Determinants of Specificity for a-Conotoxin MII on a3b2 Neuronal Nicotinic Receptors

The competitive antagonist a-conotoxin-MII (a-CTx-MII) is highly selective for the a3b2 neuronal nicotinic receptor. Other receptor subunit combinations (a2b2, a4b2, a3b4) are .200fold less sensitive to blockade by this toxin. Using chimeric and mutant subunits, we identified amino acid residues of a3 and b2 that participate in determination of a-CTx-MII sensitivity. Chimeric a subunits, constructed from the a3 and a4 subunits, as well as from the a3 and a2 subunits, were expressed in combination with the b2 subunit in Xenopus laevis oocytes. Chimeric b subunits, formed from the b2 and b4 subunits, were expressed in combination with a3. Determinants of a-CTx-MII sensitivity on a3 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 a2 and a3. When this sequence of a2 was replaced with the corresponding a3 sequence, the resulting chimera formed receptors only 26-fold less sensitive to a-CTxMII than a3b2. Site-directed mutagenesis within segment 181– 195 demonstrated that Lys185 and Ile188 are critical in determination of sensitivity to toxin blockade. Determinants of a-CTx-MII sensitivity on b2 were mapped to sequence segments 1–54, 54–63, and 63–80. Site-directed mutagenesis within segment 54–63 of b2 demonstrated that Thr59 is important in determining a-CTx-MII sensitivity.