INTERACTION OF TRYPTAMINE AND ERGOLINE COMPOUNDS WITH THREONINE-196 IN THE LIGAND-BINDING SITE OF THE 5-HYDROXYTRYPTAMINE(6) RECEPTOR

We examined the ligand-binding site of the 5-hydroxytryptamine(6) (5-H T6) receptor using site-directed mutagenesis. Interactions with residu es in two characteristic positions of transmembrane region V are impor tant for ligand binding in several bioamine receptors. In the 5-HT6 re ceptor, one of these residues is a threonine (Thr196), whereas in most other mammalian 5-HT receptors, the corresponding residue is alanine. After transient expression in human embryonic kidney 293 cells, we de termined the effects of the mutation T196A on [H-3]d-lysergic acid die thylamide (LSD) binding and adenylyl cyclase stimulation. This mutatio n produced a receptor with a 10-fold reduced affinity for [H-3]LSD and a 6-fold reduced affinity for 5-HT. The potency of both LSD and 5-HT for stimulation of adenylyl cyclase was also reduced by 18- and 7-fold , respectively. The affinity of other N1-unsubstituted ergolines (e.g. , ergotamine, lisuride) was reduced 10-30-fold, whereas the affinity o f N1-methylated ergolines (e.g., metergoline, methysergide, mesulergin e) and other ligands, such as methiothepine, clozapine, ritanserin, am itriptyline, and mianserin, changed very little or increased. This ind icates that in wild-type 5-HT6 receptor, Thr196 interacts with the N1 of N1-unsubstituted ergolines and tryptamines, probably forming a hydr ogen bond. Based on molecular modeling, a serine residue in transmembr ane region IV of the 5-HT2A receptor has previously been proposed to i nteract with the N1-position of 5-HT. When the corresponding residue o f the 5-HT6 receptor (Ala154) was converted to serine, no change in th e affinity of twelve 5-HT6 receptor ligands or in the potency of 5-HT and LSD could be detected, suggesting that this position does not cont ribute to the ligand binding site of the 5-HT6 receptor.