Dr Sasha Gartisde Institute of Neuroscience Newcastle University Neuroscience

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

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

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

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

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

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

Ligand gated ion channels AMPA GABA A       BDZ BARBS GABA Na + Cl - NMDA Na + Glu GluR2 GluR1 GluN1 GluN2 Glycine binding site Ca 2+ The NMDA receptor admits Ca 2+ as well as Na +. It is blocked by Mg 2+ at low potentials. Glycine is a co-agonist. Na +

Ligand gated ion channels  5-HT3A 5-HT3B-E 5-HT 3 Na + K+K+  δ K+K+     Nicotinic ACh ACh Ca 2+ The 5-HT 3 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 Ca 2+ permeable.

G protein linked receptors-adenylate cyclase 7-transmembrane structure binding site Intracellular loops α γ β G protein α AC -ve GDP GTP out in 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 adenylate cyclase

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

G protein linked receptors-phosphatidyl inositol Intracellular loops α γ β G protein α PIP2IP3 & DAG GDP GTP out in Some G protein linked receptors stimulate phosphatidyl inositol turnover Phosphatidylinositol 4,5-biphosphate (PIP2) is cleaved into inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). IP3 causes releases Ca 2+ from the ER DAG and Ca 2+ activate protein kinase C and other kinases.

G protein linked receptors- ion channels Intracellular loops α γ β G protein GDP GTP out in 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-HT 1A autoreceptor is coupled to a K + channel α GTP K+K+ K+K+ K+K+

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

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

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 K+K+ K+K+ K+K+ K+K+ K+K+ K+K+ Na + K+K+ K+K+ The resting membrane potential Na + A-A- A-A- A-A- A-A- A-A- A-A- A-A- Cl - K+K+ K+K+ K+K+ K+K+ ATPase

mM K+K+ 5 Na Cl A-A- 0 mM K+K+ 100 Na + 15 Cl - 13 A-A- 385 Inside Outside The resting membrane potential (RMP) The unequal distribution of ions leads to a negative charge inside the cell RMP ≈70 mV

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

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+K+ K+K+ K+K+ K+K+ K+K+ Na + K+K+ K+K+ A-A- A-A- A-A- A-A- A-A- A-A- A-A- Cl - K+K+ K+K+ K+K+ K+K+ ATPase Na +

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 Membrane potential (mV) RMP time Small positive deflections in the membrane potential caused by receptor activation and cation influx induce an action potential -55

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

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+K+ K+K+ K+K+ K+K+ K+K+ Na + K+K+ K+K+ A-A- A-A- A-A- A-A- A-A- A-A- A-A- Cl - K+K+ K+K+ K+K+ K+K+ ATPase Cl - Na + Cl -

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

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+K+ K+K+ K+K+ K+K+ K+K+ Na + K+K+ K+K+ A-A- A-A- A-A- A-A- A-A- A-A- A-A- Cl - K+K+ K+K+ K+K+ ATPase Cl - Na + K+K+ K+K+ K+K+

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

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

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

Monoamine receptors DA -all G-protein linked D 2 - like inhibit AC, open K + channels D 1 - like stimulate AC NA –all G protein linked  1 -stimulate PI cycle  2 -inhibit AC, open K + channels  -stimulate AC 5-HT -mixed 5-HT 1 - inhibit AC, open K + channels 5-HT 2 - stimulate PI cycle 5-HT 3 - ligand gated ion channel

Cholinergic receptors Muscarinic –G protein linked M 1 – stimulates PI cycle Nicotinic –ligand gated ion channel Neuronal –α7 homomer /αβ heteromers Ganglionic NMJ

Receptor families 5-HT5-HT1 5- HT1A 5- HT1B 5- HT1D 5- HT1E 5- HT1F 5-HT2 5- HT2A 5- HT2B 5- HT2C 5-HT35-HT45-HT55-HT65-HT7 NAαα1α1α2Aα2Bα1Dα2α2α2Aα2Bα2Cα3α3ββ1β1β2β2β3β3

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

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

Postsynaptic receptors Postsynaptic receptors can be excitatory or inhibitory Sometimes both are found on the same cell e.g. 5-HT 2A, α 1, D 1&2, nACh, NMDA

Presynaptic receptors Presynaptic receptors are always inhibitory They inhibit neurotransmitter release by inhibiting voltage-gated Ca 2+ channels or enhancing K + -channel activation. They can also decrease release by modulating intracellular Ca 2+. e.g. 5-HT 1B, α 2, D 2, mACh 2, GABA B

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-HT 1A, α 2, D 2

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

Receptor desensitization and down regulation 1.Reduction in receptor affinity. Rapid and reversible. G-protein binding affects the receptor affinity. 2.‘Homologous desensitization’:- change of receptor coupling. Phosphorylation of GPCRs allows interaction with arrestins which prevents G protein coupling.

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

Receptor desensitization/down regulation 3.‘Down regulation’: reduction of receptor number in the membrane. – receptor internalization – enhanced receptor degradation – reduced receptor synthesis

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

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

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 5HT 1A autoreceptors E.g.2 Increased synaptic DA levels in response antipsychotics down regulate D 1 receptors

Receptor sensitization/upregulation Reduced exposure to agonists and continuous exposure to antagonists causes increased responsiveness 1.Increase in receptor affinity. Rapid and reversible. When G-protein is bound receptor affinity is greater. 2.‘Up regulation’: increase in receptor number in the membrane. – Receptor trafficking – Enhanced receptor synthesis – Reduced receptor degradation

Receptor sensitization/upregulation Denervation supersensitivity NB. 5-HT 2 receptors desensitize in response to both agonist and antagonist stimulation

Summary Receptor types Membrane & intracellular effects Locations & roles Receptor subtypes Receptor adaptation

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

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 Monoamine reuptake transporters In Out Monoamine Na + Cl - In Out

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