The Acetylcholine Receptor – An LGIC Prototype

1. The Acetylcholine Receptor – An LGIC Prototype Rose Chase February 11, 2005

2. Acetylcholine • Major neurotransmitter of the PNS • Receptors in brain, skeletal muscle, and much of parasympathetic system

3. History of ACh • 1936 Nobel in Physiology/Medicine to Otto Loewi and Sir Henry Dale • Loewi – experiment using two beating frog hearts in saline solution, a chemical secreted from Vagus nerve controlled contractions, dubbed “Vagusstuff” • Dale – 1914 postulated Ach was possible neurotransmitter

4. History of the receptor • La Torre, et al. among first to officially isolate “receptor proteolipid” specific for Ach and its competitive inhibitors MHM, TDF (1970) • Not sure of exact location of receptors, work was still being done to isolate membrane fractions

5. Localization • Prevalent throughout the body • Brain • Presynaptic boutons, regulate release of ACh, NA, DA, glutamate, and GABA  ADDICTION • Unclear function in muscle, lymphoid • Stimulates release of TNFα and interleukins 1 & 6 in macrophages • Cell proliferation in lung tissue – cancer • Angiogenesis in vascular tissue • Permeability of blood/brain barrier

6. Associated Disorders • Tourette syndrome & ADHD – Nic improves symptoms • Autism – decreased # of AChRs in parietal cortex and cerebellum • Schizophrenia – implied affect from high smoking rate (90% vs. 33%) and different symptoms • Parkinson’s – DA release release • Alzheimer’s – decrease in AChRs, Nic has positive effect (heavy smoking and otherwise)

7. Two Types • Muscarinic • Second messenger system – G protein coupled receptor • Excitatory/inhibitory • Nicotinic • LGIC – Ca+2/Na+; permeabilities differ among tissues • Excitatory • Will focus on Nicotinic…

8. General Composition • Pentamer – Hetero/Homogeneous • α,β,δ,ε,γ – various mix of different subunits • Each subunit consistently composed of a heterogeneous, antiparallel, 4 helix bundle • Labeled as M1, M2, M3, M4

9. General Scheme

10. Structure and Motion of AChR using EM • Unwin, FEBS Letters (555), 91 • In general… • ACh + AChR  rotation of protein chains  rotation of α-helices opens gate • Movements are small and energetically favorable

11. Structure continued • EM or NMR…Unwin says EM b/c: • Protein can be imaged in native environment • Transient events can be reproduced and freeze trapped • Interesting b/c: • NMR people believe otherwise…for both transient events and general structure • Kim, et al. paper

12. Structure continued • Sample preparation • Homogenization of electric organ from Torpedo • Crystallization of vesicle containing suspension • EM in short • Sample applied to glow discharged carbon support grid and negatively stained • Recording of electron micrographs to film for analysis

13. Structure continued • α-carbon trace fitted to e- density map, as viewed from synaptic cleft • Pore ~20Å long • M2 ~40Å long

14. Older Unwin work…

15. More old Unwin work…

16. Another comparison…

17. Structure continued • At narrowest part of M2 • Widening of ~3Å • Consistent with 6Å and 9Å measurements of pore • 15° rotation of each is only model that fits

18. Structure continued • Binding notes… • Two receptor binding regions, ~50Å from channel gate • Affects extended allosteric change, involving rotation of α-subunits

19. Structure continued • Gating notes… • “hydrophobic girdle” • L251 w/ neighboring A or S • F256 w/neighboring V or I • Too small/high energy for cation to pass • Binding of ACh stimulates asymmetric movement, starting w/α-subunit • Binding likened to “relaxing” of α-subunit

20. Structure continued

21. Clustering of AChR at synapse • Gally et al., Nature (431), 578 • Demonstrate necessity of lev-10 for post synaptic aggregation of nicotinic AChR in Caenorhabditis elegans

22. Clustering continued • Levamisole – nematode specific ACh agonist that activates AChRs at NMJs hypercontraction and death • Impairing function of genes affecting AChR clustering would generate subtle phenotypes: • Unclustered levamisole sensitive AChRs might still function if inserted properly • Possibly additional class of AChRs present at NMJs insensitive to levamisole • Therefore, isolate mutants weakly resistant to levamisole to probe the genes for clustering

23. Clustering continued • unc-29 mutants lack levamisole senstive AChRs • Demonstrates relative increase of resistance, with retention of some function in lev-10 mutants

24. Clustering continued • Cartoon of C.elegans physiology • http://www.wormatlas.org/MoW_built0.92/nervous_system.html#organization

25. Clustering Continued • Immunostaining and western blot for UNC-29 and UNC-17, verifying presence, but lack of clustering • UNC-29 – non α-subunit of the levamisole sensitive AChR in muscle • UNC-17 – vesicular ACh transporter

26. Clustering continued • Receptors are functional (unc-29/lev-10, no current) • Time for peak/decay increase and current decrease indicative of decreased ratio of synaptic/perisynaptic receptors • w/o antagonist, equal response, therefore, lev-10 needed for levamisole sensitive AChR clustering at the synapse

27. Clustering continued • Lev-10 localized at cholinergic NMJs in WT • Possible co-dependence of lev-10 & AChR • Lev-10 concentrated in membranes

28. Clustering continued • Additional experiments: • Fusion of extracellular part of lev-10 to human CD4 TM domain, expression rescued levamisole sensitivity, locomotion, and AChR clustering • GFP tagged, truncated LEV-10 (before TM region); was secreted from muscle to pseudocoelomic cavity and rescued mutant phenotypes (recall earlier picture of C.elegans)

29. Nicotine and the AChR • Cormier, et al., Mol. Pharm. (66) 1712 • Smoking leads to up regulation of AChRs, which modulate activity of the CNS reward centers • An effort to investigate the regulation of neuronal AChRs in smokers and mice

30. Nicotine continued • Smokers rated based on Fagerstrom Test for Nicotine Dependence • Only took smokers w/score > 2 • Fagerstrom Test has been studied by many groups for efficacy…still used.

31. Nicotine continued • Score of 6 or higher considered very dependent, less than 2, very low dependence • Score of 7 or higher – recommended support for quiting, likelihood of quitting estimated at around 8%

32. Nicotine continued • Structures of AChR binding ligands • ACh – cation/π w/Trp • Nic – hydrogen bond to backbone C=O • EB – both interactions, higher affinity

33. Nicotine continued • EB/BGT – bind hetero/homo pentamer • 18/92 smokers excluded w/no EB binding • Scatchard demonstrates 2 classes of EB binding sites • From PMN leukocytes [3H]EB and 125I-αBGT

34. Nicotine continued • PMN culture 3 days +/- Nic • Decrease in EB sites from immediate test to 3 days (recall previous figure)

35. Nicotine continued • Moderately positive correlation between EB sites and Fagerstrom index

36. Nicotine continued • Identifies only single class of binding sites in mouse splenocytes

37. Nicotine continued • Constant administration of nicotine using mini osmotic pumps • Plasma concentration of 30-35ng/ml ~30cigs/day • Nic admin – black bars • Noted increase in EB sites (C & D), vs αBGT

38. Nicotine continued

39. Nicotine continued • Ca+2 influx through AChR verified by addition of competitive antagonist, DHβE • Notable that WT+Nic is comparable to β2-/-

40. Nicotine continued • Conclusions • Increase in EB binding sites, indicative of tobacco consumption. May be more accurate and objective assay of addiction vs. standard cot./creat. • Nic is the substance in cigs generating AChR effects, and they do last several days • Heteromeric receptors are up regulated • Loss of calcium conduction, although possibly tissue dependent