Roles of amino acids and subunits in determining the inhibition of nicotinic acetylcholine receptors by competitive antagonists.
BACKGROUND
Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the alpha-delta and alpha-epsilon 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 alpha-epsilon 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 (alphaY198F, epsilonD59A, epsilonD59N, epsilonD173A, epsilonD173N, deltaD180K) 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. alphaY198F 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 epsilonD173 mutations. epsilonD59 mutations had large effects on metocurine and cisatracurium. deltaD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two delta subunits but no epsilon subunit. Differences in IC50 arose from differences in both dissociation and association rates.


CONCLUSIONS
Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the alpha-epsilon interface. Pancuronium, vecuronium, and cisatracurium were selective for the alpha-delta interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.
Dilger
James P
JP
Department of Anesthesiology, Stony Brook University, NY 11794-8480, USA, and Department of Anesthesiology, Surugadai Nihon University Hospital, Kanda, Tokyo, Japan. james.dilger@stonybrook.edu
Vidal
Ana Maria
AM
Liu
Man
M
Mettewie
Claire
C
Suzuki
Takahiro
T
Pham
Anh
A
Demazumder
Deeptankar
D
D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D051379:Mice; D016296:Mutagenesis; D003473:Neuromuscular Nondepolarizing Agents / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D010197:Pancuronium / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014403:Tubocurarine / Q000494:pharmacology
D000596:Amino Acids; D003473:Neuromuscular Nondepolarizing Agents; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D010197:Pancuronium; D014403:Tubocurarine