Identification of residues that confer α-conotoxin-PnIA sensitivity on the α3 subunit of neuronal nicotinic acetylcholine receptors

Drew Everhart, Edward Reiller, Armen Mirzoian, J. Michael McIntosh, Arun Malhotra, Charles W Luetje

Research output: Contribution to journalArticle

31 Citations (Scopus)

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 languageEnglish
Pages (from-to)664-670
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume306
Issue number2
DOIs
StatePublished - Aug 1 2003

Fingerprint

Conotoxins
Nicotinic Receptors
Acetylcholine
Isoleucine
Bungarotoxins
Xenopus
Glutamine
Proline
Inhibitory Concentration 50
Oocytes
Carrier Proteins

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 journalArticle

Everhart, Drew ; Reiller, Edward ; Mirzoian, Armen ; McIntosh, J. Michael ; Malhotra, Arun ; Luetje, Charles W. / Identification of residues that confer α-conotoxin-PnIA sensitivity on the α3 subunit of neuronal nicotinic acetylcholine receptors. In: Journal of Pharmacology and Experimental Therapeutics. 2003 ; Vol. 306, No. 2. pp. 664-670.
@article{e294a092332540608b45a4aa8616ea52,
title = "Identification of residues that confer α-conotoxin-PnIA sensitivity on the α3 subunit of neuronal nicotinic acetylcholine receptors",
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.",
author = "Drew Everhart and Edward Reiller and Armen Mirzoian and McIntosh, {J. Michael} and Arun Malhotra and Luetje, {Charles W}",
year = "2003",
month = "8",
day = "1",
doi = "10.1124/jpet.103.051656",
language = "English",
volume = "306",
pages = "664--670",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "2",

}

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.

UR - http://www.scopus.com/inward/record.url?scp=0037622708&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037622708&partnerID=8YFLogxK

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 -