Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor: Tests with novel side chains and with several agonists

Patrick C. Kearney, Mark W. Nowak, Wenge Zhong, Scott K Silverman, Henry A. Lester, Dennis A. Dougherty

Research output: Contribution to journalArticle

Abstract

Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, α93, α190, and α198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of α-Tyr93, whereas a variety of substituents are functional at the analogous position of αTyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.

Original languageEnglish (US)
Pages (from-to)1401-1412
Number of pages12
JournalMolecular Pharmacology
Volume50
Issue number5
StatePublished - Nov 1 1996
Externally publishedYes

Fingerprint

Nicotinic Receptors
Binding Sites
Amino Acids
Tyrosine
Cholinergic Agonists
Mutation
Nonsense Codon
Xenopus laevis
Sensory Receptor Cells
Transfer RNA
Nicotine
Mutagenesis
Hydroxyl Radical
Oocytes
Membrane Proteins

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor : Tests with novel side chains and with several agonists. / Kearney, Patrick C.; Nowak, Mark W.; Zhong, Wenge; Silverman, Scott K; Lester, Henry A.; Dougherty, Dennis A.

In: Molecular Pharmacology, Vol. 50, No. 5, 01.11.1996, p. 1401-1412.

Research output: Contribution to journalArticle

@article{34d2ef84fb3b447e889410cdec1bfaed,
title = "Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor: Tests with novel side chains and with several agonists",
abstract = "Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, α93, α190, and α198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of α-Tyr93, whereas a variety of substituents are functional at the analogous position of αTyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.",
author = "Kearney, {Patrick C.} and Nowak, {Mark W.} and Wenge Zhong and Silverman, {Scott K} and Lester, {Henry A.} and Dougherty, {Dennis A.}",
year = "1996",
month = "11",
day = "1",
language = "English (US)",
volume = "50",
pages = "1401--1412",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "5",

}

TY - JOUR

T1 - Dose-response relations for unnatural amino acids at the agonist binding site of the nicotinic acetylcholine receptor

T2 - Tests with novel side chains and with several agonists

AU - Kearney, Patrick C.

AU - Nowak, Mark W.

AU - Zhong, Wenge

AU - Silverman, Scott K

AU - Lester, Henry A.

AU - Dougherty, Dennis A.

PY - 1996/11/1

Y1 - 1996/11/1

N2 - Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, α93, α190, and α198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of α-Tyr93, whereas a variety of substituents are functional at the analogous position of αTyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.

AB - Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, α93, α190, and α198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of α-Tyr93, whereas a variety of substituents are functional at the analogous position of αTyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.

UR - http://www.scopus.com/inward/record.url?scp=0029811223&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029811223&partnerID=8YFLogxK

M3 - Article

C2 - 8913372

AN - SCOPUS:0029811223

VL - 50

SP - 1401

EP - 1412

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 5

ER -