Extracellular Ligand-Gated Receptors

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Extracellular Ligand-Gated Receptors
Lecture note series II. 2007 Pharmacology 4407B

• Dr. Melanie Kelly
• Department of Pharmacology
• Dalhousie University.
• melanie.kelly_at_dal.ca

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Extracellular Ligand-Gated Receptors

• Belong to a family of homologous multi-pass
  transmembrane proteins
• Activation of ELGR causes opening of the ion
  channel which forms a central pore through the
  receptor structure.

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Properties of ELGR

• They are activated in response to
  neurotransmitters and combine ion-selective
  functions with those for agonist binding.
• They conduct ions through the impermeable cell
  membrane.
• They select among different ions (specificity).
• Changes in ion permeability following channel
  activation
• alter the excitability of cells.
• Excitation of cells is associated with opening of
  cation-influx (depolarizing)
• channels, while inhibition of neuronal firing is
  generally associated with
• increased anion (Cl-) ion permeability and
  hyperpolarization.

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ELGR ACTIVATION
Ligand-gated channels open in response to binding
of a ligand. The pore of the ACh receptor channel
is closed in the absence of ACh. Binding of the
ligand (ACh) produces a conformational change
that results in opening of the channel pore. Ions
then move through the pore, resulting in
single-channel currents.
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Organization of ELGR

• The ligand-gated ion channel family consists of
  receptors composed of multiple subunits.
• Each subunit has four transmembrane (TM)
  domains. Subunits exhibit sequence identities
  from 25 to 75 with a similar distribution of
  hydrophilic and hydrophobic domains.

Topology of ELGR family A,B,C regions involved in
agonist binding. N-terminus and C-terminus are
extracellular Examples of ELGR receptors are
nAChR, 5HT3, Glycine receptors, GABAA receptor
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Architecture of ELG Receptors
Subunit topology (left) and subunit organization
(right) of two families of ELGR. Possible ligand
binding sites are labelled with asterisks. The
left hand diagram is of a single homologous
subunit and the right hand diagram represents the
subunit composition of the receptor
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Functions of ELG Receptors

• Fast excitatory transmission (nAChR) - Primary
  excitatory receptors in skeletal muscle and
  peripheral nervous system.
• In central nervous system, nAChR are present in
  smaller numbers than glutamate receptors which
  mediate excitatory neurotransmission in both the
  central and peripheral nervous system.
• GABA and Glycine are the main inhibitory
  neurotransmitters in the nervous system. Both
  ligands activate a receptor associated with
  chloride-selective channel.

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Neuronal Nicotinic Acetylcholine Receptors (nAChR)

• Sixteen genes encode the nAChR. 5 genes encode
  nAChR at the NMJ and the remaining 11 genes
  encode genes widely expressed in CNS and PNS.

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Correlating Structure and Function of the nAChR

• Structure of nAChR highly conserved, each
  receptor is pentamer with 5 subunits surrounding
  ionic pore.
• Each subunit consist of aprox. 600 amino acids
  organized to span the membrane 4X. N- and C-
  termini lie in the synaptic cleft. The 2nd TM
  domain of each subunit forms the pore.
• Neurotransmitter binding site is at interface of
  two adjacent subunits.

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Structure of nAChR at the Neuromuscular Junction
(NMJ)
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Activation of nAChRs at NMJ
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Pharmacology of nAChR

• Drugs activating nAChR - drugs with structure
  similar to the endogenous neurotransmitter such
  that they can interact with binding site on the
  receptor e.g synthetic esters such as carbachol
  and nicotine.
• Drugs blocking nAChR - bind to receptor on
  extracellular site and/or permeate the channel
  and bind in pore. Prevent ACh interacting with
  receptor e.g Tubocurare

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Diseases Involving nACh Receptors

• Myasthenia Gravis - autoimmune disease,
  prevalence of 7-9 cases/100,000 people.
• 2/3 patients are women, develops in early life
  with peak age of onset in 30s.
• Characterized by profound weakness of skeletal
  muscle which increases with exercise. Manifest
  initially in facial and eye muscles. Difficulty
  holding head up, chewing,
• swallowing, weakness in limbs.
• Caused by autoantibodies directed at
  postsynaptic nACh receptors leading to loss
• of function. Mortality rare, treated with drugs
  that prolong the life-time of the
  neurotransmitter, ACh, in the synapse.

