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

The Acetylcholine The Acetylcholine Receptor – An LGIC Receptor – An LGIC PrototypePrototype

Rose ChaseRose Chase

February 11, 2005February 11, 2005

AcetylcholineAcetylcholine

Major neurotransmitter of the PNSMajor neurotransmitter of the PNS Receptors in brain, skeletal muscle, Receptors in brain, skeletal muscle,

and much of parasympathetic systemand much of parasympathetic system

History of AChHistory of ACh

1936 Nobel in Physiology/Medicine to 1936 Nobel in Physiology/Medicine to Otto Loewi and Sir Henry DaleOtto Loewi and Sir Henry Dale

Loewi – experiment using two beating Loewi – experiment using two beating frog hearts in saline solution, a frog hearts in saline solution, a chemical secreted from Vagus nerve chemical secreted from Vagus nerve controlled contractions, dubbed controlled contractions, dubbed “Vagusstuff”“Vagusstuff”

Dale – 1914 postulated Ach was Dale – 1914 postulated Ach was possible neurotransmitterpossible neurotransmitter

History of the receptorHistory of the receptor

La Torre, et al. among first to La Torre, et al. among first to officially isolate “receptor officially isolate “receptor proteolipid” specific for Ach and its proteolipid” specific for Ach and its competitive inhibitors MHM, TDF competitive inhibitors MHM, TDF (1970)(1970)

Not sure of exact location of Not sure of exact location of receptors, work was still being done receptors, work was still being done to isolate membrane fractionsto isolate membrane fractions

LocalizationLocalization

Prevalent throughout the bodyPrevalent throughout the body BrainBrain

– Presynaptic boutons, regulate release of Presynaptic boutons, regulate release of ACh, NA, DA, glutamate, and GABA ACh, NA, DA, glutamate, and GABA ADDICTIONADDICTION

Unclear function in muscle, lymphoidUnclear function in muscle, lymphoid Stimulates release of TNFStimulates release of TNFαα and and

interleukins 1 & 6 in macrophagesinterleukins 1 & 6 in macrophages Cell proliferation in lung tissue – cancerCell proliferation in lung tissue – cancer Angiogenesis in vascular tissueAngiogenesis in vascular tissue Permeability of blood/brain barrierPermeability of blood/brain barrier

Associated DisordersAssociated Disorders

Tourette syndrome & ADHD – Nic improves Tourette syndrome & ADHD – Nic improves symptomssymptoms

Autism – decreased # of AChRs in parietal Autism – decreased # of AChRs in parietal cortex and cerebellumcortex and cerebellum

Schizophrenia – implied affect from high Schizophrenia – implied affect from high smoking rate (90% vs. 33%) and different smoking rate (90% vs. 33%) and different symptomssymptoms

Parkinson’s – DA release releaseParkinson’s – DA release release Alzheimer’s – decrease in AChRs, Nic has Alzheimer’s – decrease in AChRs, Nic has

positive effect (heavy smoking and positive effect (heavy smoking and otherwise)otherwise)

Two TypesTwo Types

MuscarinicMuscarinic– Second messenger system – G protein Second messenger system – G protein

coupled receptorcoupled receptor– Excitatory/inhibitory Excitatory/inhibitory

NicotinicNicotinic– LGIC – Ca+2/Na+; permeabilities differ LGIC – Ca+2/Na+; permeabilities differ

among tissuesamong tissues– ExcitatoryExcitatory

Will focus on Nicotinic…Will focus on Nicotinic…

General CompositionGeneral Composition

Pentamer – Hetero/HomogeneousPentamer – Hetero/Homogeneous– αα,,ββ,,δδ,,εε,,γγ – various mix of different – various mix of different

subunitssubunits Each subunit consistently Each subunit consistently

composed of a heterogeneous, composed of a heterogeneous, antiparallel, 4 helix bundleantiparallel, 4 helix bundle– Labeled as M1, M2, M3, M4Labeled as M1, M2, M3, M4

General SchemeGeneral Scheme

Structure and Motion Structure and Motion of AChR using EMof AChR using EM Unwin, FEBS Letters (555), 91Unwin, FEBS Letters (555), 91

– In general…In general…– ACh + AChR ACh + AChR rotation of protein rotation of protein

chains chains rotation of rotation of αα-helices opens -helices opens gate gate

– Movements are small and Movements are small and energetically favorableenergetically favorable

Structure continuedStructure continued

EM or NMR…Unwin says EM b/c:EM or NMR…Unwin says EM b/c:– Protein can be imaged in native Protein can be imaged in native

environmentenvironment– Transient events can be reproduced and Transient events can be reproduced and

freeze trappedfreeze trapped Interesting b/c:Interesting b/c:

