Postsynaptic Aspects of Synaptic Transmission

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Post-synaptic Aspects of Synaptic Transmission

• October 29, 2008

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Generation of Post-Synaptic Potentials

• Action potentials produces very brief, very large
  increase in exocytosis rate
• synchronizes release at active zones
• Exocytosis releases vesicles (quanta) of
  neurotransmitter into synaptic cleft (100 nm
  wide)
• Diffuses across cleft within 2 ms concentration
  reaches 1 mM
• Neurotransmitter binds to and opens channels
• Ion influx produces depolarization

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Generation of Post-Synaptic Potentials
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Neurotransmitter Receptors

• Response to neurotransmitter release
  (post-synaptic current) depends on
• Receptor Type
• Structure
• Function
• How channel structure is related to function
• Post-synaptic Potentials
• Effect of synaptic current on membrane potential
• Electrical synapses

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Criteria for Identification of Neurotransmitter
Acting at Neuron

• Synthesized by Neuron
• Neuron contains neurotransmitter
• Neuron contains synthesizing enzymes
• Released from nerve terminal by stimulation
• Putative neurotransmitter applied exogenously
  reproduces effect on post-synaptic cell that
  occurs with nerve stimulation
• Post-synaptic response depends on transmitter
  released, and post-synaptic receptor

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Response Depends on Receptor

• Type
• Direct (ionotropic)
• Modulatory (metabotropic G protein coupled)
• Magnitude depends on
• Number of receptors
• State of receptors
• Amount of neurotransmitter
• Sign
• Inhibitory or excitatory

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Receptor Types

• Ionotropic
• Electrical response to neurotransmitter binding
• Large, 4 to 5 subunit protein forms channel
• Impermeable in the absence of transmitter
• Millisecond timescale
• Rapid onset, rapidly reversible
• G protein coupled receptor (GPCR)
• Single polypeptide receptor
• Enzyme that activates GTP binding protein
• Slow onset, long duration

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Receptor Types
Ionotropic
Metabotropic
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Receptor Types Example

• Acetylcholine receptors
• Nicotinic Ach (ionotropic)
• Predominant type in muscle
• Blocked by hexamethonium, curare
• Muscarinic Ach (GPCR)
• Predominant type in heart and CNS
• Blocked by atropine, scopolamine
• Receptor subtypes, e.g. m1-m4, exist within the
  major classes

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Acetylcholine Receptors in Periphery
The Neuron, Levitan and Kaczmarek
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Terminology (Receptors discriminated
pharmacologically)

• Agonist
• Molecule that binds to and activates receptor or
  channel
• Antagonist
• Molecule that binds to and inhibits receptor or
  channel
• Desensitization
• Transition to closed state in presence of
  neurotransmitter
• Limits ion influx
• Allosteric binding sites
• Different than binding site of ligand
• Modulates receptor or channel properties

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Pharmacological Characterization

• Pharmacological profile
• Concentration of various agonists and antagonists
  that produce effect
• Different ligands have different affinities for
  different receptors
• Measured with radioligand assay
• Maximum amount of ligand bound number of
  binding sites
• Concentration for half binding affinity

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The Neuron, Levitan and Kaczmarek
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Pharmacological Characterization

• Assay for affinity of other agonists or
  antagonists
• Mix various concentrations of agonist or
  antagonist with saturating concentration of
  ligand
• Affinity of agonist/antagonist is concentration
  at which half of ligand is displaced

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Curves for different competitve antagonists A1 is
high affinity (easily displaces bound ligand) A4
is low affinity (high concentration required to
displace bound ligand)
The Neuron, Levitan and Kaczmarek
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Ionotropic Receptors

• Two main families, subdivided by agonist
• Nicotinic Acetylcholine Receptor (nAChR) Family
• nAChR
• GABAA
• Glycine
• Serotonin
• Glutamate Receptor Family
• NMDA
• Non-NMDA

