Photolabeling the Torpedo Nicotinic Acetylcholine Receptor with 4-Azido-2,3,5,6-tetrafluorobenzoylcholine, a Partial Agonist
Author(s)
Nirthanan, S
Ziebell, MR
Chiara, DC
Hong, F
Cohen, JB
Griffith University Author(s)
Year published
2005
Metadata
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The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (Keq = 12 卩 for the two agonist binding sites. Upon UV irradiation ...
View more >The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (Keq = 12 卩 for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR a, ?, and d subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled ?Leu-109/dLeu-111, ?Tyr-111, and ?Tyr-117 in binding site segment E as well as aTyr-198 in a subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.
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View more >The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (Keq = 12 卩 for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR a, ?, and d subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled ?Leu-109/dLeu-111, ?Tyr-111, and ?Tyr-117 in binding site segment E as well as aTyr-198 in a subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.
View less >
Journal Title
Biochemistry
Volume
44
Issue
41
Subject
Medicinal and biomolecular chemistry
Biochemistry and cell biology
Receptors and membrane biology
Medical biochemistry and metabolomics
Basic pharmacology