Allosterically linked noncompetitive antagonist binding sites in the resting nicotinic acetylcholine receptor ion channel
Section snippets
Materials and methods
Materials. [Piperidyl-3,4-(N)]-(N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine) ([]TCP; 57.6 Ci/mmol) was obtained from New England Nuclear Research Products (Boston, MA), 3-trifluoromethyl-3-(m-[]iodophenyl)diazirine ([]TID; ∼10 Ci/mmol) from Amersham Pharmacia Biotech (Piscataway, NJ), and both were stored in ethanol at −20 and 4 °C, respectively. []Tetracaine (36 Ci/mmol) was a gift from Dr. Jonathan Cohen (Harvard Medical School, Boston, MA), []amobarbital (50 mCi/mmol) was
Potentiation of []TID photoincorporation into the resting AChR by ketamine and TCP
Because the interaction of 3-trifluoromethyl-3-(m-[]iodophenyl) diazirine with the resting AChR has been very well characterized, including identification of a high-affinity binding site within the ion channel pore [9] (reviewed in [2], [4]), we began our studies by examining the effect of ketamine and TCP on []TID photoincorporation into the resting receptor. AChR native membranes, in the absence of agonist, were equilibrated with ∼430 nM []TID and various concentrations of
Discussion
In order to characterize and potentially localize the binding site for the dissociative anesthetics ketamine and TCP in the resting AChR, we have taken into consideration previous studies that have established the binding sites for the well-known NCAs TID [9], [10], tetracaine [11], and barbiturates [8] in the resting AChR channel. In the present study, we examined the effect of ketamine and TCP on the pattern of photolabeling of []TID and of []amobarbital, []TCP, and []tetracaine
Acknowledgements
We thank Dr. Jay Ponder (Washington University School of Medicine, St. Louis, MO) for use of his computer workstation, molecular graphics software, and invaluable assistance. This research was supported in part by National Institutes of Health Grant R29-NS35786 (M.P.B.).
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