Forty-five structurally diverse 5-hydroxytryptamine(6) receptor (5-HT(6)R) antagonists were selected to develop a 3D pharmacophore model with the Catalyst software. The structural features for antagonism at this receptor are a positive ionizable atom interacting with Asp(3.32), a hydrogen bond acceptor group interacting with Ser(5.43) and Asn(6.55), a hydrophobic site interacting with residues in a hydrophobic pocket between transmembranes 3, 4, and 5, and an aromatic-ring hydrophobic site interacting with Phe(6.52).

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We previously demonstrated that D3.49(164)Y or T6.34(279)K mutation in the rat mu opioid receptor (MOPR) resulted in agonist-independent activation. Here, we identified the cysteine(s) within the transmembrane domains (TMs) of the D3.49(164)Y mutant that became accessible in the binding-site crevice by use of methanethiosulfonate ethylammonium (MTSEA) and inferred conformational changes associated with receptor activation. While the C7.38(321)S mutant was insensitive to MTSEA, the D3.49(164)Y/C7.38(321)S mutant showed similar sensitivity as the D3.49(164)Y, suggesting that, in the D3.49(164)Y mutant, C7.38(321) becomes inaccessible while other cysteines are accessible in the binding-site crevice. Each of the other seven cysteines in the TMs was mutated to serine on the background of D3.49(164)Y/C7.38(321)S, and the resulting triple mutants were evaluated for [3H]diprenorphine and [d-Ala2,NMe-Phe4,Gly5-ol]-enkephalin (DAMGO) binding and effect of MTSEA on [3H]diprenorphine binding. The D3.49(164)Y/C7.38(321)S mutant and the triple mutants, except the C6.47(292)S triple mutant, retained similar affinities for [3H]diprenorphine and DAMGO as the D3.49(164)Y mutant. The second-order rate constants for MTSEA reactions showed that C3.44(159)S, C4.48(190)S, C5.41(235)S, and C7.47(330)S significantly reduced sensitivity to MTSEA, compared with the D3.49(164)Y/C7.38(321)S. These results suggest that the four cysteines may be rotated and/or tilted to become accessible. While the D3.49(164)Y/C7.38(321)S was similarly sensitive to MTSEA as the D3.49(164)Y mutant, the T6.34(279)K/C7.38(321)S was much less sensitive to MTSEA than the T6.34(279)K mutant, suggesting that the two constitutively active mutants assume different conformations and/or possess different dynamic properties. Molecular models of the MOPR monomer and homodimer, using the crystal structures of rhodopsin, the beta2-adrenergic receptor, and the ligand-free opsin, which contains several features characteristic of the active state, were employed to analyze these experimental results in a structural context.