Abstract

The structurally divergent agents gallamine and hexamethylene-bis-[dimethyl-(3-phthalimidopropyl)ammonium]dibromide (W84) are known to interact competitively at a common allosteric site on muscarinic receptors. Previous studies reported that the M₂ selectivity of gallamine depended largely on the EDGE (172-175) sequence in the second outer loop (o2) and on ⁴¹⁹Asn near the junction of o3 and the seventh transmembrane domain (TM7), whereas the selectivity of W84 depended on nearby residues ¹⁷⁷Tyr and ⁴²³Thr. However, it has so far proven difficult to confer the high sensitivity for allosteric modulation of the M₂ subtype onto the weakly sensitive M₅ subtype by substituting these key residues. We now have found that M₂ ⁴²³Thr, not ⁴¹⁹Asn, is the dominant residue in the o3/TM7 region for gallamine's high potency, although ⁴¹⁹Asn can substitute for ⁴²³Thr in some contexts; in contrast, the presence of ⁴¹⁹Asn reduces the potency of W84 in every context we have studied. In addition, the orientation of ¹⁷⁷Tyr is crucial to high sensitivity toward W84, and it seems that the proline residue at position 179 in M₅ (corresponding to M₂ ¹⁷²Glu) may interfere with that orientation. Consistent with these observations, a mutant M₅ receptor with these three key mutations, M₅P179E, Q184Y, and H478T, showed dramatically increased sensitivity for W84 (>100-fold), compared with the wild-type M₅ receptor. This same mutant receptor approached M₂ sensitivity toward gallamine. Thus, gallamine and W84 derive high potency from the same receptor domains (epitopes in o2 and near the junction between o3 and TM7), even though these allosteric agents have quite different structures.