The Nicotine Receptor ebooksclub.org Tobacco or Health Physiological and Social Damages Caused by Tobacco Smoking Second Edition

Langley฀and฀Anderson฀ [1] ฀and฀Dale฀ [2] .฀Detailed฀analyses฀of฀the฀receptor฀and฀its฀subunits฀ were฀made฀possible฀in฀the฀wake฀of฀studies฀of฀the฀electric฀organ฀of฀the฀Torpedo฀and฀the฀ identiication฀of฀a-neurotoxins. nAChRs฀consist฀of฀pentameric฀LGICs,฀are฀found฀in฀the฀central฀nervous฀system฀CNS฀ and฀in฀peripheral฀nerve฀structures,฀and฀comprise฀an฀a-฀and฀a฀b-subunit฀with฀a฀large฀number฀ of฀variants฀a2–a9,฀b2–b4,฀g,฀d,฀e฀Fig.฀ 4.1 ฀ [3,฀4] .฀In฀contrast฀to฀their฀central฀role฀in฀auto- nomic฀neurotransmission฀and฀the฀triggering฀of฀muscle฀contraction,฀nAChRs฀in฀the฀CNS฀ display฀several฀modulatory฀reactions฀ [3] .฀The฀importance฀of฀nAChRs฀in฀the฀pathophysiol- ogy฀of฀various฀disorders,฀such฀as฀Alzheimer’s฀disease,฀Parkinson’s฀disease,฀schizophrenia,฀ Tourette฀syndrome,฀etc.,฀is฀now฀established฀ [5] .฀Furthermore,฀they฀also฀play฀a฀key฀role฀in฀ smoking฀cessation,฀analgesia,฀anxiolysis฀and฀neuroprotection฀ [5] .฀Important฀subtypes฀of฀ nAChR฀are฀found฀in฀the฀central฀and฀peripheral฀nervous฀system฀and฀another฀subtype฀is฀ found฀in฀both฀systems฀Fig.฀ 4.1 . The฀nAChR฀of฀mammalian฀brain฀consists฀predominantly฀about฀90฀of฀2a 4 ฀and฀3b 2 ฀ subunits฀ [6] ฀and฀binds฀[H]-cytisine฀and฀nicotine฀with฀high฀afinity฀ [7,฀8] ฀Table฀ 4.1 [10] .฀ Another฀form฀of฀nAChR฀occurring฀both฀in฀the฀CNS฀and฀the฀peripheral฀nervous฀system฀is฀ made฀up฀exclusively฀of฀a 7 ฀subunits฀ [11] ฀Fig.฀ 4.1 .฀The฀a฀subunits฀contain฀a฀cysteine฀pair฀ in฀positions฀192–193฀of฀the฀C฀loop฀Figs.฀ 4.2 ฀and฀ 4.3 .฀These฀loops฀accommodate฀the฀bind- ing฀site฀for฀agonists฀and฀also฀contain฀aromatic฀side-chains฀tryptophan,฀tyrosine,฀which฀ trigger฀cationic฀p-interactions฀with฀the฀agonists฀ [13] .฀The฀polypeptide฀chain฀of฀the฀nAChR฀ subunits฀contains฀four฀hydrophobic฀transmembrane฀domains฀M1–M4,฀which฀span฀the฀ plasma฀membrane฀Fig.฀ 4.2 .฀M2฀is฀an฀a-helix฀that฀includes฀the฀cation฀channel. Binding฀afinity฀K i ฀nM Functional฀potency฀EC 50 ฀µM a 4b2 a 7 a 3b4 a 4b2 a 7 a 3b4 –-Nicotine 1–11 400–8,900 300–475 0.3–15 18–91 5–410 –-Cytisine 0.14–2.7 1,400–3,883 56–195 0.019–71.4 n.d. 72–134 –-Epibatidine 0.01–0.06 3.1–350 n.d. 0.004–0.02 1.1–2.2 0.009 Acetylcholine 6.8–57 4,000–10,830 560–881 0.48–3 79–316 53–210 Choline 112,000 2,380,000 n.d. n.d. 1,600 n.d. Lobeline 