– NMR people believe otherwise…for both NMR people believe otherwise…for both transient events and general structuretransient events and general structure

– Kim, et al. paperKim, et al. paper


Sample preparationSample preparation– Homogenization of electric organ from Homogenization of electric organ from

TorpedoTorpedo– Crystallization of vesicle containing Crystallization of vesicle containing

suspensionsuspension EM in shortEM in short

– Sample applied to glow discharged carbon Sample applied to glow discharged carbon support grid and negatively stainedsupport grid and negatively stained

– Recording of electron micrographs to film Recording of electron micrographs to film for analysisfor analysis


αα-carbon -carbon trace fitted to trace fitted to e- density e- density map, as map, as viewed from viewed from synaptic cleftsynaptic cleft

Pore ~20Å Pore ~20Å longlong

M2 ~40Å longM2 ~40Å long

Older Unwin work…Older Unwin work…

More old Unwin work…More old Unwin work…

Another comparison…Another comparison…


At narrowest part of M2At narrowest part of M2 Widening of ~3ÅWidening of ~3Å Consistent with 6Å and 9Å measurements of poreConsistent with 6Å and 9Å measurements of pore 15° rotation of each is only model that fits15° rotation of each is only model that fits


Binding notes…Binding notes…– Two receptor binding regions, ~50Å Two receptor binding regions, ~50Å

from channel gatefrom channel gate– Affects extended allosteric change, Affects extended allosteric change,

involving rotation of involving rotation of αα-subunits-subunits


Gating notes…Gating notes…– ““hydrophobic girdle”hydrophobic girdle”

L251 w/ neighboring A or SL251 w/ neighboring A or S F256 w/neighboring V or IF256 w/neighboring V or I

– Too small/high energy for cation to passToo small/high energy for cation to pass– Binding of ACh stimulates asymmetric Binding of ACh stimulates asymmetric

movement, starting w/movement, starting w/αα-subunit-subunit– Binding likened to “relaxing” of Binding likened to “relaxing” of αα--

subunitsubunit


Clustering of AChR at Clustering of AChR at synapsesynapse Gally et al., Nature (431), 578Gally et al., Nature (431), 578 Demonstrate necessity of Demonstrate necessity of lev-10lev-10

for post synaptic aggregation of for post synaptic aggregation of nicotinic AChR in nicotinic AChR in Caenorhabditis Caenorhabditis eleganselegans

Clustering continuedClustering continued

Levamisole – nematode specific ACh agonist Levamisole – nematode specific ACh agonist that activates AChRs at NMJs that activates AChRs at NMJs hypercontraction and deathhypercontraction and death

Impairing function of genes affecting AChR Impairing function of genes affecting AChR clustering would generate subtle phenotypes:clustering would generate subtle phenotypes:– Unclustered levamisole sensitive AChRs might still Unclustered levamisole sensitive AChRs might still

function if inserted properlyfunction if inserted properly– Possibly additional class of AChRs present at NMJs Possibly additional class of AChRs present at NMJs

insensitive to levamisoleinsensitive to levamisole Therefore, isolate mutants weakly resistant Therefore, isolate mutants weakly resistant

to levamisole to probe the genes for to levamisole to probe the genes for clusteringclustering


unc-29 mutants unc-29 mutants lack levamisole lack levamisole senstive AChRssenstive AChRs

Demonstrates Demonstrates relative increase relative increase of resistance, of resistance, with retention of with retention of some function in some function in lev-10 mutantslev-10 mutants


- Cartoon of Cartoon of C.elegans C.elegans physiologyphysiology

- http://www.wormahttp://www.wormatlas.org/MoW_builttlas.org/MoW_built0.92/nervous_syst0.92/nervous_system.html#organizaem.html#organizationtion

Clustering ContinuedClustering Continued

Immunostaining and western blot for UNC-29 and UNC-17, Immunostaining and western blot for UNC-29 and UNC-17, verifying presence, but lack of clusteringverifying presence, but lack of clustering

UNC-29 – non UNC-29 – non αα-subunit of the levamisole sensitive AChR in -subunit of the levamisole sensitive AChR in musclemuscle

UNC-17 – vesicular ACh transporterUNC-17 – vesicular ACh transporter


Receptors are functional Receptors are functional (unc-29/lev-10, no (unc-29/lev-10, no current)current)

Time for peak/decay Time for peak/decay increase and current increase and current decrease indicative of decrease indicative of decreased ratio of decreased ratio of synaptic/perisynaptic synaptic/perisynaptic receptorsreceptors

w/o antagonist, equal w/o antagonist, equal response, therefore, lev-response, therefore, lev-10 needed for levamisole 10 needed for levamisole sensitive AChR clustering sensitive AChR clustering at the synapseat the synapse


