TY - JOUR
T1 - Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [125I]epibatidine and 5[125I]A-85380
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.
N1 - Funding Information:
This research was supported in part by an American Heart Association South Central Affiliate Grant-In-Aid 0755029Y (M.P.B.), the South Plains Foundation (M.P.B.), and by a United States Public Health Services grant NS-22356 (J.H.S.). JHS is the Russell and Mary Shelden Professor of Anesthesiology. We would like to thank Drs. Jonathan B. Cohen and David C. Chiara (Department of Neurobiology, Harvard Medical School) for performing the amino acid sequencing experiments and for their valuable contributions during the writing of this manuscript. We also thank Sarah Hiyari for technical assistance.
PY - 2009/9
Y1 - 2009/9
N2 - 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.
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.
KW - Affinity-purification
KW - Cys-loop receptor
KW - HEK-293 cell
KW - Neuronal nicotinic receptor
KW - Photoaffinity labeling
KW - Protein sequencing
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U2 - 10.1016/j.bbamem.2009.06.012
DO - 10.1016/j.bbamem.2009.06.012
M3 - Article
C2 - 19545536
AN - SCOPUS:68749095924
SN - 0005-2736
VL - 1788
SP - 1987
EP - 1995
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 9
ER -