Critical amino acid residues of the common allosteric site on the M 2 muscarinic acetylcholine receptor

More similarities than differences between the structurally divergent agents gallamine and bis(ammonio)alkane-type hexamethylene-bis-[dimethyl-(3-phthalimidopropyl) ammonium]dibromide

Xi Ping Huang, Stefanie Prilla, Klaus Mohr, John Ellis

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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 M 2 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.

Original languageEnglish (US)
Pages (from-to)769-778
Number of pages10
JournalMolecular Pharmacology
Volume68
Issue number3
DOIs
StatePublished - Sep 1 2005

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Gallamine Triethiodide
Allosteric Site
Alkanes
Muscarinic Receptors
Ammonium Compounds
Amino Acids
Proline
Epitopes
Mutation

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

@article{9a28617d1aba4869b2029a78c006d725,
title = "Critical amino acid residues of the common allosteric site on the M 2 muscarinic acetylcholine receptor: More similarities than differences between the structurally divergent agents gallamine and bis(ammonio)alkane-type hexamethylene-bis-[dimethyl-(3-phthalimidopropyl) ammonium]dibromide",
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 M 2 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.",
author = "Huang, {Xi Ping} and Stefanie Prilla and Klaus Mohr and John Ellis",
year = "2005",
month = "9",
day = "1",
doi = "10.1124/mol.105.014043",
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T1 - Critical amino acid residues of the common allosteric site on the M 2 muscarinic acetylcholine receptor

T2 - More similarities than differences between the structurally divergent agents gallamine and bis(ammonio)alkane-type hexamethylene-bis-[dimethyl-(3-phthalimidopropyl) ammonium]dibromide

AU - Huang, Xi Ping

AU - Prilla, Stefanie

AU - Mohr, Klaus

AU - Ellis, John

PY - 2005/9/1

Y1 - 2005/9/1

N2 - 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 M 2 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.

AB - 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 M 2 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.

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