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Nicotinic AChR

• Studied as prototype of ionotropic receptors
• Torpedo electric organ AChR is model
• Acetylcholine receptor
• Nicotine is an agonist
• a-bungarotxoin binds with high specificity
• Used to purify the receptor
• Affinity 10-15
• 290 kDa

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Nicotinic AChR structure

• Heteropentamer
• Each channel has 2 a, 1 b, g, d
• Assembles into a ring forming central pore
• Channel structure
• Funnel extends 100 Å from outer leaflet
• Funnel has 20-25 Å inside diameter, narrows near
  center of bilayer to form gate
• Intracellular domains associate with proteins
  determining subcellular localization

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Space filling model Side Top
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Nicotinic AChR structure

• Subunit Structure
• 40-65 kDa each
• 4 transmembrane segments
• N terminus and C terminus are extracellular
• TM2-TM3 loop is extracellular
• TM1-TM2 and TM3-TM4 loops intracellular

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Pore Domain and Ion Selectivity

• TM2 regions line pore
• 9-10 Å diameter (images of open state)
• 8.5 Å diameter (estimated)

• Three rings of negatively charged amino acids
  surround pore
• Contributed to by TM2 of all subunits
• Anions excluded due to charge repulsion
• Cations permitted
• monovalents preferred

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Pore Region
Three rings of negatively charged amino acids
Figure 11.5, Byrne and Roberts
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Binding Sites

• Two binding sites per receptor complex
• 30 Å from outer membrane
• Composed of 6 amino acids of a subunit
• Amino acids of g and d subunits contribute
• Adjacent cysteines form disulfide link
• Present in most ionotropic receptors
• Binding sites not equivalent
• Cooperative
• Binding of 1st site facilitates binding of 2nd
  site

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Binding Sites

• Disulfide bond
• C192-C193
• D174/180 and E183/189 contact positively charged
  part of ACh

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Binding Sites

• Access to binding sites is through small channels
  that open into mouth of pore

Figure 11.6, Byrne and Roberts
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Channel Opening

• Rotation of TM2 segment
• TM2 is a helix with kink
• Leucine forms tight ring if kink rotated in
• Binding causes rotation out
• Rotation moves leucines apart
• Serine and threonine align in central pore
• Aides water-solvated ion permeation

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Channel Opening
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Summary of nAChR Function/Structure

• ACh binding relaxes central pore, allows
  expansion of holes in lateral walls
• Rapsyn anchors nACh to synapse

Figure 11.8, Byrne and Roberts
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Types of nAChR

• Muscle predominant form in periphery
• Similar to torpedo
• Adult has a2bed
• Embryonic form has a2bgd
• Subunit assembly tightly controlled to prevent
  other types from forming
• Subunits assemble with the ER
• includes glycosylation

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nAChR Anchoring

• Rapsyn bindsd to cytoplasmic tail and other
  proteins

• MuSK (tyrosine kinase)
• Activation recruites Src family kinases to
  phsophorylate the nAChR
• Dystrophin-utrophin glycoprotein complex
• Mutation leads to muscular dystrophy

Figure 11.9, Byrne and Roberts
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Channel Kinetics

• Open almost instantly after binding
• Time constant 20 microseconds
• Desensitization 50 - 100 ms
• Kinetics controlled by g/e, d subunits
• Mean open time 1 ms
• Channel conductance controlled by g/e
• Channel conductance 25 pS

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The Neuron, Levitan and Kaczmarek
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Types of nAChR

• Neuronal
• Only two subunits
• a ?2 - ?9 (muscle is ?1)
• ACh binding site
• b ?2 - ?4 (muscle is ?1)
• Many different subunit compositions exist

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Neuronal nAChR

• Functional receptors can be homomeric (a only) or
  heteromeric (a and b)
• Neuronal b differ from muscle b1 by lacking
  adjacent Cys residues
• Subunit composition is diverse, and influences
  trafficking (subcellular location) and function
• Modulate synaptic transmission through
  pre-synaptic action
• May play a role in learning and memory
• Stimulation of nicotinic receptors aids memory
• Nicotinic receptors are decreased in Alzheimers

