Study of Ligand−Receptor Interactions by Fluorescence Correlation Spectroscopy with Different Fluorophores: Evidence That the Homopentameric 5-Hydroxytryptamine Type 3AsReceptor Binds Only One Ligand†

T. Wohland, K. Friedrich, R. Hovius, H. Vogel
1999 Biochemistry  
The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-aminopropyl)-4H-carbazol-4-one (GR-H) was labeled with the fluorophores rhodamine 6G,
more » ... rhodamine 6G, fluorescein, N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl], and the cyanine dye Cy5. These labels cover a large part of the visible electromagnetic spectrum. It is shown that the photophysical and chemical properties have a direct influence on the measurement quality (duration of measurement, signal-to-noise ratio) and the ligandreceptor interactions (dissociation constants), respectively. This makes it necessary to choose a suitable label or a combination of labels for receptor studies. The affinities of the fluorescently labeled ligands determined by FCS were virtually identical to the values obtained by radioligand binding experiments. Moreover, the dissociation constant of a nonfluorescent receptor ligand was determined successfully by an FCS competition assay. The experimental results showed that only one antagonist binds to the receptor, in agreement with measurements previously published [Tairi et al. (1998) Biochemistry 37, 15850-15864]. Fluorescence correlation spectroscopy (FCS) 1 measures statistical fluctuations of the fluorescence intensity in a small illuminated sample volume to obtain information about the chemical and photophysical properties of individual fluorescent molecules. This technique was first described more than two decades ago (1-4) and has recently regained interest for the investigation of and screening for molecular properties and interactions (5-10). Since then, several ligand-receptor systems have been investigated (11, 12), but so far no systematic comparative studies have been conducted on how a particular type of fluorophore influences the FCS measurement itself or the reactions between the interacting molecules. However, because FCS requires suitable fluorophores that, with a few exceptions, are extrinsic labels, it is essential to select a fluorescent probe that allows accurate measurements without disturbing the interaction between the molecules under investigation. In the present communication we investigate the specific interaction between fluorescently labeled ligands and the short spliced form of the purified serotonin ()hydroxytryptamine) type 3A receptor (5HT 3As receptor). This receptor is a typical example of a neuroreceptor functioning as a ligand-gated ion channel. Membrane receptors, in particular channel proteins, are important targets for therapeutic agents (13). In this respect, the investigation of ligand-receptor interactions is equally important for fundamental and applied research. It provides necessary information on the molecular mechanism of receptor function and, in addition, is the basis for the screening of potential pharmaceutical active compounds. FCS promises, in this context, novel opportunities with respect to sensitivity, miniaturization, and testing large numbers of ligands or receptors. The 5HT 3As receptor is ideally suited to investigate the applicability of this analytical technique in the field of neuroreceptors. First, this receptor is of medical interest as a target of several important therapeutic agents. Such compounds have an influence, for instance, on depression, †
doi:10.1021/bi990366s pmid:10393542 fatcat:jae7xv2dvnc2jh3iokpni5aj6y