TY - JOUR
T1 - [3H]Epibatidine photolabels non-equivalent amino acids in the agonist binding site of Torpedo and α4β2 nicotinic acetylcholine receptors
AU - Srivastava, Shouryadeep
AU - Hamouda, Ayman K.
AU - Pandhare, Akash
AU - Duddempudi, Phaneendra K.
AU - Sanghvi, Mitesh
AU - Cohen, Jonathan B.
AU - Blanton, Michael P.
PY - 2009/9/11
Y1 - 2009/9/11
N2 - Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (-) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111 respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation with the α-γ transmitter binding site, whereas it binds in two distinct orientations in α4β2 nAChR.
AB - Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α2βγδ and expressed human α4β2 nAChRs with [3H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr198 in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu109 and γTyr117 in Segment E of the complementary (-) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [3H]epibatidine photolabeled α4Tyr195 (equivalent to Torpedo αTyr190) in Segment C as well as β2Val111 and β2Ser113 in Segment E (equivalent to Torpedo γLeu109 and γTyr111 respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation with the α-γ transmitter binding site, whereas it binds in two distinct orientations in α4β2 nAChR.
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U2 - 10.1074/jbc.M109.019083
DO - 10.1074/jbc.M109.019083
M3 - Article
C2 - 19620239
AN - SCOPUS:69949185815
SN - 0021-9258
VL - 284
SP - 24939
EP - 24947
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 37
ER -