Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors

Ann L. Gnagey, Margaret Seidenberg, John Ellis

Research output: Contribution to journalArticle

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Abstract

Gallamine allosterically modulates the binding of classical muscarinic ligands with a potency order of M2 > M1,M4 > M3,M5. We have suggested previously that the M2/M5 and M2/M3 selectivities are attributable to an epitope in the sixth transmembrane region or third outer loop (o3) region of the receptor. In this study, analysis of numerous point mutations in this region of the M5 receptor found that a mutation of V → N resulted in an increased affinity toward gallamine, suggesting that the asparagine residue at M2419 is responsible for gallamine's M2/M5 selectivity. Mutations in the other subtypes indicated that the acidic residues found at this position in M1 and M4 are associated with slightly higher affinity toward gallamine, whereas the valine and lysine residues of M5 and M3, respectively, are associated with significantly lower affinity. In the o2 region, replacement of an acidic sequence of M2 (EDGE) by the corresponding neutral sequence of M1 (LAGQ) reduced the affinity toward gallamine, as reported previously by others; the converse substitution of the acidic sequence into M1 significantly increased affinity for gallamine. Substitution of the M1 sequence into this region of M5 markedly reduced affinity toward gallamine, whereas substitution into M4 had no effect. All of the above mutations are consistent with gallamine binding with a similar orientation at each subtype, such that it interacts with acidic residues in the o2 region of M3 and M5 and with acidic residues in the o3 region of M1 and M4; gallamine appears to interact with both regions of the M2 subtype.

Original languageEnglish (US)
Pages (from-to)1245-1253
Number of pages9
JournalMolecular pharmacology
Volume56
Issue number6
DOIs
StatePublished - Jan 1 1999

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Gallamine Triethiodide
Muscarinic Receptors
Site-Directed Mutagenesis
Epitopes
Mutation
Asparagine
Valine
Point Mutation
Cholinergic Agents
Lysine
Ligands

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

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title = "Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors",
abstract = "Gallamine allosterically modulates the binding of classical muscarinic ligands with a potency order of M2 > M1,M4 > M3,M5. We have suggested previously that the M2/M5 and M2/M3 selectivities are attributable to an epitope in the sixth transmembrane region or third outer loop (o3) region of the receptor. In this study, analysis of numerous point mutations in this region of the M5 receptor found that a mutation of V → N resulted in an increased affinity toward gallamine, suggesting that the asparagine residue at M2419 is responsible for gallamine's M2/M5 selectivity. Mutations in the other subtypes indicated that the acidic residues found at this position in M1 and M4 are associated with slightly higher affinity toward gallamine, whereas the valine and lysine residues of M5 and M3, respectively, are associated with significantly lower affinity. In the o2 region, replacement of an acidic sequence of M2 (EDGE) by the corresponding neutral sequence of M1 (LAGQ) reduced the affinity toward gallamine, as reported previously by others; the converse substitution of the acidic sequence into M1 significantly increased affinity for gallamine. Substitution of the M1 sequence into this region of M5 markedly reduced affinity toward gallamine, whereas substitution into M4 had no effect. All of the above mutations are consistent with gallamine binding with a similar orientation at each subtype, such that it interacts with acidic residues in the o2 region of M3 and M5 and with acidic residues in the o3 region of M1 and M4; gallamine appears to interact with both regions of the M2 subtype.",
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Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors. / Gnagey, Ann L.; Seidenberg, Margaret; Ellis, John.

In: Molecular pharmacology, Vol. 56, No. 6, 01.01.1999, p. 1245-1253.

Research output: Contribution to journalArticle

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AU - Gnagey, Ann L.

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