Kinetic binding radioligand

In the following, an example of this new kind of MS binding experiment is presented as a straightforward alternative to conventional radioligand binding assays and suitable for the performance of saturation, competition and kinetic binding assays [80]. 

Direct quantitation of receptor concentrations and dmg—receptor interactions is possible by a variety of techniques, including fluorescence, nmr, and radioligand binding. The last is particularly versatile and has been appHed both to sophisticated receptor quantitation and to dmg screening and discovery protocols (50,51). The use of high specific activity, frequendy pH]- or p lj-labeled, dmgs bound to cmde or purified cellular materials, to whole cells, or to tissue shces, permits the determination not only of dmg—receptor saturation curves, but also of the receptor number, dmg affinity, and association and dissociation kinetics either direcdy or by competition. Complete theoretical and experimental details are available (50,51). 

For these reasons, kinetic measurements are now usually done with isolated cells (e.g., a single neuron or a muscle fiber) or even a patch of cell membrane held on the tip of a suitable microelectrode. Another approach is to work with a cell membrane preparation and examine direcdy the rate at which a suitable radioligand combines with, or dissociates from, the receptors that the membrane carries. Our next task is to consider what binding kinetics might be expected under such conditions. 

Both the onset of binding, when the radioligand is first applied, and offset, when dissociation is promoted, can be studied directly. The relevant kinetic equations relating to the simple bimolecular interaction of ligand with receptor are presented in Chapter 1, Section 1.3. 

Figure 2 Time course of radioligand binding kinetics. Radioligand is introduced to the system (A) and allowed to bind (B) until equilibrium is reached (Q. Free radioligand is removed or outcompeted by an excess of orthosteric cold ligand (D) which results in a time-dependent dissociation of radioligand (E).

Without any calcium added, specific binding increased with the radioligand concentration, but saturation was not reached and meaningful kinetics could not be calculated. None of the known RyR ligands like ryanodine, calmodulin, methylxanthines, ATP, cADP-ribose, nor dantrolene affected flubendiamide binding. 

Since the kinetics of receptor binding, and therefore the sensitivity of a radioreceptor assay, may vary with tissue concentration or incubation conditions, it is prudent to calculate a standard curve for the displacement of radioligand during each experiment. To this end, the amount of radioligand bound in the presence and absence of various concentrations... 

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