Determinants of specificity for α-conotoxin MII on α3β2 neuronal nicotinic receptors

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 welt 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 181195 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.