Roles of amino acids and subunits in determining the inhibition of nicotinic acetylcholine receptors by competitive antagonists

James P. Dilger, Ana Maria Vidal, Man Liu, Claire Mettewie, Takahiro Suzuki, Anh Pham, Deeptankar DeMazumder

Research output: Contribution to journalArticle

Abstract

BACKGROUND: Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the α-δ and α-ε subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the α-ε interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. METHODS: HEK293 cells were transfected with wild-type or mutant (αY198F, εD59A, εD59N, εD173A, εD173N, δD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. RESULTS: Effects of mutations were antagonist specific. αY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to εD173 mutations. εD59 mutations had large effects on metocurine and cisatracurium. δD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two δ subunits but no ε subunit. Differences in IC50 arose from differences in both dissociation and association rates. CONCLUSION: Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the α-ε interface. Pancuronium, vecuronium, and cisatracurium were selective for the α-δ interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.

Original languageEnglish (US)
Pages (from-to)1186-1195
Number of pages10
JournalAnesthesiology
Volume106
Issue number6
DOIs
StatePublished - Jun 2007
Externally publishedYes

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Nicotinic Receptors
Pancuronium
Amino Acids
Vecuronium Bromide
Tubocurarine
Mutation
Inhibitory Concentration 50
Cholinergic Receptors
Neuromuscular Blocking Agents
HEK293 Cells
Acetylcholine
Complementary DNA
Binding Sites
Muscles
cisatracurium

ASJC Scopus subject areas

  • Anesthesiology and Pain Medicine

Cite this

Roles of amino acids and subunits in determining the inhibition of nicotinic acetylcholine receptors by competitive antagonists. / Dilger, James P.; Vidal, Ana Maria; Liu, Man; Mettewie, Claire; Suzuki, Takahiro; Pham, Anh; DeMazumder, Deeptankar.

In: Anesthesiology, Vol. 106, No. 6, 06.2007, p. 1186-1195.

Research output: Contribution to journalArticle

Dilger, James P. ; Vidal, Ana Maria ; Liu, Man ; Mettewie, Claire ; Suzuki, Takahiro ; Pham, Anh ; DeMazumder, Deeptankar. / Roles of amino acids and subunits in determining the inhibition of nicotinic acetylcholine receptors by competitive antagonists. In: Anesthesiology. 2007 ; Vol. 106, No. 6. pp. 1186-1195.
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abstract = "BACKGROUND: Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the α-δ and α-ε subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the α-ε interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. METHODS: HEK293 cells were transfected with wild-type or mutant (αY198F, εD59A, εD59N, εD173A, εD173N, δD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. RESULTS: Effects of mutations were antagonist specific. αY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to εD173 mutations. εD59 mutations had large effects on metocurine and cisatracurium. δD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two δ subunits but no ε subunit. Differences in IC50 arose from differences in both dissociation and association rates. CONCLUSION: Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the α-ε interface. Pancuronium, vecuronium, and cisatracurium were selective for the α-δ interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.",
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AU - Dilger, James P.

AU - Vidal, Ana Maria

AU - Liu, Man

AU - Mettewie, Claire

AU - Suzuki, Takahiro

AU - Pham, Anh

AU - DeMazumder, Deeptankar

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N2 - BACKGROUND: Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the α-δ and α-ε subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the α-ε interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. METHODS: HEK293 cells were transfected with wild-type or mutant (αY198F, εD59A, εD59N, εD173A, εD173N, δD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. RESULTS: Effects of mutations were antagonist specific. αY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to εD173 mutations. εD59 mutations had large effects on metocurine and cisatracurium. δD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two δ subunits but no ε subunit. Differences in IC50 arose from differences in both dissociation and association rates. CONCLUSION: Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the α-ε interface. Pancuronium, vecuronium, and cisatracurium were selective for the α-δ interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.

AB - BACKGROUND: Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the α-δ and α-ε subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the α-ε interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors. METHODS: HEK293 cells were transfected with wild-type or mutant (αY198F, εD59A, εD59N, εD173A, εD173N, δD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcholine in the absence and presence of antagonist. Concentration-response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates. RESULTS: Effects of mutations were antagonist specific. αY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to εD173 mutations. εD59 mutations had large effects on metocurine and cisatracurium. δD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two δ subunits but no ε subunit. Differences in IC50 arose from differences in both dissociation and association rates. CONCLUSION: Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the α-ε interface. Pancuronium, vecuronium, and cisatracurium were selective for the α-δ interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.

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