Figure shows minature excitatory potentials
recorded from normal (A) and Myasthenic
human intercostal muscle (B). In the diseased
muscle excitatory potentials due to nAChR
activation are abnormally small and decreased in
amplitude further with repetitive stimulation.
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Neuronal nACHR and Epilepsy

• Autosomal dominant frontal lobe epilepsy (ADFLE)
  is associated with a mutation in the CHRNA4 gene
  that encodes the ?4 subunit of the high-affinity
  nACHR.
• Mutation results in instability in neuronal
  cicuitary with excess excitation and abnormal
  synchrony. ADNFLE seizures arise mainly during
  stage II sleep in the frontal cortex and can
  progress to tonic-clonic seizures.
• Penetrance of ADNFLE is incomplete with 70 of
  persons carrying the mutation displaying typical
  sleep pattern.
• Mutant receptor has decreased channel open time,
  reduced single channel conductance and increased
  rate of desensitization. The reduction in nAChR
  function results in decreased activation of
  neurons releasing inhibitory neurotransmitter and
  results in enhanced excitability of postsynaptic
  neurons and lower seizure threshold.

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Glutamate Receptors

• L-glutamate, L-aspartate and other acidic amino
  acids act as excitatory neurotransmitters at
  synapses in the peripheral and central nervous
  system.
• Glutamate primary excitatory neurotransmitter in
  CNS.
• Two types of receptors ionotropic and
  metabotropic.
• Ionotropic are extracellular ligand-gated
  receptors (channels).
• Metabotropic receptors do not have an integral
  ion channel and mediate their effects via G
  protein activation of second messenger cascade.

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Ionotropic Excitatory Amino Acid Receptors

• Different ionotropic glutamate receptors co-exist
  on many neurones classified according to their
  preferred agonist. Glutamate activates all
  channel subtypes.
• N-methyl-D-aspartate receptors (NMDA)-slow
  kinetics, high Ca permeability. Important in
  generation of slow synaptic potentials and forms
  of activity-dependent synaptic plasticity
• ?-amino-3-hydroxy-5-methyl-4-isoxazole propriate
  receptors (AMPA) -fast kinetics,Na permeability,
  usually low Ca permeability, rapid
  desensitization. Important in mediating rapid
  synaptic transmission (similarly for Kainate
  Receptors).
• Kainate receptors - slow currents. Na
  permeability, Low Ca permeability.

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Relationship between Genes Encoding different
Glutamate Receptor Subunits

• Line lengths are proportional to mean number of
  differences per residue along each branch.

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Mammalian Non-NMDA Glutamate Receptor Genes
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Putative Topology of GluR Subunits

• AMPA and Kainate subunits consist of three
  transmembrane domains (TMs 1,3,4), a large
  extracellular N-terminus and intracellular
  C-terminus. Pore is lined by hairpin loop (TM2)
  which enters from cytosolic side.
• S1 and S2 domains are involved in agonist
  binding.
• Flip/Flop domain is alternatively spliced
  module.
• Q/R and R/G sites are subject to RNA editing
  (1st letter gives ?? encoded by genomic DNA 2nd
  letter indicates edited residue).

