When an allosteric modulator binds to its site on the receptor protein, the resulting conformational change yields a protein with different properties. Allosteric modulators will cause a change in the association and/or dissociation characteristics of the orthosteric ligand at its receptor. A change in the dissociation characteristics of an orthosteric ligand in the presence of a second agent can only occur as a consequence of a conformational change in the receptor. Consequently, the dissociation kinetic assay has become a standard technique for the detection of allosteric modulation. The aim of this study was to search for allosteric interactions between CB₁ receptor ligands by using a radioligand binding assay to investigate the kinetics of dissociation of these ligands from CB₁ receptors in the brain.

We utilised the 'isotopic dilution' method to measure the dissociation rate constant for [³H]-CP55940 from brain membranes. [³H]-CP55940 (0.5nM) was incubated with mouse (adult, male MF1) brain membranes (100µg) for 60 minutes at 25°C in Tris (50mM) containing 1mg/ml BSA. Dissociation was initiated by the addition of 1µM CP55940 in the presence and absence of test compounds. Dissociation times of 0.5 to 120 minutes were used. To determine the non-specific binding, some experiments were also performed in the presence of 1µM CP55940.

The dissociation curve for [³H]-CP55940 was best-fit to a biphasic exponential, the dissociation rate constants (k_-1) for the two "agonist dissociation states" being 0.00780 ± 0.0024 and 0.1435 ± 0.040 min^-1 (n = 6). In the presence of SR141716A (10µM), the corresponding k_-1 values for [³H]-CP55940 of 0.0102 ± 0.0026 and 0.0908 ± 0.061 min^-1 were not significantly different (P>0.05, Students' unpaired t-test) (n = 6). However, SR141716A (10µM) significantly altered (P<0.05, Student's unpaired t-test) the relative proportion of the two "agonist dissociation states". Thus 77 ± 4.90 % of the specific binding dissociated slowly in the presence of SR141716A compared to 39 ± 6.25% in its absence. The non-hydrolysable GTP analogue, Gpp(NH)p, is known to uncouple complexes between receptors and G-protein. Like SR141716A, Gpp(NH)p did not alter the k_-1 values for [³H]CP55940 which were 0.00696 ± 0.00305 and 0.1448 ± 0.0342 min^-1. However, in contrast to SR141716A, Gpp(NH)p reduced the proportion of the slower "agonist dissociation state" to 15 ± 3.16 %.

In conclusion, [³H]-CP55940 exhibits distinctly biphasic dissociation from CB₁ receptors, supporting the hypothesis that CB₁ receptors can exist in different "agonist dissociation states". In addition, we have found that Gpp(NH)p and the inverse agonist SR141716A, have opposite effects on the relative proportion of slow and fast "agonist dissociation states" of the CB₁ receptor.

Supported by NIDA and The Wellcome Trust.