was analyzed quantitatively at the membrane of single rat hippocampal neurons. The values obtained for the dissociation constant Kd = (9.9 +/- 1.9) nm and the rate constant for ligand-receptor dissociation kdisS = (1.28 +/- 0.08) x 10(-3) s(-1) show that there is a specific and high affinity interaction between the dye-labeled ligand (Ro-Alexa) and the receptor site. The binding was saturated at approx. 100 nM and displacement of 10 nM Ro-Alexa, with a 1,000-fold excess of midazolam, showed a non-specific binding of 7-10%. Additionally, two populations of the benzodiazepine receptor that differed in their lateral mobility were detected in the membrane of rat neurons. The diffusion coefficients for these two populations [D(bound1) = (1.32 +/- 0.26) microm2/s; D(bound2) = (2.63 +/- 0.63) x 10(-2) microm2/s] are related to binding sites, which shows a mono-exponential decay in a time-dependent dissociation of the ligand-receptor complex.

Transport of a hyaluronan-binding protein in brain tissue

“…The signal was auto-correlated online to data acquisition with a digital hardware correlator (ALV-5000, ALV; Langen, Germany). Prior to the experiments, the volume element of observation was calibrated using a defined concentration of rhodamine 6G as described (Hegener et al, 2002). Hyaluronan from rooster comb was bound to glass surfaces by drying saturated solutions of the glycosaminoglycan on coverslips.…”

“…Measurements were performed in the presence of nanomolar concentrations of the fluorescent proteins in the solution. Autocorrelation curves were fitted with the FCS Access software (Evotec) allowing for up to three different species as described previously (Hegener et al, 2002;Meissner and Häberlein, 2003).…”

Hepatoma-derived Growth Factor

“…All cells were tested for binding of the fluorescent ligands at the concentrations given under "Results." In addition, for all cells and ligands, laser scans were recorded as described by Hegener et al (34,35).…”

Structure-Activity Relationship Studies: Methods and Ligand Design for G-Protein Coupled Peptide Receptors

“…An introduction to the theoretical and experimental background is given by a number of publications [41,42]. Application of FCS was shown by researching galanine receptors in cultured cells [43,44]. The appearance of different diffusion times of rhodamine labeled galanine (GAL) at cells and cell membranes led to the suggestion of different receptor subtypes present in the researched cells.…”