Lecture 7: Transporters and Targets of Drug Action

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will induce a CC and activate the IC G- prtein signalling cascade f events. The imprtant difference t transprters is that the ligand unbinds after binding rather than being itself transprted.

1 Lecture 7: Transprters and Targets f Drug Actin Receptrs, whether intrpic (e.g. AMPA) r metabtrpic (e.g. mglur), are passive. Intrpic channels bind a ligand/are affected by vltage changes and then pen a pre in the channel whereby ins flw dwn against their electrchemical gradients, while metabtrpic will bind a ligand which will induce a CC and activate the IC G- prtein signalling cascade f events. The imprtant difference t transprters is that the ligand unbinds after binding rather than being itself transprted. Transprters n the ther hand can be either passive r active and have a cmpletely different functin i.e. t mve substances acrss the membrane int r ut f the cell e.g. EAAT. A substrate will bind t the transprter which will then mve it int the cell. Membrane prteins cntrl mvement f everything acrss the cell membrane: nutrient uptake, waste dispsal, cell t cell cmmunicatin (NT) Passive transprters (facilitatrs e.g. Na + independent glucse transprter) Active transprters (pumps) require energy in rder t mve a substance against its cncentratin gradient Primary active transprters: energy in the frm f light, ATP hydrlysis e.g. Na + /K + /ATPase pump which mves 2 K + ins in and 3 Na + ins ut and is pwered by the hydrlysis f ATP t ADP + P. This results in high Na + utside the cell and high K + in the cell. Secndary active transprters: energy in the frm f pre-existing in gradients f Na and K e.g. all NT transprters such as EAAT which mves Glu int the cell via a cupling with the favurable mvements f Na + and K + There are tw main families f NT transprters fund n pre- and pst-synaptic neurns and glial cells. Their rle is t clear NT frm the cleft. The tw families are: Glutamate transprter family (aka excitatry amin acid family) t remve glutamate and aspartate Neurtransmitter Sdium Symprter (NSS) family cntaining transprters t remve GABA, glycine, DA, NA, 5-HT imprtant fr antidepressants and anticnvulsants Bth are secndary active transprters cupled t pre-existing in gradients. An advantage t understanding hw these wrk is that we have crystal structures frm bacteria t use as mdels and mre recently has been mdelled ff mammalian and human structures. Glutamate transprter family

2 Excitatry Amin Acid Transprter (subtypes EAAT1-5): fund in pstsynaptic (EAAT 3 and 4) and astrcytic (EAAT 1 and 2) membranes Transprt L-glutamate and L-aspartate with similar affinities (2-20μM), EAAT3 als transprts L-cysteine Substrate inhibitr (taken thrugh transprter int cell) is D-aspartate fr all and additinally 4-Methyl- Glutamate (4MG) fr EAAT1 and 3; blckers (i.e. binds but prduces n effect) are TBOA fr all and additinally 4MG fr EAAT 2 and 4 and dihydrkainate (DHK) fr EAAT2 EAAT1 and 2 widely expressed thrughut glial cells EAAT3 widely expressed thrughut neurns EAAT4 expressed in cerebellar neurns EAAT5 expressed in retinal neurns EAAT3 and 4 are fund further away frm the synapse as their rle is t stp stray wandering glutamate frm diffusing t far away t anther synapse their rle is mre t d with verflw EAAT2 is ne f the mst abundantly expressed prteins in the brain and it des mst f the clearing [glutamate] at the synapse varies hugely frm 10mM t 10nM frm when released t when cleared They are very pwerful as they have very high rder cmplex cupling mechanisms: ne glutamate mlecule cuple t three Na + ins and ne H + prtn which all have a large driving frce int the cell and fr rerientatin a K + in flws ut but there is still a net transfer f tw + charges can use electrphysilgy t measure functin There are n therapeutic drugs that target glutamate transprters as inhibiting them wuld elevate EC glutamate which is severely excittxic Structures Bacterial transprter frm Pyrccccus hrikshii (Glt Ph ): three identical but independent subunit prtmers that cme tgether t frm trimer with a deep bwl facing utside the cell each subunit is capable f transprting. It is an Na + dependent aspartate transprter whereby aspartate binds in cnjunctin with Na + TBOA: an analgue f aspartate but with a benzyl ring; the mlecule binds in the same place as aspartate but the benzyl ring pushes ne subunit int the ther s the lid can t clse and the transprt prcess can t cntinue thus a cmpetitive inhibitr Excittxicity leads t multiple shrt term cnditins such ischaemia/strke and als and lng term such as neurdegenerative diseases, Alzheimer s, Parkinsn s, Mtr Neurn Disease Ischaemia (lack f xygen supply t brain), depletin f glucse, reductin in ATP failure f Na + /K + /ATPase t maintain gradients rundwn f gradients and thus membrane ptential uncrdinated AP generatin excessive glut release and glut transprter failure excessive stimulatin f GlutR excessive Ca 2+ influx activatin f prteases, lipases, NO synthase, endnucleases cell death by necrsis r apptsis EAATs culd be gd drug targets t minimise excittxicity if enhancers culd be made t wrk Neurtransmitter Sdium Symprter (NSS) family Cupled t c-transprt f Na + and Cl - Target fr many current and prspective therapies including drugs f abuse Crystal structures have been btained frm a bacterial hmlgue but the family is very big and brad s this mdel has prven t be nt as useful Bacterial hmlgue is LeuT Aa which is a mnmer has 12 tm dmains and is a sht glass shape, the substrate (leucine) is bund within the subunits and is c-transprted with tw Na + ins (substrate and Na + binding sites are highly cnserved acrss whle family) NT Transprter Therapeutic drugs Drugs f abuse targeting transprters Glycine GlyT1 and GlyT2 Pain, antipsychtics GABA GAT1-4 Anticnvulsants (tiagabine) Dpamine DAT Antidepressants (TCAs) Amphetamine, meth, ccaine, MTPT NA NET Antidepressants (TCAs, SNRIs) Amphetamine, meth, ccaine 5-HT SERT Antidepressants (TCAs, SSRIs) MDMA, ccaine Glycine: GlyT1 cupled t 2 Na + and 1 Cl - while GlyT2 cupled t 3 Na + and 1 Cl - thus they have slightly different cncentrating capacities becmes apparent in terms f their lcatin as it can be inhibitry at inhibitry neurns and als excitatry at excitatry synapses as it is an essential c-agnist at the NMDA receptr GlyT2 is nly fund at inhibitry neurns and have a strnger cncentrating capacity s there is lw glycine in the area