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Neuronal Nicotinic AchR

• Blocked by neuronal- but not a- bungarotoxin
• Channel kinetics and permeability controlled by
  subunit subtype
• Neuronal nAChR conductance from 5 50 pS
• Calcium permeability larger than for muscle
• CaNa 1-1.5 for most subtypes
• High calcium permeability conveyed by a7 CaNa
  20
• Desensitization varies
• 100 - 500 ms for some
• 2-20 s for others

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Post-translation Modification

• Both muscle and neuronal types
• Glycosylation of each subunit in ER during
  assembly of subunits
• Required for production of receptor
• Phosphorylation
• kinases
• PKA g, d
• PKC d
• Tyrosine kinase b, g, d
• Intracellular loop between TM3 and TM4
• Increase rapid phase of desensitization

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Receptor Function

• Presynaptic AP produces
• Brief depolarization at excitatory synapse
• Brief hyperpolarization at inhibitory synapse

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Receptor Function

• Neurotransmitter causes channels to open
• Opening is all or none for single channels
• Neurotransmitter increases the probability that
  channels will open
• Does not increase the conductance of single
  channels
• Single channel currents are tiny
• Macroscopic currents sum of single channel
  currents
• Mean number of open channels number of channels
  ? open probability

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Acetylcholine Receptor Function

• ACh binding produces current flow
• Channel transitions to open state
• Each opening is independent
• Prolonged ACh application allows multiple
  openings of the same channel

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Acetylcholine Receptor Function

• Physiological neurotransmitter release is
  transient
• ACh that does not bind immediately diffuses away
• Single openings of variable duration observed
• Repeated single openings sum to macroscopic
  current
• Decay time constant (2.7 ms) is mean open duration

1000 channels
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Acetylcholine Receptor Function

• Channel conductance is independent of voltage
• Both macroscopic conductance AND open probability
• IV relationship is linear
• Isyn(t) Gsyn(t) (VM-ES)
• Channel is permeable to sodium and potassium
• Reversal potential is 0 mV

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Synaptic Channel Function

• Effect of synaptic current on membrane potential
  depends on
• reversal potential of channel
• synaptic conductance
• Other channels, e.g. Leak conductance
• At steady state (no additional change in membrane
  potential), synaptic current equals leak current
  (all other channels)
• Isyn(t) Gsyn(t) (VM-ES) -Ileak(t) -Gleak(t)
  (VM-Eleak)

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Synaptic Channel Function

• Can solve for membrane potential
• Gsyn (VM-ES) -Gleak (VM-Eleak)
• VM (GsynES GleakEleak) / (Gsyn Gleak)
• VM is weighted sum of reversal potential and cell
  resting potential, Eleak
• Effect of channel opening depends on reversal
  potential
• Greater than rest produces depolarization
• Less than rest produces hyperpolarization

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Synaptic Channel Function

• Steady State effect of synaptic channel
• When GSyn0, VMEleak
• When GSyn gtgt Gleak, VMES
• Transients
• Membrane doesnt depolarize instantly
• If GS is present briefly, membrane potential will
  not reach ES

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glycine
Ionotropic nAChR Family
GABA

• Major subdivision is permeant ions
• Next set of subdivisions based on physiological
  agonist

Anion
5-HT3
Cation
ACh
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Serotonin Receptor, type 3

• Ionotropic
• In contrast to all other 5HT receptors
• Homopentamer
• Subunit most similar to a7
• 7.6 Å diameter pore
• Na/K permeable
• Impermeable to calcium
• Kinetics
• Binding and/or opening is 10x slower than other
  ionotropic
• Desensitization rate 1 to 5 s
• Blocked by turbocurarine

