Abstract
Neuronal nicotinic receptors composed of the α3 and β2 subunits are at least 1000-fold more sensitive to blockade by α-conotoxin-PnIA than are α2β2 receptors. A series of chimeric subunits, formed from portions of α2 and α3, were coexpressed with βp2 in Xenopus oocytes and tested for toxin sensitivity. We found determinants of toxin sensitivity to be widely distributed in the extracellular domain of α3. Analysis of receptors formed by a series of mutant α3 subunits, in which residues that differ between α3 and α2 were changed from what occurs in α3 to what occurs in α2, allowed identification of three determinants of α-conotoxin-PnIA sensitivity: proline 182, isoleucine 188, and glutamine 198. Comparison with determinants of α-conotoxin-MII and κ-bungarotoxin sensitivity on the α3 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 α-conotoxin-PNIA blockade was 25 ± 4 nM for α3β2, 84 ± 7 nM for α3P182Tβ2, 700 ± 92 nM for α3I188Kβ2, and 870 ± 61 nM for α3Q198Pβ2. To examine the location of these residues within the receptor structure, we generated a homology model of the α3β2 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 α3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.
| Original language | English |
|---|---|
| Pages (from-to) | 664-670 |
| Number of pages | 7 |
| Journal | Journal of Pharmacology and Experimental Therapeutics |
| Volume | 306 |
| Issue number | 2 |
| DOIs | |
| State | Published - Aug 1 2003 |
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ASJC Scopus subject areas
- Pharmacology
Cite this
Identification of residues that confer α-conotoxin-PnIA sensitivity on the α3 subunit of neuronal nicotinic acetylcholine receptors. / Everhart, Drew; Reiller, Edward; Mirzoian, Armen; McIntosh, J. Michael; Malhotra, Arun; Luetje, Charles W.
In: Journal of Pharmacology and Experimental Therapeutics, Vol. 306, No. 2, 01.08.2003, p. 664-670.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification of residues that confer α-conotoxin-PnIA sensitivity on the α3 subunit of neuronal nicotinic acetylcholine receptors
AU - Everhart, Drew
AU - Reiller, Edward
AU - Mirzoian, Armen
AU - McIntosh, J. Michael
AU - Malhotra, Arun
AU - Luetje, Charles W
PY - 2003/8/1
Y1 - 2003/8/1
N2 - Neuronal nicotinic receptors composed of the α3 and β2 subunits are at least 1000-fold more sensitive to blockade by α-conotoxin-PnIA than are α2β2 receptors. A series of chimeric subunits, formed from portions of α2 and α3, were coexpressed with βp2 in Xenopus oocytes and tested for toxin sensitivity. We found determinants of toxin sensitivity to be widely distributed in the extracellular domain of α3. Analysis of receptors formed by a series of mutant α3 subunits, in which residues that differ between α3 and α2 were changed from what occurs in α3 to what occurs in α2, allowed identification of three determinants of α-conotoxin-PnIA sensitivity: proline 182, isoleucine 188, and glutamine 198. Comparison with determinants of α-conotoxin-MII and κ-bungarotoxin sensitivity on the α3 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 α-conotoxin-PNIA blockade was 25 ± 4 nM for α3β2, 84 ± 7 nM for α3P182Tβ2, 700 ± 92 nM for α3I188Kβ2, and 870 ± 61 nM for α3Q198Pβ2. To examine the location of these residues within the receptor structure, we generated a homology model of the α3β2 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 α3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.
AB - Neuronal nicotinic receptors composed of the α3 and β2 subunits are at least 1000-fold more sensitive to blockade by α-conotoxin-PnIA than are α2β2 receptors. A series of chimeric subunits, formed from portions of α2 and α3, were coexpressed with βp2 in Xenopus oocytes and tested for toxin sensitivity. We found determinants of toxin sensitivity to be widely distributed in the extracellular domain of α3. Analysis of receptors formed by a series of mutant α3 subunits, in which residues that differ between α3 and α2 were changed from what occurs in α3 to what occurs in α2, allowed identification of three determinants of α-conotoxin-PnIA sensitivity: proline 182, isoleucine 188, and glutamine 198. Comparison with determinants of α-conotoxin-MII and κ-bungarotoxin sensitivity on the α3 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 α-conotoxin-PNIA blockade was 25 ± 4 nM for α3β2, 84 ± 7 nM for α3P182Tβ2, 700 ± 92 nM for α3I188Kβ2, and 870 ± 61 nM for α3Q198Pβ2. To examine the location of these residues within the receptor structure, we generated a homology model of the α3β2 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 α3 subunit, with isoleucine 188 nearest the acetylcholine-binding pocket.
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U2 - 10.1124/jpet.103.051656
DO - 10.1124/jpet.103.051656
M3 - Article
VL - 306
SP - 664
EP - 670
JO - Journal of Pharmacology and Experimental Therapeutics
JF - Journal of Pharmacology and Experimental Therapeutics
SN - 0022-3565
IS - 2
ER -