Dr Sasha Gartisde Institute of Neuroscience Newcastle University

1. Dr Sasha GartisdeInstitute of NeuroscienceNewcastle University Neuroscience

2. Drugs, receptors, and transporters • Most psychoactive drugs interfere with neurotransmission • The main targets are enzymes, transporters and receptors

3. Drugs, receptors, and transporters • Enzymes • monoamine oxidase inhibitors, L-DOPA, anticholinesterases • Transporters- • SSRIs antidepressants, cocaine, buproprion • Receptors • antipsychotics, anxiolytics (BDZs),

4. Neurotransmitter receptors • Specialized proteins • Embedded in the cell membrane • Bind neurotransmitter (or drug) • Induce intracellular response in response to extracellular event

5. Neurotransmitter receptors • 2 types • Ligand gated ion channel (ionotropic) • G-protein linked (metabotropic)

6. Ligand gated cation channels: e.g. The glutamate AMPA receptor GluR2 Na+ Na+ Na+ GluR2 GluR2 Cation channel closed Allosteric change opens Na+ channel Tetrameric structure Dimers of GluR2 and GluR1, GluR3 or GluR4

7. Cl- Cl- Ligand gated anion channels e.g. The GABAA receptor complex BDZ     GABA    GABA     GABA    GABA    BARBS Other modulators affect GABAA function Neurosteroids Z hypnotics Pentamer Chloride channel Allosteric change opens Cl- channel

8. Cl- Ligand gated ion channels Glycine binding site BDZ Na+ Na+ Glu  GluR1  GluN1 Ca2+ GluR2 Glu GABA GluN2 GABA  Na+  GluR1  GluN2  GluR2 GluN1 Glu Glu BARBS AMPA GABAA NMDA The NMDA receptor admits Ca2+ as well as Na+. It is blocked by Mg2+ at low potentials.Glycine is a co-agonist.

9. Ligand gated ion channels Na+ Na+ Ca2+ 5-HT3B-E   5-HT3A ACh 5-HT3B-E  δ  5-HT3B-E   5-HT3B-E K+ K+ Nicotinic ACh 5-HT3 The 5-HT3 receptor is a pentamer. The open receptor is permeable to Na+ and K+ The nicotinic acetyl choline receptor is a pentamer. The open receptor is permeable to Na+ and K+. Some forms are also Ca2+ permeable.

10. G protein linked receptors-adenylatecyclase 7-transmembrane structure G-protein linked receptors are a single protein chain There is a binding site outside and a G-protein binding site inside When activated, the G-protein hydrolyses GDP to GTP The GTP activated α subunit interacts with the enzyme adenylatecyclase binding site out AC in -ve α GTP Intracellular loops α γ β G protein GDP GTP

11. Bi-directional regulation of adenylatecyclase Regulation of adenylatecyclase is bi-directional Some receptors inhibit adenylatecyclase Some receptors activate adenylatecyclase cAMP activates protein kinase A binding site out AC in -ve +ve α α GTP Intracellular loops α γ β ATP cAMP G protein GDP GTP

12. G protein linked receptors-phosphatidylinositol Some G protein linked receptors stimulate phosphatidylinositol turnover Phosphatidylinositol 4,5-biphosphate (PIP2) is cleaved into inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). IP3 causes releases Ca2+ from the ER DAG and Ca2+ activate protein kinase C and other kinases. out in α GTP Intracellular loops α γ β PIP2 IP3 & DAG G protein GDP GTP

13. G protein linked receptors- ion channels Some G protein linked receptors are coupled to ion channels Activation of the receptor opens the K+ channel K+ leaves the cell causing hyperpolarization The 5-HT1Aautoreceptor is coupled to a K+ channel out K+ in α K+ GTP Intracellular loops α γ β K+ G protein GDP GTP

14. Summary: receptors • Neurotransmitter receptors are membrane bound • Ligand gated ion channel or G-protein linked • Multiple subtypes/ isoforms

15. What do neurotransmitter receptors do? • Receptors transfer the external signal (neurotransmitter) to the target cell • Ligand gated ion channels • have direct effects on membrane excitability • G-protein linked receptors • have indirect effects on membrane excitability • mediate other intracellular responses • modulate responses to ligand-gated ion channels