Inhibitrs f this transprter will nly target these inhibitry receptrs as they are nly fund in the SC, s inhibitin f receptrs will result in enhanced glycinergic inhibitin thus a reductin in pain

3 Inhibitrs f this transprter will nly target these inhibitry receptrs as they are nly fund in the SC, s inhibitin f receptrs will result in enhanced glycinergic inhibitin thus a reductin in pain signals frm SC t brain and a reduced perceptin f pain Example f inhibitr: ALX1393 (a nn-cmpetitive inhibitr that binds differently t the substrate) which prevents ne helix frm mving dwn int the active site which verall prevents the whle transprt prcess frm ccurring Lipid-based drugs may bind at the prtein-lipid interface GlyT1 is fund at bth e and i neurns but nly plays a rle in excitatry neurns, it als has a lwer cncentrating capacity meaning that there is mre glycine at these synapses, thus a higher basal cnc. f glycine s when glutamate is released frm vesicles the glycine is already there at the synapse ready t be a c-agnist There are currently n GlyT1 inhibitrs in the market but they are a prmising target because f the NMDAR hypfunctin hypthesis in schizphrenia there is reduced NMDA activity s if GlyT1 is inhibited there will be higher [Gly] at excitatry synapses, further stimulating NMDAR e.g. Sarcsine, NFPS GABA transprters: GAT1-4 expressed in neurns and glial cells thrughut the CNS GABA is cupled t 2 Na + and 1 Cl - and drive GABA int the cell Drug mdulatin: particularly useful as anticnvulsants as inhibitin f transprters will elevate GABA and stimulate GABA neurtransmissin prducing a sedative effect e.g. tiagabine which is selective fr GAT1 and is based n nipectic acid DAT, NET, SERT: mst widely studied transprters due t their ptential as drug targets, mve their respective substrates int the cell via cupling f inward-mving ins Amphetamine and Methamphetamine 1890s first synthesised frm ephedrine 1950s prescribed fr narclepsy, Parkinsn s, alchlism, depressin, besity 1960s used as recreatinal drug 1980s made illegal but use escalated Since 2000 crystal methamphetamine has becme the mre cmmn street frm and is a drug f abuse Generally methamphetamine prduces mre exaggerated effects related t the higher effective dses used and greater prpensity fr addictin Amphetamine: arusal, euphria, alertness, capable f cnducting ver learned tasks Methamphetamine: euphria, increased energy and attentiveness, diarrhea and nausea, excessive sweating, lss f appetite, insmnia, tremr, jaw-clenching, agitatin, talkativeness, irritability, panic attacks, increased libid Chrnic use f methamphetamine leads t drug craving, withdrawal-related depressin, rapid tth decay meth muth, degeneratin f the dpaminergic neurns, weight lss, psychsis Overdse f methamphetamine leads t brain damage, sensatin f flesh crawling with bugs, parania, delusins, hallucinatins, muscle breakdwn leading t kidney failure, death due t strke, heart failure, cardiac arrest r hyperthermia MOA f methamphetamine: a false substrate (binds, inhibits dpamine receptr and enters cell via this receptr) leading t increased efflux f dpamine = much larger [dpamine] utside the cell; als binds t and affects the NA transprter Accumulates in presynaptic neurn t inhibit mnamine xidase, cmpetes with VMAT t deplete and replace strage f DA in vesicles

