Effects of the N343A mutation in TM6 of the human serotonin 5-HT2A Receptor

The human serotonin 2A receptor (h5-HT2A R) plays an essential role in cognition, and is the principal target for serotonergic hallucinogens as well as atypical antipsychotics. Utilizing our in silico -activated h5-HT2A R homology model, an interaction was predicted between Asn343(6.55) in TM6 and the carbonyl of LSD. Virtual docking simulations of a new class of 5-HT2 agonists, substituted N -benzyl phenethylamines (“ N -Benzyls”), indicated that interaction with N343 also might contribute to the high binding affinities of those compounds. It was hypothesized that an N343A mutation would have a detrimental effect on binding and activity of ergolines and N -Benzyl compounds with polar ortho-substituents. Surprisingly, affinity was relatively unaffected for most ergolines and N -Benzyls. LSD actually had 2-fold increased affinity for the N343A mutant, and other tryptamines had mixed effects. Dramatic decreases in binding affinity were observed with an N -Benzyl analogue that lacked a polar ortho-substituent ( N -naphthyl; >10-fold loss) and serotonin (>10-fold loss). Using phosphoinositide accumulation to assess function, LSD, DET, and DMT showed 3- to 4-fold increased potency, psilocin and 5-MeO-DMT were 2- and 5-fold less potent, respectively, and serotonin was 60-fold less potent. The N343A mutation also decreased the intrinsic activity of all drugs tested. Thus, our original hypothesis appears incorrect. We now hypothesize that N343 either stabilizes a high-energy ground state of the helical bundle, or else may stabilize an activated receptor conformation once the ligand has bound. Supported by NIDA grant DA02189.