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Serotonin Receptor, type 3

• Peripheral Nervous System
• Superior cervical ganglion and Vagus nerve
• Substantia gelatinosa
• Facilitates release of substance P
• Modulate nociceptive mechanisms?
• Cortical and limbic areas
• Anxiolytic, antidepressant, cognitive effects
• Post-synaptic (modifies activity)
• Predominantly GABAergic neurons
• Modulate dopaminergic neuron activity

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GABA receptor

• Heteropentamer
• a, b, g, d, r subunits
• Multiple subtypes
• r seen only in retina (called GABAC)
• Channel properties depend on subunit composition
• a
• Two required for functional channel
• Has high affinity GABA binding site

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GABA receptor

• Selective for anions
• Chloride ions flow in
• Hyperpolarization (reversal near -70 mV in adult)
• Shunts excitatory currents
• Inhibits action potential initiation
• Also permeable to bicarbonate (HCO3-)
• 27-30 pS is most common conductance

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Modulation of GABA Properties

• Allosteric binding sites that increase current
• sedative, anti-epileptic effects
• Benzodiazapines (Valium)
• Binds to g subunit at interface with a
• Potentiates GABA binding
• Barbiturates (phenobarbitol)
• Steroid metabolites of testosterone,
  corticosterone, progesterone

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Potentiation of GABA binding
The Neuron, Levitan and Kaczmarek
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Modulation of GABA Properties

• Allosteric binding sites that decrease current
• Seizure inducing or convulsants
• Decrease of GABA binding
• Bicuculline
• Open channel blocker
• Binds within pore
• Prevents ion flow
• Picrotoxin
• penicillin

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Modulation of GABA Properties

• Phosphorylation
• In vitro
• Protein kinase A
• Protein kinase C
• Calcium-calmodulin kinase
• In vivo
• Unknown kinases
• Unknown function

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Glycine Receptor

• Major inhibitory receptor in spinal cord
• Chloride permeable
• Heteropentamer
• 3 a (pore forming subunit) and 2 b (modulatory
  role)
• 3 molecules bind to open channel
• Conductance is 35 - 50 pS
• Blocked by Strychnine (convulsant)

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Purinergic Receptors

• Bind ATP (co-released with neurotransmitters) or
  adenosine
• Most are metabotropic
• P2x and P2z are ionotropic
• Non-specific cation channel
• Depolarizing
• Subunits have two transmembrane domains

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Glutamate Receptor Family

• Different than Nicotinic receptor family
• Receptors distinguished by their affinity for
• NMDA, AMPA, Kainate, Quisqualate

Kainate KA1, KA2 (high affinity), GluR5-7 (low
affinity)
NMDA NMDAR1 NMDAR2A-D
AMPA GluR1-GluR4
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Glutamate Receptor Agonists
Kainate

• Quisqualic Acid also activates mGluR
• Trans-ACPD activates mGluR

Quisqualate
a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic
acid
N-methyl-D-aspartic acid
The Neuron, Levitan and Kaczmarek
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Glutamate Receptor Structure

• Heterotetramer (1 fewer than nAChR)
• 600 kDa (larger than nAChR)
• Each subunit has 900 amino acids, four
  transmembrane domains
• Large extracellular N terminal forms binding site
• Responsible for excitatory synaptic transmission
  in the CNS and spinal cord
• Permeable to Na/K reversal near 0 mV

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Glutamate Receptor Function

• AMPA subunits
• GluR1, GluR3 subunits
• Channels are permeable to calcium
• Non-linear (inward rectifying) IV curve
• GluR2 subunit
• Homomeric channels have small conductance
• Heterotrimeric (with GluR1 and GluR3) produces
  channels impermeable to calcium
• Heterotrimeric channels have linear IV plot

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Glutamate Receptors

• Critical amino acids
• Gln vs Arg on TM2
• GluR2 has Arg which prevents calcium permeability
• GluR1, 3, 4 have Gln (Q) which conveys calcium
  permeability
• flip/flop modules
• 38 amino acids preceding TM4
• Flop (dominates in CA1, DG) produces larger
  desensitization
• Flip dominates in CA3 larger currents

