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CELLULAR AND MOLECULAR

Identification of Residues That Confer -Conotoxin-PnIA Sensitivity on the 3 Subunit of Neuronal Nicotinic Acetylcholine Receptors

Departments of Molecular and Cellular Pharmacology (D.E., A.M., C.W.L.), Biochemistry and Molecular Biology (A.M.), and Neuroscience Graduate Program (E.R.), University of Miami School of Medicine, Miami, Florida; and Departments of Psychiatry and Biology, University of Utah, Salt Lake City, Utah (J.M.M.)

Neuronal nicotinic receptors composed of the 3 and 2 subunits are at least 1000-fold more sensitive to blockade by -conotoxin-PnIA than are 22 receptors. A series of chimeric subunits, formed from portions of 2 and 3, were coexpressed with 2 in Xenopus oocytes and tested for toxin sensitivity. We found determinants of toxin sensitivity to be widely distributed in the extracellular domain of 3. Analysis of receptors formed by a series of mutant 3 subunits, in which residues that differ between 3 and 2 were changed from what occurs in 3 to what occurs in 2, allowed identification of three determinants of -conotoxin-PnIA sensitivity: proline 182, isoleucine 188, and glutamine 198. Comparison with determinants of -conotoxin-MII and -bungarotoxin sensitivity on the 3 subunit revealed overlapping, but distinct, arrays of determinants for each of these three toxins. When tested against an EC₅₀ concentration of acetylcholine, the IC₅₀ for -conotoxin-PnIA blockade was 25 ± 4 nM for 32, 84 ± 7 nM for 3P182T2, 700 ± 92 nM for 3I188K2, and 870 ± 61 nM for 3Q198P2. To examine the location of these residues within the receptor structure, we generated a homology model of the 32 receptor extracellular domain using the structure of the acetylcholine binding protein as a template. All three residues are located on the C-loop of the 3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.

Address correspondence to: Dr. Charles W. Luetje, Department of Molecular and Cellular Pharmacology (R-189), P.O. Box 016189, University of Miami School of Medicine, Miami, FL 33101. E-mail: cluetje{at}chroma.med.miami.edu

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