The Acetylcholine The Acetylcholine Receptor – An LGIC Receptor – An LGIC Prototype Prototype Rose Chase Rose Chase February 11, 2005 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
Structure continuedStructure continued
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
Structure continuedStructure continued
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…
Structure continuedStructure continued
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
Structure continuedStructure continued
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
Structure continuedStructure continued
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
Structure continuedStructure continued
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
Clustering continuedClustering continued
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
Clustering continuedClustering continued
- 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
Clustering continuedClustering continued
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
Clustering continuedClustering continued
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
Clustering continuedClustering continued
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.
Nicotine continuedNicotine continued
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%
Nicotine continuedNicotine continued
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
Nicotine continuedNicotine continued
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
Nicotine continuedNicotine continued
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)
Nicotine continuedNicotine continued
Moderately positive correlation Moderately positive correlation between EB sites and Fagerstrom between EB sites and Fagerstrom indexindex
Nicotine continuedNicotine continued
Identifies only single class of Identifies only single class of binding sites in mouse splenocytesbinding sites in mouse splenocytes
Nicotine continuedNicotine continued
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
Nicotine continuedNicotine continued
Nicotine continuedNicotine continued
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-/--/-
Nicotine continuedNicotine continued
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