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Functional Diversity

• Heteromerization Native AMPARs are
  predominantly heteromeric. Homomeric AMPARs are
  significantly more Ca2 permeable than
  heteromeric channels.
• mRNA editing editing of mRNA results in
  substitution of arginine (R) residue for
  glutamine (Q). Designated Q/R site. When
  glutamine is present at Q/R site results in
  significant increases in Ca permeability of
  AMPARs.
• Alternative Splicing GluR1-GluR4 may exist in
  flip or flop version. Results from use of
  alternative exons to code for a region of 38 ??
  that are extracellular and preceed TM4. Flip/flop
  module affects desensitization rate of the
  channel.
• Differential expression of flip/flop modules
  during development as well as between CNS and
  peripheral tissue.

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NMDA Receptors

• Binding of two glutamate (NMDA) and two glycine
  is required to activate NMDA channels.
• Two major gene families encode NMDA receptor
  subunits NR1 and NR2.
• NR1 aprox 900 ??, 25 identity with AMPA and
  Kainate receptors, and is subject to alternative
  splicing with at 8 splice variants identified.
• NR2 subunits (NR2A and NR2D) have longer
  C-terminal sequence and are larger (130 to 170
  kDa).
• Expression of NR1 and NR2 subunits is necessary
  for functional receptor and native NMDA receptors
  are thought to be composed of two NR1 and two NR2
  subunits.

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Activation of NMDA Receptors

• Binding of glutamate or agonist (NMDA) to site
  on extracellular surface of receptor (thought to
  be NR2).
• Glycine binding (to NR1) results in enhancement
  of the ability of glutamate or NMDA to open the
  channel through allosteric interaction.
• Following binding of the agonist, depolarization
  results in removal of Mg2 block and influx of
  Na and Ca2 ions.
• NMDARs more permeable to Ca2 than other
  cations.
• Influx of cations produces further
  depolarization.

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Influx of Ca2 through NMDARs can trigger
long-lasting changes in synaptic efficacy.
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Glutamate Receptors and Disease

• Overactivation of GluR and excitotoxicity can
  occur due to pathological recurrent neuronal
  excitation i.e epilepsy, trauma,
  neurodegenerative diseases, ischemia or
  neurological toxicity - Demoic acid (kainate
  receptor agonist) found in mussels feeding on
  domoate-rich phytoplankton.
• Seeds of chickling pea Lathyrus sativus - contain
  potent AMPA receptor agonist causes lathyrism,
  neurological disease with muscle ridgidity and
  spasm
• Flour made from seeds of cycad, Cycas
  Circulnalis -contains NMDA agonist causes Guam
  disease with symptoms of Parkinsons and ALS.

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GABA Type A Receptor

• ?-aminobutyric acid (GABA) is the main inhibitory
  transmitter in the CNS.
• Stimulation of inhibitory neurons in the CNS
  releases GABA onto adjacent neurons
• Binding of GABA to receptors on the postsynaptic
  membrane causes a transient increase in
  permeability in Cl- via channel opening
• The inhibitory actions of GABA are enhanced by
  the presence of barbiturates or benzodiazepines
  (allosteric modulators).
• The GABAA Receptor is composed of ? and ?
  subunits and ? and/or ? subunits forming a
• functional receptor.

The influx of Cl- in neurons causes
hyperpolarization or inhibitory potentials which
move the membrane potential of the neuron away
from its firing threshold.
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Inhibitory Neurotransmission

• Antagonists of GABA receptors such as
  bicuculline and picrotoxin act as convulsants
  because they prevent GABA receptor activation.
• Potentiators/agonists - enhance GABA receptor
  activation and depress the CNS, therefore are
  used as sedatives, anesthetics and
  anticonvulsants e.g barbiturates,
  benzodiazepines, volatile anesthetics.

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Diseases/Syndromes Associated With GABA Channels

• Angelman Syndrome - characterized by severe
  mental retardation, absence of speech,
  puppet-like ataxic movements and seizures.
• Incidence is 1 in 20,000 and majority of
  patients appear to be sporadic cases.
• Disease in 70-80 of these patients associated
  with a deletion in region of maternal chromosome
  15, such that the gene encoding GABAA subunit is
  deleted. Loss of GABAA subunit function
  contributes to neurological pathogenesis in this
  disease.