Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [125I]epibatidine and 5[125I]A-85380

Ayman K. Hamouda, Xiaochun Jin, Mitesh Sanghvi, Shouryadeep Srivastava, Akash Pandhare, Phaneendra K. Duddempudi, Joe Henry Steinbach, Michael P. Blanton

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [125I]epibatidine and 5[125I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [125I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [125I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [125I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

Original languageEnglish (US)
Pages (from-to)1987-1995
Number of pages9
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1788
Issue number9
DOIs
StatePublished - Sep 1 2009
Externally publishedYes

Fingerprint

epibatidine
A 85380
Nicotinic Receptors
Labeling
Cholinergic Agonists
HEK293 Cells
Protein Sequence Analysis
Nervous System Diseases
Binding Sites
Rats
Amino Acids
Chemical activation

Keywords

  • Affinity-purification
  • Cys-loop receptor
  • HEK-293 cell
  • Neuronal nicotinic receptor
  • Photoaffinity labeling
  • Protein sequencing

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [125I]epibatidine and 5[125I]A-85380. / Hamouda, Ayman K.; Jin, Xiaochun; Sanghvi, Mitesh; Srivastava, Shouryadeep; Pandhare, Akash; Duddempudi, Phaneendra K.; Steinbach, Joe Henry; Blanton, Michael P.

In: Biochimica et Biophysica Acta - Biomembranes, Vol. 1788, No. 9, 01.09.2009, p. 1987-1995.

Research output: Contribution to journalArticle

Hamouda, Ayman K. ; Jin, Xiaochun ; Sanghvi, Mitesh ; Srivastava, Shouryadeep ; Pandhare, Akash ; Duddempudi, Phaneendra K. ; Steinbach, Joe Henry ; Blanton, Michael P. / Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [125I]epibatidine and 5[125I]A-85380. In: Biochimica et Biophysica Acta - Biomembranes. 2009 ; Vol. 1788, No. 9. pp. 1987-1995.
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abstract = "The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [125I]epibatidine and 5[125I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [125I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [125I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [125I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.",
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AU - Hamouda, Ayman K.

AU - Jin, Xiaochun

AU - Sanghvi, Mitesh

AU - Srivastava, Shouryadeep

AU - Pandhare, Akash

AU - Duddempudi, Phaneendra K.

AU - Steinbach, Joe Henry

AU - Blanton, Michael P.

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AB - The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [125I]epibatidine and 5[125I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [125I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [125I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [125I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.

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KW - Photoaffinity labeling

KW - Protein sequencing

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