Ccaine Islated frm leaves f the cca plant, been used fr thusands f years by Suth American Indians but was purified in 1855 Stimulant and appetite suppressant, tpical anaesthetic as a Na + channel

system cause it t be addictive Was added t Cca Cla between 1886 and 1906 Last year a new mdel was released which was better than the LeuT Aa mdel frm the drsphilia dpamine transprter (ddat).

4 Ccaine Islated frm leaves f the cca plant, been used fr thusands f years by Suth American Indians but was purified in 1855 Stimulant and appetite suppressant, tpical anaesthetic as a Na + channel blcker MOA: Cmpetitive inhibitr/blcker f DAT, NET and SERT transprters at the surface t increase these [NT] in the synapse rather than entering the cell like methamphetamines Actins n the meslimbic system cause it t be addictive Was added t Cca Cla between 1886 and 1906 Last year a new mdel was released which was better than the LeuT Aa mdel frm the drsphilia dpamine transprter (ddat). Lecture 8: Mnamines Transmitter substances: Synaptic events f mnamines: Mnamines are catechlamines (NA, A, DA) with a catechl ring and indleamines (5-HT, melatnin) with an indl ring. They are thus structurally different mlecules. All mnamines are synthesised frm decarbxylated AAs, the reactin f which is catalysed by cytslic enzymes. Synthesising enzyme determines the characteristics f the neurn e.g. dpamine fr dpaminergic neurns. They activate mainly GPCRs thus attractive drug targets because they mderate functin rather than mediate it. The six targets fr drugs at the synapse: synthesis, strage, release, receptr actin, reuptake and degradatin Synthesis D, NA and A in their respective neurns frm ne precursr: L-tyrsine (AA) L-DOPA via tyrsine hydrxylase fund in cytplasm dpamine via L-armatic acid decarbxylase/dpa decarbxylase in cytplasm NA via dpamine β-hydrxylase in vesicles A via PNMT in cytplasm

5-HT: tryptphan (AA) is an essential AA fund nly in diet (mre tryptphan eaten = mre 5-HT synthesised) Drugs affecting mnamine synthesis: dietary tryptphan increases 5-HT synthesis (in bananas) MDMA

regulates mnamine cnc. in the cell and synapse) Release: traditinal synaptic release.

5 5-HT: tryptphan (AA) is an essential AA fund nly in diet (mre tryptphan eaten = mre 5-HT synthesised) Drugs affecting mnamine synthesis: dietary tryptphan increases 5-HT synthesis (in bananas) MDMA inhibits tryptphan hydrxylase thus inhibits 5-HT synthesis L-DOPA is used in Parkinsn s disease t increase synthesis f dpamine (it can x the BBB since dpamine can t) Strage: actively transprted int vesicles (1.1M) via vesicular mnamine transprter (VMAT) and stred reversibly as bund cmplex with cnstituents such as ATP, prtein, Ca 2+, Mg 2+ t prevent leakage f the mnamine frm the vesicle t the cytplasm (thus regulates mnamine cnc. in the cell and synapse) Release: traditinal synaptic release. 5-HT and NA are als called diffusely prjecting NTs as they can als be released frm varicsities alng the axns f the neurn, frming cncentratin gradients in these prjectin areas, as well as at synapses int the EC space Drugs affecting strage and release: Amphetamines such as dexamphetamine, methamphetamine (which are substrates fr VMAT) cmpete with mnamines fr vesicular strage, they displace mnamines thus enhancing their release, reverse vesicular uptake and plasma membrane uptake prcess, but can be used therapeutically e.g. in treatment f ADHD Pathways: NA: riginate frm lcus ceruleus (where synthesis enzymes are fund) and reticular frmatin in brainstem, prjected t amygdala, hypthalamus, thalamus, mst f crtex, cerebellum, SC (fr pain mdulatin) 5-HT: similar t NA, mainly in raphe nuclei and prject t cerebellum and dwn SC, t amygdala, hippcampus, hypthalamus, septum, crtex, striatum

Cellular Neurophysiology Membrane Ionic Gradients Concentration gradients and diffusion Membrane transport:

Cellular Neurophysiology Membrane Ionic Gradients Concentration gradients and diffusion Membrane transport: Cellular Neurphysilgy Membrane Inic Gradients Fluid-msaic mdel f plasma membrane: lipid bilayer separating intracellular and extracellular fluids Biplar phsphlipids Hydrphilic head grups and hydrphbic