16. The resting membrane potential The cell membrane is impermeable to Na+ but permeable to K+ Na+/K+ATPase pumps 3Na+ out and 2K+in Large anions are fixed to cellular components Extracellular Cl- ions balance the large anions There are concentration gradients and an electrochemical gradient A- A- K+ A- K+ Cl- A- K+ Na+ K+ Cl- K+ K+ Cl- Na+ Na+ Na+ K+ K+ Cl- Na+ Na+ A- Na+ A- K+ Na+ Na+ Na+ K+ Na+ ATPase Cl- A- Na+ K+ K+ Cl- Cl-

17. The resting membrane potential (RMP) Inside Outside • The unequal distribution of ions leads to a negative charge inside the cell • RMP ≈70 mV

18. Ligand gated (cat)ion channels When a ligand gated cation channel is activated Na+ channels in the membrane open K+ A- A- K+ A- K+ Cl- A- K+ K+ Cl- K+ Cl- Na+ Na+ K+ K+ Cl- Na+ A- Na+ A- K+ Na+ Na+ Na+ K+ Na+ ATPase Cl- A- Na+ K+ Cl- Cl-

19. Ligand gated (cat)ion channels • Na+ rushes in down its concentration gradient • The Na+ carries positive charge • This increases the membrane potential to a more positive value K+ K+ K+ A- A- K+ A- K+ K+ Na+ Na+ Cl- A- K+ Na+ Cl- Na+ Cl- Na+ Na+ Na+ Na+ K+ Cl- A- Na+ A- Na+ K+ Na+ K+ ATPase Cl- A- Na+ Na+ K+ K+ Cl- Cl-

20. Membrane depolarization • If the membrane potential reaches -55mV • Voltage-gated Na+ channels open • Huge quantities of Na+ are allowed to enter the cell and an action potential occurs +30 -15 Membrane potential (mV) -55 -70 RMP time Small positive deflections in the membrane potential caused by receptor activation and cation influx induce an action potential

21. Ligand gated anion channels When a ligand gated anion channel is activated Cl- channels in the cell membrane open K+ A- A- K+ A- K+ Cl- Cl- Cl- K+ A- K+ Na+ Cl- K+ Cl- Cl- Na+ Na+ Na+ K+ K+ Cl- Na+ A- Na+ A- K+ Na+ Na+ Na+ K+ Na+ ATPase Cl- A- Na+ K+ K+ Cl- Cl-

22. Ligand gated anion channels • Some Cl- moves in down its concentration gradient • Cl- carries negative charge • The membrane potential is decreased to a more negative value K+ A- A- K+ A- K+ K+ Cl- Cl- Cl- Cl- A- K+ K+ Cl- Cl- Cl- Cl- Cl- Cl- Na+ Na+ K+ Cl- Na+ A- Na+ A- Na+ K+ Na+ Na+ Na+ K+ Na+ ATPase Cl- A- Na+ Na+ K+ K+ Cl- Cl-

23. Membrane hyperpolarization • Negative deflections offset any excitatory potentials • The cell is less likely to fire an action potential +30 -15 Membrane potential (mV) -55 -70 RMP time Small negative deflections in the membrane potential caused by receptor activation and chloride ion influx reduce the probability of an action potential

24. G-protein linked K+channels • Some GPCRs open K+ channels • K+ moves out down its concentration gradient • The membrane potential is decreased to a more negative value K+ A- A- K+ A- K+ K+ Na+ K+ A- K+ K+ K+ Cl- Cl- K+ Na+ K+ K+ Na+ Cl- A- Na+ A- K+ Na+ Na+ Na+ K+ ATPase Cl- A- Na+ Na+ Cl- Cl- Cl- Cl-

25. Membrane hyperpolarization • Negative deflections offset any excitatory potentials • The cell is less likely to fire an action potential +30 -15 Membrane potential (mV) -55 -70 RMP time Small negative deflections in the membrane potential caused by receptor activation and K+ efflux reduce the probability of an action potential

26. Neurotransmitter receptors • All neurotransmitters interact with multiple receptor subtypes • Subtypes mediate different effects and have different distributions • Drugs (but not the neurotransmitter) can distinguish between them

27. GABA and Glutamate receptors • GABAAligand gated Cl- ion channel (complex) • GABAB G-protein linked ↓AC , opens K+ channel • NMDA • AMPA ligand gated cation channel • Kainate • mGluR1-5 –metabotropic (G protein linked)

