Abstract
The competitive antagonist α-conotoxin-MII (α-CTx-MII) is highly selective for the α3β2 neuronal nicotinic receptor. Other receptor subunit combinations (α2β2, α4β2, α3β4) are >200-fold less sensitive to blockade by this toxin. Using chimeric and mutant subunits, we identified amino acid residues of α3 and β2 that participate in determination of α-CTx-MII sensitivity. Chimeric α subunits, constructed from the α3 and α4 subunits, as well as from the α3 and α2 subunits, were expressed in combination with the β2 subunit in Xenopus laevis oocytes. Chimeric β subunits, formed from the β2 and β4 subunits, were expressed in combination with α3. Determinants of α-CTx-MII sensitivity on α3 were found to be within sequence segments 121–181 and 181–195. The 181–195 segment accounted for approximately half the difference in toxin sensitivity between receptors formed by α2 and α3. When this sequence of α2 was replaced with the corresponding α3 sequence, the resulting chimera formed receptors only 26-fold less sensitive to α-CTx-MII than α3β2. Site-directed mutagenesis within segment 181–195 demonstrated that Lys185 and Ile188 are critical in determination of sensitivity to toxin blockade. Determinants of α-CTx-MII sensitivity on β2 were mapped to sequence segments 1–54, 54–63, and 63–80. Site-directed mutagenesis within segment 54–63 of β2 demonstrated that Thr59 is important in determining α-CTx-MII sensitivity.
Footnotes
- Received June 14, 1996.
- Accepted October 19, 1996.
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Send reprint requests to: Dr. Charles W. Luetje, Department of Molecular and Cellular Pharmacology (R-189), University of Miami, P.O. Box 016189, Miami, FL 33101. E-mail:cluetje{at}newssun.med.miami.edu
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This work was supported by grants from the National Institute on Drug Abuse (RO1-DA08102), the American Heart Association, Florida Affiliate, and the Pharmaceutical Research and Manufacturers of America Foundation (C.W.L.) and from the National Institute of Mental Health (K20-MH00929, MH53631, GM48677) (J.M.M.). C.W.L. was an Initial Investigator of the American Heart Association, Florida Affiliate.
- The American Society for Pharmacology and Experimental Therapeutics
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