4–50 11,000–13,100 480 n.d. no฀activ. n.d. Carbachol 207–582 18,000–580,000 3,839 2.5–29 296 n.d. DMPP 9.4–400 160–2,300 1,300 0.07–18 19–75 10–92 Table 4.1 ฀฀฀Binding฀ afinities฀ K i ฀ and฀ functional฀ potencies฀ EC 50 ฀ reported฀ at฀ brain฀ thalamic฀ synaptosomes฀a 4 b 2 -nAChRs฀or฀heterogenously฀expressed฀nAChRs฀for฀nicotinic฀agonists฀at฀native฀ or฀recombinant฀a 4 b 2 -฀and฀a 7 -nAChRs฀and฀recombinant฀a 3 b 4 -nAChRs฀ [9] DMPP฀dimethylphenylpiperazine;฀n.d.฀not฀determined;฀no฀activ.฀no฀activation CNSPNS CNS PNS α4 α4 β2 β2 β2 α3 α5 α3 β4 βx α7 α7 α7 α7 α7 Fig. 4.1 ฀฀฀Schematic฀ ฀illustration฀of฀the฀structure฀of฀ nAChRs฀in฀the฀central฀and฀ peripheral฀nervous฀system NH 2 N-Terminal Glycosylation C-Terminal COOH 128 142 M1 M2 M4 Phosphorylation s s 192-193 M3 s-s Fig. 4.2 ฀฀฀Structure฀of฀a฀ nAChR฀subunit฀segments฀ M1฀to฀M4฀ [12] Channel mouth and outer vestibule Non-competitive antagonist interacting with extracellular domain Positive allosteric modulator Protein-lipid Interface: Local anesthetic blockage, allosteric steroid blockage Channel blocking non-competive antagonist Modulatory sites e. g. Pi: Abnormal aggregation, defective interaction with nonreceptor proteins Channel inner vestibule Cytoplasmatic domain Channel “synaptic” half Agonist binding site AgonistCompetive antagonist Extracellular domain: ligand recognition sites Ca2+Na+ K+ Fig. 4.3 ฀฀฀Structural฀ overview฀ of฀ a฀ nAChR฀ with฀ one฀ subunit฀ removed฀ revealing฀ channel฀ lumen.฀ Demonstrated฀are฀binding฀sites฀for฀agonists,฀competitive฀and฀non-competitive฀antagonists฀and฀positive฀ allosteric฀modulators฀e.g.฀ACh฀esterase฀inhibitors฀ [5] A฀study฀published฀in฀2004฀in฀Science฀analysed฀the฀identity฀of฀nicotinic฀receptor฀฀subtypes฀ suficient฀to฀elicit฀both฀the฀acute฀and฀chronic฀effects฀of฀nicotine฀dependence฀is฀unknown.฀ The฀researchers฀engineered฀mutant฀mice฀with฀a4฀nicotinic฀subunits฀containing฀a฀single฀ point฀mutation,฀Leu9’→Ala9’,฀in฀the฀pore-forming฀M2฀domain,฀rendering฀a4฀receptors฀ hypersensitive฀ to฀ nicotine.฀ Selective฀ activation฀ of฀ a4฀ nicotinic฀ acetylcholine฀ receptors฀ with฀low฀doses฀of฀agonist฀recapitulates฀nicotine฀effects฀thought฀to฀be฀important฀in฀depen- dence,฀including฀reinforcement฀in฀response฀to฀acute฀nicotine฀administration,฀as฀well฀as฀ tolerance฀and฀sensitisation฀elicited฀by฀chronic฀nicotine฀administration.