Lev-10 localized Lev-10 localized at cholinergic at cholinergic NMJs in WTNMJs in WT

Possible co-Possible co-dependence of dependence of lev-10 & AChRlev-10 & AChR

Lev-10 Lev-10 concentrated in concentrated in membranesmembranes


Additional experiments:Additional experiments:– Fusion of extracellular part of lev-10 to Fusion of extracellular part of lev-10 to

human CD4 TM domain, expression human CD4 TM domain, expression rescued levamisole sensitivity, locomotion, rescued levamisole sensitivity, locomotion, and AChR clusteringand AChR clustering

– GFP tagged, truncated LEV-10 (before TM GFP tagged, truncated LEV-10 (before TM region); was secreted from muscle to region); was secreted from muscle to pseudocoelomic cavity and rescued pseudocoelomic cavity and rescued mutant phenotypes (recall earlier picture mutant phenotypes (recall earlier picture of C.elegans)of C.elegans)

Nicotine and the AChRNicotine and the AChR

Cormier, et al., Mol. Pharm. (66) Cormier, et al., Mol. Pharm. (66) 17121712

Smoking leads to up regulation of Smoking leads to up regulation of AChRs, which modulate activity of AChRs, which modulate activity of the CNS reward centersthe CNS reward centers

An effort to investigate the An effort to investigate the regulation of neuronal AChRs in regulation of neuronal AChRs in smokers and micesmokers and mice

Nicotine continuedNicotine continued

Smokers rated based on Smokers rated based on Fagerstrom Test for Nicotine Fagerstrom Test for Nicotine DependenceDependence

Only took smokers w/score Only took smokers w/score > > 22 Fagerstrom Test has been studied Fagerstrom Test has been studied

by many groups for efficacy…still by many groups for efficacy…still used.used.


Score of 6 or higher Score of 6 or higher considered very considered very dependent, less dependent, less than 2, very low than 2, very low dependencedependence

Score of 7 or higher Score of 7 or higher – recommended – recommended support for quiting, support for quiting, likelihood of quitting likelihood of quitting estimated at around estimated at around 8%8%


Structures of AChR binding ligandsStructures of AChR binding ligands ACh – cation/ACh – cation/ππ w/Trp w/Trp Nic – hydrogen bond to backbone C=ONic – hydrogen bond to backbone C=O EB – both interactions, higher affinityEB – both interactions, higher affinity


EB/BGT – bind hetero/homo pentamerEB/BGT – bind hetero/homo pentamer 18/92 smokers excluded w/no EB binding18/92 smokers excluded w/no EB binding Scatchard demonstrates 2 classes of EB Scatchard demonstrates 2 classes of EB

binding sitesbinding sites From PMN leukocytes [From PMN leukocytes [33H]EB and H]EB and 125125I-I-ααBGTBGT


PMN culture 3 PMN culture 3 days +/- Nicdays +/- Nic

Decrease in EB Decrease in EB sites from sites from immediate test to immediate test to 3 days (recall 3 days (recall previous figure)previous figure)


Moderately positive correlation Moderately positive correlation between EB sites and Fagerstrom between EB sites and Fagerstrom indexindex


Identifies only single class of Identifies only single class of binding sites in mouse splenocytesbinding sites in mouse splenocytes


Constant Constant administration of administration of nicotine using mini nicotine using mini osmotic pumpsosmotic pumps

Plasma concentration Plasma concentration of 30-35ng/ml of 30-35ng/ml ~30cigs/day~30cigs/day

Nic admin – black barsNic admin – black bars Noted increase in EB Noted increase in EB

sites (C & D), vs sites (C & D), vs ααBGTBGT



CaCa+2+2 influx through influx through AChR verified by AChR verified by addition of addition of competitive competitive antagonist, DHantagonist, DHββEE

Notable that Notable that WT+Nic is WT+Nic is comparable to comparable to ββ22-/--/-


ConclusionsConclusions– Increase in EB binding sites, indicative of Increase in EB binding sites, indicative of

tobacco consumption. May be more tobacco consumption. May be more accurate and objective assay of addiction accurate and objective assay of addiction vs. standard cot./creat. vs. standard cot./creat.

– Nic is the substance in cigs generating Nic is the substance in cigs generating AChR effects, and they do last several daysAChR effects, and they do last several days

– Heteromeric receptors are up regulatedHeteromeric receptors are up regulated– Loss of calcium conduction, although Loss of calcium conduction, although

possibly tissue dependentpossibly tissue dependent

Documents

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