[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Phaneendra K. Duddempudi,Ayman K. Hamouda,Akash Pandhare,Jonathan B. Cohen,Mitesh Sanghvi,Shouryadeep Srivastava,Michael P. Blanton

doi:10.1074/jbc.m109.019083

Abstract

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 alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), 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 within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

Highlights

Gated ion channels that mediate the actions of the neurotransmitter acetylcholine [1]. Nicotinic acetylcholine receptor (nAChR) from vertebrate skeletal muscle and the electric organs of Torpedo rays are heteropentamers of homologous subunits with a stoichiometry of 2␣:␤: ␥(⑀):␦ that are arranged pseudosymmetrically around central cation-selective ion channels [1, 2]
Photoaffinity labeling provides an alternative means to identify amino acids contributing to a drug binding site [18, 19] and has been used to determine the orientation of drugs bound in the agonist binding sites (ABS) of Torpedo nAChR [20]
We use the intrinsic photoreactivity of the nAChR agonist [3H]epibatidine to compare its mode of binding in the Torpedo and neuronal ␣4␤2 nAChRs

Summary

EXPERIMENTAL PROCEDURES

Materials—[3H]Epibatidine (45 Ci/mmol) was obtained from PerkinElmer Life Sciences and stored in 95% ethanol at 4 °C. HEK-h␣4␤2 cell membranes (typically 2 g of protein from ϳ600 dishes) at 1 mg/ml protein in VDB containing 0.2 ␮l/ml protease inhibitor mixture III were solubilized with 1% CHAPS, centrifuged (91,500 ϫ g for 1 h) to pellet insoluble material, and dialyzed for 5 h against 1% cholate. [3H]Epibatidine Photolabeling of Torpedo nAChR—For analytical labelings, ϳ50-␮g samples of affinity-purified Torpedo nAChRs in 1 ml of VDB were incubated with 0.13 ␮M [3H]epibatidine in the absence or presence of epibatidine (40 ␮M) or increasing concentrations of dTC (final concentrations 0.2–20 ␮M). For labeling on a preparative scale, affinity-purified Torpedo nAChRs (ϳ3 mg of protein in 8 ml) were labeled with 550 nM [3H]epibatidine, and the ␣ and ␥ subunits were isolated by SDSPAGE and subjected to in-gel digestion with V8 protease. Nonspecific binding was determined in the presence of 1 mM carbamylcholine

RESULTS

Findings

DISCUSSION