฀The฀data฀indicated฀ that฀activation฀of฀a4฀receptors฀is฀suficient฀for฀nicotine-induced฀reward,฀tolerance฀and฀ sensitisation฀ [14] . M3฀and฀M4฀are฀separated฀from฀each฀other฀by฀a฀long฀intracellular฀loop฀that฀contains฀ centres฀for฀the฀phosphorylation฀of฀serinethreonine฀kinases฀ [15] .฀During฀binding฀of฀the฀ agonist฀e.g.฀nicotine,฀the฀nAChRs฀undergo฀allosteric฀modulation฀ [16] ฀in฀which฀there฀is฀a฀ transition฀from฀the฀resting฀conformation฀to฀an฀open฀state฀in฀which฀the฀cations฀Na + ,฀K + ฀and฀ Ca 2+ ฀are฀transported.฀When฀nAChRs฀are฀in฀the฀open฀state,฀agonists฀are฀bound฀with฀low฀ afinity฀Figs.฀ 4.2 ฀and฀ 4.3 .฀The฀permanent฀presence฀of฀an฀agonist฀results฀in฀channel฀closure฀ and฀desensitisation฀of฀the฀receptor,฀which฀becomes฀refractory฀for฀activation฀Fig.฀ 4.4 ฀ [17] .฀ The฀various฀nAChR฀subtypes฀differ฀considerably฀in฀terms฀of฀extent฀of฀desensitisation฀and฀ recovery:฀the฀a 7 ฀nAChR฀becomes฀desensitised฀very฀rapidly฀ [18] ฀and฀the฀permanent฀presence฀ of฀an฀agonist฀results฀in฀inactivation฀followed฀by฀only฀slow฀recovery.฀The฀neuronal฀a 4 b 2 ฀ nAChR฀is฀very฀susceptible฀to฀inactivation฀in฀response฀to฀chronic฀nicotine฀exposure฀ [19] .฀ The฀transitions฀from฀the฀resting,฀open฀and฀inactive฀states฀are฀reversible,฀and฀various฀ligands฀ are฀able฀to฀stabilise฀the฀conformational฀receptor฀state.฀Agonists฀initially฀stabilise฀the฀acti- vated฀ open฀ state,฀ whereas฀ competitive฀ antagonists฀ preferentially฀ stabilise฀ the฀ closed฀ resting฀or฀inactive฀state฀Fig.฀ 4.4 . Tables฀ 4.1 ฀and฀ 4.2 ฀present฀details฀of฀the฀activity฀of฀a฀small฀number฀of฀agonists฀and฀ antagonists฀on฀various฀nAChR฀subtypes. As฀well฀as฀the฀muscle฀nicotine฀receptor,฀the฀neuronal฀nicotine฀receptor฀has฀also฀been฀ largely฀characterised.฀The฀nAChRs฀in฀the฀various฀brain฀regions฀display฀differences฀in฀their฀ R Resting Channel closed Fast msec Nicotine binds with low affinity mM-µM A Active Channel open There are multiple desensitised states D Desensitised channel closed I Inactive Nicotine binds with High affintiy µM-nM Slow sec - min Fig. 4.4 ฀฀฀Relationship฀ between฀major฀states฀ occupied฀by฀a฀nAChR binding฀kinetics฀to฀nicotine฀and฀in฀their฀reactivity฀to฀electrophysiological฀stimuli฀ [3,฀4,฀20] .฀ Permanent฀desensitisation฀of฀the฀receptor฀may฀explain฀the฀occurrence฀of฀tachyphylaxis฀ Fig.฀ 4.4 .฀In฀addition,฀up-regulation฀of฀nicotine฀receptors฀occurs฀in฀response฀to฀prolonged฀ nicotine฀exposure฀ [21,฀22] .

