Determinants of specificity for alpha-conotoxin MII on alpha3beta2 neuronal nicotinic receptors

The competitive antagonist alpha-conotoxin-MII (alpha-CTx-MII) is highly selective for the alpha3beta2 neuronal nicotinic receptor. Other receptor subunit combinations (alpha2beta2, alpha4beta2, alpha3beta4) are >200-fold less sensitive to blockade by this toxin. Using chimeric and mutant subunits, we identified amino acid residues of alpha3 and beta2 that participate in determination of alpha-CTx-MII sensitivity. Chimeric alpha subunits, constructed from the alpha3 and alpha4 subunits, as well as from the alpha3 and alpha2 subunits, were expressed in combination with the beta2 subunit in Xenopus laevis oocytes. Chimeric beta subunits, formed from the beta2 and beta4 subunits, were expressed in combination with alpha3. Determinants of alpha-CTx-MII sensitivity on alpha3 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 alpha2 and alpha3. When this sequence of alpha2 was replaced with the corresponding alpha3 sequence, the resulting chimera formed receptors only 26-fold less sensitive to alpha-CTx-MII than alpha3beta2. Site-directed mutagenesis within segment 181-195 demonstrated that Lys185 and Ile188 are critical in determination of sensitivity to toxin blockade. Determinants of alpha-CTx-MII sensitivity on beta2 were mapped to sequence segments 1-54, 54-63, and 63-80. Site-directed mutagenesis within segment 54-63 of beta2 demonstrated that Thr59 is important in determining alpha-CTx-MII sensitivity.