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Volume 16, Number 12,
Issue of June 15, 1996
pp. 3798-3806
Copyright ©1996 Society for Neuroscience
Determinants of Competitive Antagonist Sensitivity on Neuronal
Nicotinic Receptor  Subunits
Received Nov. 6, 1995; revised Feb. 15, 1996; accepted April 2, 1996.
Scott C. Harvey and
Charles W. Luetje
Department of Molecular and Cellular Pharmacology, University of
Miami School of Medicine, Miami, Florida 33101
We constructed a series of chimeric and mutant neuronal nicotinic
acetylcholine receptor  subunits to map amino acid residues that
determine sensitivity to competitive antagonists. The 2 and 4
subunits form pharmacologically distinct receptors when expressed in
combination with the  3 subunit in Xenopus oocytes. At
equipotent acetylcholine concentrations, 32 is 56-fold more
sensitive to blockade by dihydro--erythroidine than is 34. The
32 combination is also sensitive to long-term blockade by
neuronal bungarotoxin, whereas 34 is not. Pharmacological
analysis of receptors formed by chimeric subunits reveals that
amino acid residues that determine both dihydro--erythroidine and
neuronal bungarotoxin sensitivity are located within several sequence
segments. The major determinant of sensitivity to both competitive
antagonists is located between residues 54 and 63. A minor determinant
of sensitivity to both antagonists lies between residues 1 and 54, whereas a minor determinant of NBT sensitivity lies between residues 74 and 80. Within region 54-63 of 2, mutant 2 subunits were used to
identify threonine 59 as a residue critical in determining competitive
antagonist sensitivity. Changing threonine 59 to lysine, as occurs in
4, causes a 9-fold decrease in dihydro--erythroidine sensitivity
and a 71-fold decrease in neuronal bungarotoxin sensitivity. Changing
polar threonine 59 to negatively charged aspartate causes a 2.5-fold
increase in neuronal bungarotoxin sensitivity and has no effect on
dihydro--erythroidine sensitivity.
Key words:
nicotinic receptor;
neuronal;
antagonists;
mutant;
chimera;
neuronal bungarotoxin;
dihydro--erythroidine
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