4.2 Agonists and Antagonists of the nAChR and its Subtypes

Nicotine฀agonists฀react฀with฀the฀different฀nAChR฀subtypes฀by฀attaching฀themselves฀to฀the฀ various฀binding฀sites฀and฀causing฀allosteric฀modulation฀of฀the฀pentameric฀complex฀with฀ opening฀of฀the฀ion฀channel.฀The฀changes฀measured฀for฀some฀agonists฀are฀summarised฀in฀ Table฀ 4.1 .฀Apart฀from฀nicotine,฀naturally฀occurring฀agonists฀include฀cytisine,฀anatoxin฀A,฀ epibatidine฀and฀anabasine฀Box฀ 4.1 .฀The฀binding฀afinity฀of฀these฀substances฀to฀the฀a4b2฀ nAChR฀subtypes฀is฀100–1,000฀times฀higher฀than฀to฀the฀a7฀subtypes฀Table฀ 4.1 .฀Their฀ binding฀afinities฀K i ฀are฀100–1,000฀times฀higher฀than฀their฀functional฀potencies฀EC 50 ฀for฀ the฀activation฀of฀the฀nAChR฀subtypes฀Table฀ 4.1 .฀Nicotine฀is฀the฀prototype฀nAChR฀ago- nist:฀it฀binds฀with฀high฀afinity฀to฀the฀a4b2฀nAChR฀Table฀ 4.1 ,฀while฀the฀a7฀nAChR฀reacts฀ 1,000฀times฀less฀sensitively฀ [23] .฀The฀binding฀activities฀of฀the฀nicotine฀metabolite,฀coti- nine,฀are฀scarcely฀measurable฀ [24] . Competitive฀antagonists฀act฀reversibly฀with฀the฀nAChR฀by฀stabilising฀the฀conformation฀of฀ the฀binding฀site฀and฀thereby฀blocking฀the฀action฀of฀agonists.฀However,฀this฀effect฀can฀be฀abol- ished฀by฀high฀doses฀of฀the฀agonist.฀ d -tubocurarine฀d-TC;฀Box฀ 4.2 ฀and฀dihydro-b-฀erythroidine฀ are฀classic฀examples฀of฀such฀substances฀see฀Table฀ 4.2 .฀This฀group฀includes฀various฀snake฀ venoms฀ such฀ as฀ bungarotoxin฀ and฀ a฀ range฀ of฀ a-conotoxins.฀ d-TC฀ does฀ not฀ discriminate฀ between฀the฀various฀nAChR฀subtypes,฀and฀is฀active฀at฀concentrations฀of฀10฀µM฀ [25] . Non-competitive฀antagonists฀produce฀their฀effect฀away฀from฀the฀binding฀centre฀and฀ therefore,฀do฀not฀interact฀with฀agonists.฀Their฀effect฀is฀achieved฀as฀a฀result฀of฀binding฀in฀ the฀vicinity฀of฀the฀ion฀channel,฀and฀consequently,฀these฀substances฀can฀also฀be฀used฀to฀ predict฀possible฀concentrations฀at฀the฀receptor.฀In฀this฀category,฀the฀classic฀example฀is฀ Table 4.2 ฀฀฀Binding฀afinities฀K i ฀for฀nicotinic฀antagonists฀in฀competition฀binding฀assays฀at฀native฀or฀ recombinant฀a4b2฀and฀a7-nAChRs฀and฀recombinant฀a3b4-nAChRs nAChR-subtype antagonist K i ฀nM a 4b2 a 7 a 3b4 d-Tubocurarine 1,000–25,000 3,400–7,700 22,929 Dihydro-b-erythroidine 13.9–1,900 25,000–57,900 218,622 Methyllycoconitine฀ MLA 3,700–6,100 0.69–10.3 3,700 Decamethonium 460–120,000 124,000–200,000 n.d. Mecamylamine 822,000–1,000,000 1,000,000 1,000,000 Binding฀afinities฀reported฀for฀competition฀binding฀assays฀of฀[H]-agonist฀binding฀to฀brain฀mem- branes฀a4b2-nAChRs฀and฀[?H]-agonist฀binding฀to฀heterologously฀expressed฀a4b2-nAChRs฀ [9] .