Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor by reducing the microscopic agonist dissociation constant.
BACKGROUND
Isoflurane increases the apparent agonist affinity of ligand-gated ion channels. This action reflects a reduction in the receptor's agonist dissociation constant and/or the preopen/open channel state equilibrium. To evaluate the effect of isoflurane on each of these kinetic constants in the nicotinic acetylcholine receptor, the authors analyzed isoflurane's actions on (1) the binding of the fluorescent agonist Dns-C6-Cho to the nicotinic acetylcholine receptor's agonist self-inhibition site and (2) the desensitization kinetics induced by the binding of the weak partial agonist suberyldicholine.


METHODS
The dissociation constant for Dns-C6-Cho binding to the self-inhibitory site was determined using stopped-flow fluorescence spectroscopy. The values of the kinetic constants for agonist binding, channel gating, and desensitization were determined by modeling the suberyldicholine concentration-dependence of the apparent rate of desensitization.


RESULTS
Isoflurane did not significantly alter the dissociation constant for Dns-C6-Cho binding to the self-inhibitory site even at a concentration as high as 1.5 mM, the highest concentration studied. At this concentration, isoflurane substantially reduced the dissociation constant for suberyldicholine binding to its channel opening site by 97% from 17 +/- 5 microM to 0.5 +/- 0.2 microM, whereas the preopen/open channel state equilibrium was reduced only from 19.1 to 5 +/- 1.


CONCLUSIONS
Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor primarily by reducing the agonist dissociation constant of the site responsible for channel opening rather than altering channel gating kinetics.
Raines
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Harvard Medical School, Massachusetts General Hospital, Boston 02114, USA. Raines@etherdome.mgh.harvard.edu
Zachariah
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VT
D000465:Algorithms; D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000818:Animals; D002794:Choline / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D018678:Cholinergic Agents / Q000378:metabolism / Q000494:pharmacology; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007530:Isoflurane / Q000494:pharmacology; D007700:Kinetics; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D018722:Nicotinic Agonists / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds; D011978:Receptors, Nicotinic / Q000187:drug effects*; D013050:Spectrometry, Fluorescence; D014101:Torpedo
D018685:Anesthetics, Inhalation; D018678:Cholinergic Agents; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D018722:Nicotinic Agonists; D000644:Quaternary Ammonium Compounds; D011978:Receptors, Nicotinic; C009335:subecholine; C027933:(1-(5-dimethylaminonaphthalene)sulfonamido)-n-hexanoic acid-beta-N-trimethylammonium ethyl ester; D007530:Isoflurane; D002794:Choline