Photoaffinity labeling the agonist binding domain of nicotinic acetylcholine receptors



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Nicotinic acetylcholine receptors (nAChRs) are pentameric cationic ion channels which belong to the superfamily of ligand-gated ion channels (LGICs). nAChRs are implicated in patho-physiological conditions such as myasthenia gravis, Alzheimer’s & Parkinson’s disease, schizophrenia, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), attention deficit hyperkinetic disorder (ADHD) and nicotine addiction, which makes them key therapeutic target for these diseases. Additionally, studies have shown that nicotinic ligands have the potential of being used to alleviate pain and inflammation. Recent advances have helped us in understanding the role of different nAChR subtypes in different disease conditions (eg. α4β2 nAChR- nicotine addiction, Alzheimer’s disease, ADHD, ADNFLE; α7 nAChR- schizophrenia). However, the therapeutic drugs that are available to treat these conditions are either non-specific ligands (eg. nicotine used for smoking cessation) or affect the cholinergic system globally (eg. acetylcholinesterase inhibitors used to treat Alzheimer’s disease), which might explain many of the side-effects of these drugs. In order to reduce the adverse effects of the drugs and to improve the patient compliance, we need to develop nicotinic ligands which target nAChR subtype(s) specifically involved in a particular disease condition. For example, Varenicline/CHANTIX (Pfizer), a high-affinity partial agonist of the α4β2 nAChR, has a better therapeutic profile than nicotine when treating patients with nicotine addiction. In addition, CHANTIX treatment has a higher abstinence from smoking and lesser craving and withdrawal symptoms. Nicotinic ligands such as agonists, partial agonists, and competitive antagonists act at the agonist binding site (ABS) of the nAChR. The amino-acid residues involved in ligand binding at the ABS have been identified by a variety of methods and most of the identified amino-acid residues are conserved across the nAChR family. We hypothesize that the relatively few non-conserved residues at the ABS are responsible for conferring the unique physiological and pharmacological properties of ligands that bind to the ABS of various nAChRs subtypes. The work presented in this dissertation can be divided into two main parts. The first part focuses on ligand-ABS interactions of nAChRs. Previous studies have established that photoaffinity labeling studies in conjunction with site-directed mutagenesis, electrophysiological, and modeling studies have helped us develop more refined models that reflect the subtle structural differences among the ABS of different nAChR subtypes. In this study, we have photoaffinity labeled the ABS of Torpedo and neuronal nAChRs (α4β2 and α4β4) and the acetylcholine binding proteins (AChBPs) with the high-affinity agonists epibatidine and cytisine with the goal to obtaining a more complete understanding of the ligand-ABS interactions at the various nAChR subtypes. The results of these studies indicate: 1) The agonists [125I]epibatidine and 5[125I]A-85380 photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively; 2) The labeling of [125I]epibatidine in the β4 subunit was mapped to Loop E (β4I109–P120) of the ABS; 3) Amino-acid residues β2F119/β4Q117 at the ABS act as an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation; 4) In the Torpedo nAChR, [3H]epibatidine binds to both the α−γ and α−δ ABS with high affinity (~11 nM) and it does not have the high selectivity between these two sites; 5) Despite its high affinity binding to the α−γ and α−δ ABS in the Torpedo nAChR, [3H]epibatidine photoincorporates into the α and γ subunits with little, if any, in the δ subunit; 6) Within the α and γ subunits, [3H]epibatidine labeled αTyr198 (28 cpm/pmol), γLeu109 (23 cpm/ pmol), and γTyr117 (13 cpm/pmol) in the Loops C (α) and E (γ) of the ABS; 7) In the α4β2 nAChR, [3H]epibatidine photolabeled the α4 subunit at ~3-fold higher efficiency than the β2 subunit, and consistent with this, [3H]epibatidine photolabeled α4Tyr195 (10 cpm/pmol; equivalent to Torpedo αTyr190) in Loop C more efficiently than the two labeled amino acids in the β2 subunit: β2Val111 (2 cpm/pmol) and β2Ser113 (~0.6 cpm/pmol) in Loop E (equivalent to Torpedo γLeu109 and γTyr111, respectively); 8) The homology models of the Torpedo and α4β2 nAChRs based upon the structure of the epibatidine-bound form of Aplysia AChBP and the results of ligand docking simulations suggest that epibatidine binds in a single preferred orientation within the α-γ ABS of Torpedo nAChR, whereas it binds in two distinct orientations in the α4β2 ABS of α4β2 nAChR; 9) In the Torpedo nAChR, [3H]cytisine binds to both the α−γ and α−δ ABS with low affinity (~1.3 μM) and labels both the α−γ and α−δ sites with incorporating mainly α- and γ-subunits; 10) Within the α subunit, [3H]cytisine labeled αCys192/193 and αTyr198 in the Loop C of the ABS. Within the γ subunit, [3H]cytisine labeled γTyr117 in the Loop E and γTrp55 in the Loop D of the ABS; 11) In the A-AChBP, [3H]cytisine labeled Cys190 (equivalent to Torpedo αCys192) in the Loop C of the ABS. Additional studies are in progress to identify amino-acid residues photolabeled by [3H]Cytisine in the L-AChBP and the α4β2 nAChR, and to further determine cytisine orientation in the ABS of nAChRs and AChBPs using homology modeling. We believe the results of these studies will prove to be valuable in understanding the ligand-ABS interactions and designing new nAChR-specific ligands for future therapeutic and diagnostic applications. The second part of this dissertation focuses on affinity-purification of α7 nAChR. In order to aid in the development of more specific and efficacious α7 receptor ligands for therapeutic use, a detailed structural and functional characterization of the α7 receptor protein is an important goal. In the present study, SH-SY5Y neuroblastoma cell line stably transfected with human α7 receptors was cultured on a large scale and the expression level of the human α7 receptor was determined by [125 I]α-BgTx (α7 receptor antagonist) binding. Once sufficient quantities of SH-SY5Y neuroblastoma cell membranes (~1 g from 1000 dishes) were obtained, we affinity-purified the receptor from detergent-solubilized membranes with α-BgTx column. The preliminary results of these studies include: 1) The expression of α7 nAChR (Bmax) was ~4 pmol binding sites/mg protein and the [125I]α-bungarotoxin affinity was (Kd) ~ 2 nM; 2) Unlike α4β2 nAChRs expressed in the HEK293 cells, exposure to nicotine does not significantly upregulate α7 nAChR expression in this SH-SY5Y neuroblastoma cell line; 3) Consistent with low expression level, only a small amount of α7 nAChR protein (~150 μg) eluted from the α-BgTx column. Therefore insufficient amounts of α7 nAChR protein were available for structural studies. Additional studies are in progress to develop a stable CHO cell line that robustly expresses α7 nAChRs so that adequate amounts of α7 nAChR protein can be purified for structural studies.



Nicotinic acetylcholine receptors (nAChRs), Photoaffinity labels