฀ n.d.฀not฀determined mecamylamine฀Box฀ 4.2 ;฀Table฀ 4.2 ,฀which฀has฀IC 50 ฀values฀in฀the฀lower฀µM฀range฀ [25] ;฀ in฀this฀case,฀the฀a7฀nAChR฀subtypes฀react฀somewhat฀less฀sensitively฀than฀the฀a–b฀heter- omers.฀In฀addition,฀N-methyl- d -aspartate฀NMDA฀receptors฀are฀blocked฀at฀concentra- tions฀in฀the฀higher฀µM฀range฀ [26] .฀Similar฀effects฀are฀also฀produced฀by฀other฀ganglionic฀ blocking฀agents,฀such฀as฀hexamethonium,฀decamethonium฀and฀chlorisondamine฀Table฀ 4.2 .฀At฀concentrations฀in฀the฀low฀µM฀range,฀the฀antidepressant฀bupropion฀also฀inhibits฀ various฀nAChR฀subtypes฀a3b2,฀a4b2,฀a7฀in฀the฀rat฀as฀well฀as฀nAChR-mediated฀rubid- ium฀eflux฀of฀a฀human฀cell฀line฀SH-SY5Y฀ [27,฀28] .฀The฀effect฀of฀bupropion฀on฀human฀ cells฀was฀voltage-independent,฀with฀the฀result฀that฀an฀effect฀via฀the฀channel฀lumen฀may฀ be฀ excluded.฀ Further฀ substances฀ with฀ non-competitive฀ effects฀ include฀ the฀ neuroleptic฀ drug฀chlorpromazine฀and฀the฀anaesthetics฀phencyclidine฀and฀ketamine฀ [29] .฀Various฀ste- roid฀hormones,฀such฀as฀฀corticosterone,฀aldosterone,฀oestradiol฀and฀cortisol,฀are฀able฀to฀ inhibit฀neuronal฀nAChR฀subtypes฀from฀a฀human฀cell฀line฀SH-SY5Y฀at฀concentrations฀in฀ the฀upper฀nM฀to฀lower฀µM฀range฀ [30] .฀Progesterone฀inhibits฀the฀a4b2฀nAChR฀subtype฀at฀ concentrations฀of฀only฀9฀µM฀IC 50 ฀ [31,฀32] .฀A฀b-amyloid฀polypeptide 1–42 ฀also฀inhibits฀the฀ a 7฀nAChR฀subtype฀in฀the฀pM฀range฀ [33] ,฀prompting฀speculation฀concerning฀an฀associa- tion฀with฀the฀pathogenesis฀of฀Alzheimer’s฀disease฀and฀the฀role฀of฀nAChRs฀in฀its฀develop- ment.฀Section฀ 7.1 ฀in฀ Chap.฀7 ฀includes฀a฀discussion฀of฀the฀association฀between฀smoking฀ and฀slowing฀of฀the฀progression฀of฀Alzheimer’s฀disease.

4.3 Pharmacology

Like฀acetylcholine,฀nicotine฀stimulates฀receptors฀of฀the฀parasympathetic฀nervous฀system,฀ and฀a฀distinction฀is฀drawn฀between฀nicotinic฀and฀muscarinic฀receptors฀N฀and฀M฀receptors฀ and฀effects.

4.3.1 Effects of Nicotine on Receptors in Different Organs

Nicotine฀predominantly฀stimulates฀presynaptic฀nACh฀receptors,฀thereby฀producing฀an฀excit- atory฀action฀ [4,฀20] .฀If฀these฀receptors฀are฀located฀on฀dopaminergic฀neurons,฀they฀฀promote฀ Box 4.2 ฀฀฀฀Compounds฀with฀nAChR-antagonistic฀eficacy:฀mecamylamine,฀d-tubocurarine H N CH 3 2 H N OH O OCH 3 H O OH Mecamylamine d-Tubocurarine H 3 C H 3 CO CH 3 CH 3 CH 2 CH 3 NHCH 3