28. Monoamine receptors DA -all G-protein linked D2- like inhibit AC, open K+ channels • D1- like stimulate AC NA –all G protein linked 1-stimulate PI cycle 2 -inhibit AC, open K+ channels  -stimulate AC 5-HT -mixed 5-HT1 - inhibit AC, open K+ channels 5-HT2 - stimulate PI cycle 5-HT3 - ligand gated ion channel

29. Cholinergic receptors Muscarinic –G protein linked M1 – stimulates PI cycle Nicotinic –ligand gated ion channel Neuronal –α7 homomer /αβheteromers Ganglionic NMJ

30. Receptor families

31. All GABA receptors are inhibitory • Other neurotransmitters have mixture of inhibitory and excitatory receptors

32. Receptor localization • Receptors are found at postsynaptic, presynaptic and somatodendritic sites. • Some are also found extrasynaptically

33. Postsynaptic receptors • Postsynaptic receptors can be excitatory or inhibitory • Sometimes both are found on the same cell e.g. 5-HT2A, α1, D1&2, nACh, NMDA

34. Presynapticreceptors • Presynaptic receptors are always inhibitory • They inhibit neurotransmitter release by inhibiting voltage-gated Ca2+ channels or enhancing K+-channel activation. • They can also decrease release by modulating intracellular Ca2+. e.g. 5-HT1B, α2, D2, mACh2, GABAB

35. Somatodendritic receptors • Somatodendritic receptors are on the cell body (soma) and dendrites. • They respond to local levels of transmitter • Somatodendritic autoreceptors inhibit firing • Most activate GPRC- K+ channels e.g. 5-HT1A, α2, D2

36. Receptor adaptation Continuous exposure of cells to agonists causes loss of responsiveness 3 phases. 1. Reduction in receptor affinity 2. Reduction in receptor function 3. Reduction of receptor number

37. Receptor desensitization and down regulation • Reduction in receptor affinity. Rapid and reversible. G-protein binding affects the receptor affinity. • ‘Homologous desensitization’:- change of receptor coupling. Phosphorylation of GPCRs allows interaction with arrestins which prevents G protein coupling.

38. Desensitization/uncoupling • G-protein coupled receptors must be coupled to their intracellular G-protein out out AC AC in in -ve -ve α α GTP GTP α γ P β β-arrestin G protein GDP GTP α γ β Phosphorylated receptor binds β-arrestin G protein cannot bind

39. Receptor desensitization/down regulation • ‘Down regulation’: reduction of receptor number in the membrane. • receptor internalization • enhanced receptor degradation • reduced receptor synthesis

40. Receptor down regulation • There is a constant turn over of receptors • Receptors are synthesised in the nucleus, trafficked to the membrane, inserted in the membrane, internalized and degraded

41. Internalization • Receptors which are bound to β-arrestin are subject to internalization P β-arrestin P β-arrestin

42. Receptor adaptation in psychopharmacology • Adaptation in response to increased agonist concentration • E.g.1 Increased somatodendritic 5-HT levels in response to SSRI down regulate somatodendritic 5HT1Aautoreceptors • E.g.2 Increased synaptic DA levels in response antipsychotics down regulate D1 receptors

43. Receptor sensitization/upregulation Reduced exposure to agonists and continuous exposure to antagonists causes increased responsiveness • Increase in receptor affinity. Rapid and reversible. When G-protein is bound receptor affinity is greater. • ‘Up regulation’: increase in receptor number in the membrane. • Receptor trafficking • Enhanced receptor synthesis • Reduced receptor degradation

44. Receptor sensitization/upregulation Denervationsupersensitivity NB. 5-HT2 receptors desensitize in response to both agonist and antagonist stimulation

45. Summary • Receptor types • Membrane & intracellular effects • Locations & roles • Receptor subtypes • Receptor adaptation

46. Drugs, receptors, and transporters • Most psychoactive drugs interfere with neurotransmission • The main targets are enzymes, transporters and receptors

47. Monoamine reuptake transporters Out 12 transmembrane spanning protein Transport driven by concentration gradients of Na+ and Cl- DAT, NAT, and SERT (5­HTT) have high sequence homology Many drugs have poor transporter selectivity In Monoamine Cl- Na+ Out In

48. Monoamine reuptake transporters Out Neurotransmitters Releasing agents (amphetamines) bind and aretransported Antidepressants (TCAs, SSRIs, NARIs) Cocaine Bupropion bind and block transport In Monoamine Cl- Na+ Out In