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GluR Structure

• TM2 does not cross membrane
• Forms kink within membrane
• Also called P element
• Highly conserved (All GluR subunits)

Figure 11.13, Byrne and Roberts
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Glutamate Receptors

• Kainate Receptors
• GluR5-7
• 40 homology to GluR1-4
• KA1, KA2
• Bind kainate with high affinity
• No functional channel when expressed alone
• Some homology to GluR5-GluR7
• Co-expression of KA2 with GluR6 produces AMPA
  receptor
• KA1 has high concentration in CA3 and DG only
• Does not form functional channels with any other
  GluR subunit

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NMDA Receptors

• Structure
• Heterotetramer
• Four TM domains per subunit
• TM2 forms the pore
• Not really transmembrane bends as in GluR
  subunits
• Asn residue (at the Gln-Arg site of AMPAs TM2)
  confers calcium permeability
• Mutation dramatically reduces calcium
  permeability
• Part of Mg binding domain

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NMDA Receptor

• Subunits
• NMDAR1
• Can form functional channels
• Formation of ion pore
• 8 splice variants
• NMDAR2A-2D
• Requires NMDAR1 to form functional channels
• Modulates receptor activity
• 5-60x larger current than NMDAR1 alone
• NMDAR2 subtype changes with development
• NMDAR2 subtypes have different calcium
  permeability
• Calmodulin binding site
• 4x decrease in open channel probability

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NMDA Receptor

• Characteristics
• Glycine is co-agonist
• NMDA has 10x less affinity than glutamate
• Time course is 5-10 x slower than AMPA
• Calcium permeability is high
• More so for 2B than for 2A
• Plays a role in synaptic plasticity via calcium
  activation of kinases
• Plays a role in neurotoxicity due to elevated
  calcium
• Conductance 50 pS

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NMDA Receptor

• Characteristics (functional significance)
• Channel opening requires glutamate and
  depolarization
• Mg2 blocks pore at hyperpolarized potentials
• Little current flow below -40 mV

• Conductance depends on voltage
• Non-linear IV curve
• Inward Rectification

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Antagonists

• NMDA Receptor
• Hallucinogenic compounds act here
• Glutamate site blocker
• AP5, AP7
• Open channel blocker (Allosteric)
• MK801 (dizocilpine)
• Phencyclidine (PCP)
• Become trapped when closed, difficult to wash out
• AMPA receptor
• DNQX
• CNQX

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Anchoring of Glutamate Receptors

• Also called Scaffolding
• PSD-95
• Post-synaptic density 95
• PDZ domains allow interaction with NMDA subunits
  and other proteins
• Stargazin
• Member of TARP family
• Interacts with AMPA receptors and PSD-95
• SAP-97

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Glutamate Receptor Comparison
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Glutamate Receptor Comparison
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Basis for Cooperativity

• Weak stimulus
• AMPA weakly activated
• NMDA remains blocked
• Strong Stimulus
• AMPA causes depolarization
• NMDA receptor unblocked

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Dual component PSPs

• Both excitatory and inhibitory may be seen
• one early (inward)
• one late (outward)

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Summary

• Appreciate diversity of Receptors
• Know general structural features
• Heteropentamer
• Four transmembrane (or membrane) domains
• Definitions of agonist, antagonist,
  desensitization
• Distinguish ionotropic vs metabotropic
• Distinguish mechanisms of open channel blocker
  from binding site antagonist

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Summary

• Know major ionotropic subtypes
• Glutamate
• AMPA
• NMDA
• GABAA
• nAChr
• Know permeant ions, reversal potential, IV
  characteristics, effect on membrane potential
• Definition of excitatory vs inhibitory synapse
• Excitatory increases probability of firing
• Inhibitory decreases probability of firing