EurekaMag
+ Most Popular
Cunninghamia lanceolata plantations in China
Mammalian lairs in paleo ecological studies and palynology
Studies on technological possibilities in utilization of anhydrous milk fat for production of recombined butter-like products
Should right-sided fibroelastomas be operated upon?
Large esophageal lipoma
Apoptosis in the mammalian thymus during normal histogenesis and under various in vitro and in vivo experimental conditions
Poissons characoides nouveaux ou non signales de l'Ilha do Bananal, Bresil
Desensitizing efficacy of Colgate Sensitive Maximum Strength and Fresh Mint Sensodyne dentifrices
Administration of fluid by subcutaneous infusion: revival of a forgotten method
Tundra mosquito control - an impossible dream?
Schizophrenia for primary care providers: how to contribute to the care of a vulnerable patient population
Geochemical pattern analysis; method of describing the Southeastern limestone regional aquifer system
Incidence of low birth weights in a hospital of Mexico City
Tabanidae
Graded management intensity of grassland systems for enhancing floristic diversity
Microbiology and biochemistry of cheese and fermented milk
The ember tetra: a new pygmy characid tetra from the Rio das Mortes, Brazil, Hyphessobrycon amandae sp. n. (Pisces, Characoidei)
Risk factors of contrast-induced nephropathy in patients after coronary artery intervention
Renovation of onsite domestic wastewater in a poorly drained soil
Observations of the propagation velocity and formation mechanism of burst fractures caused by gunshot
Systolic blood pressure in a population of infants in the first year of life: the Brompton study
Haematological studies in rats fed with metanil yellow
Studies on pasteurellosis. I. A new species of Pasteurella encountered in chronic fowl cholera
Dormancy breaking and germination of Acacia salicina Lindl. seeds
therapy of lupus nephritis. a two-year prospective study

Structure of the agonist-binding site of the nicotinic acetylcholine receptor. [3H]acetylcholine mustard identifies residues in the cation-binding subsite


Structure of the agonist-binding site of the nicotinic acetylcholine receptor. [3H]acetylcholine mustard identifies residues in the cation-binding subsite



Journal of Biological Chemistry 266(34): 23354-23364



ISSN/ISBN: 0021-9258

PMID: 1744130

To characterize the structure of the agonist-binding site of the Torpedo nicotinic acetylcholine receptor (AChR), we have used [3H]acetylcholine mustard [( 3H]AChM), a reactive analog of acetylcholine, to identify residues contributing to the cation-binding subsite. Reaction of [3H]AChM, in its aziridinium form, with AChR-rich membrane suspensions, resulted initially in reversible, high affinity binding (K approximately 0.3 microM) followed by slow alkylation of the acetylcholine-binding site. Incorporation of label into AChR alpha-subunit was inhibited by agonists and competitive antagonists, but not by noncompetitive antagonists, and reaction with 3 microM [3H]AChM for 2 h resulted in specific alkylation of 0.6% of alpha-subunits. Within the alpha-subunit, greater than 90% of specific incorporation was contained within an 18-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Val-46 and containing N-linked carbohydrate. To identify sites of specific alkylation, [3H]AChM-labeled alpha-subunit was digested with trypsin, and the digests were fractionated by reverse phase high pressure liquid chromatography. Specifically labeled material was recovered within a single peak containing a peptide extending from Leu-80 to Lys-107. NH2-terminal amino acid sequencing revealed specific release of 3H in cycle 14 corresponding to alpha-subunit Tyr-93. Identification of Tyr-93 as the site of alkylation was confirmed by radiosequence analysis utilizing o-phthalaldehyde to establish that the released 3H originated from a peptide containing prolines at residues 2 and 9. Because [3H]AChM contains as its reactive group a positively charged quaternary aziridinium, alpha-subunit Tyr-93 is identified as contributing to the cation-binding domain of the AChR agonist-binding site. The selective reaction of [3H]AChM with tyrosyl rather than acidic side chains indicates the importance of aromatic interactions for the binding of the quaternary ammonium group, and the lack of reaction with the tyrosyl or acidic side chains within alpha 190-200 emphasizes the selective orientation of acetylcholine within its binding site.

Please choose payment method:






(PDF emailed within 1 workday: $29.90)

Accession: 041451258

Download citation: RISBibTeXText

Related references

Mapping the negative subsite of the nicotinic acetylcholine receptor agonist binding site. Society for Neuroscience Abstracts 17(1-2): 23, 1991

A conformational analysis of acetylcholine resident in the agonist binding site of the nicotinic acetylcholine receptor. Biophysical Journal 86(1): 74a, 2004

Interactions of acetylcholine binding site residues contributing to nicotinic acetylcholine receptor gating: role of residues Y93, Y190, K145 and D200. Journal of Molecular Graphics and Modelling 44: 145-154, 2013

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label. Biochemistry 30(28): 6987-6997, 1991

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor : [3H]nicotitn as an agonist photoaffinity label. Biochemistry (Easton) 30(28): 6987-6997, 1991

Photoactivatable agonist of the nicotinic acetylcholine receptor: potential probe to characterize the structural transitions of the acetylcholine binding site in different states of the receptor. Molecular Pharmacology 41(6): 1100-1106, 1992

Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site. Molecular Pharmacology 70(4): 1255-1263, 2006

Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the a-bungarotoxin binding site. Biochemistry (American Chemical Society) 38(16): 12-21, 1999

Modeling Binding Modes of α7 Nicotinic Acetylcholine Receptor with Ligands: The Roles of Gln117 and Other Residues of the Receptor in Agonist Binding. Journal of Medicinal Chemistry 52(3): 890-890, 2009

Modeling binding modes of alpha7 nicotinic acetylcholine receptor with ligands: the roles of Gln117 and other residues of the receptor in agonist binding. Journal of Medicinal Chemistry 51(20): 6293-6302, 2008

Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the delta subunit within 0.9 nm of the 5 alpha subunit-binding. Journal of Biological Chemistry 270(7): 3160-3164, 1995

Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site. Biochemistry 38(16): 4912-4921, 1999

Structure of the noncompetitive antagonist-binding site of the Torpedo nicotinic acetylcholine receptor : [3H]meproadifen mustard reacts selectively with α-subunit Glu-262. The Journal of Biological Chemistry 267(15): 10489-10499, 1992

Structure of the noncompetitive antagonist-binding site of the Torpedo nicotinic acetylcholine receptor. (3H)meproadifen mustard reacts selectively with a-subunit Glu-262. The Journal of Biological Chemistry 267: 489-99, 1992

Structure of the noncompetitive antagonist-binding site of the Torpedo nicotinic acetylcholine receptor. [3H]meproadifen mustard reacts selectively with alpha-subunit Glu-262. Journal of Biological Chemistry 267(15): 10489-10499, 1992