Characterization of α(2B)-adrenoceptor ligand binding in the presence of muscarinic toxin α and delineation of structural features of receptor binding selectivity.

Muscarinic toxin α (MTα), a peptide isolated from the venom of the African black mamba, was recently found to selectively antagonize the human α(2B)-adrenoceptor. To gain more information about the binding of this peptide toxin, we studied the properties of the [³H]UK14,304 agonist and the [³H]MK-912 antagonist binding to the α(2B)-adrenoceptor in the presence of MTα. In equilibrium binding experiments, MTα decreased the binding of the orthosteric ligands, but failed to completely displace these. This effect of MTα was due to noncompetitive inhibition of B(max) without change in radioligand affinity. On the contrary, cellular signaling via the α(2B)-adrenoceptor could be titrated to zero despite the incomplete receptor blockade. To locate binding sites for MTα on the receptor protein, we generated chimeric receptors of α(2B)- and α(2A)- or α(2C)-adrenoceptors. Data based on these constructs revealed the extracellular loop two (ECL2) as the structural entity that enables MTα binding. Cumulative exchange of parts of ECL2 of α(2B) for α(2A)-adrenoceptor sequence resulted in a gradual decrease in the affinity for MTα, indicating that MTα binds to the α(2B)-adrenoceptor through multiple sites dispersed over the whole ECL2. Together the results suggest that binding of MTα to the α(2B)-adrenoceptor occludes orthosteric ligand access to the binding pocket. Putative homomeric receptor complexes as factors underlying the apparent noncompetitivity are also discussed.
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