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Ligand-Gated Ion Channels
Contents
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General Information
Ligand-Gated Ion Channels
The Acetylcholine Receptor
Neurotransmitters
Molecular Diversity and its Control
Toxin targets
Channel Families
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Voltage-gated
Extracellular ligand-gated
Intracellular ligand-gated
Inward rectifier
Intercellular
Other
Typical Ion Channels with Known Structure:
K+ channel (KCSA)
Acetylcholine receptor M2
transmembrane segment
Types of ion channels:
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Simple pores (GA, GAP junctions)
Substrate gated channels (Nicotinic receptor)
Voltage-gated channels (K-channels)
Pumps (ATP-synthase, K+,Na+-ATPase)
What are the Biochemical Changes that Lead to
Channel Gating (Opening or Closing)?
Gating involves some type of conformational
change in the protein, but other than that there
are few definitive answers to the question.
However, there are several general proposed
models for gating.
Types of Biochemical Mechanisms that
Open and Close Channels
• Conformational change occurs in a discrete
area of the channel, leading to it opening.
• The entire channel changes conformation (e.g.,
electrical synapses).
• Ball-and-chain – type mechanism.
• Nt or hormone binding causes the channel to
open.
Types of Biochemical Mechanisms that
Open and Close Channels (Cont’d)
• Nt or hormone binding to receptor causes a 2nd
messenger to activate a protein kinase that
phosphorylates a channel and thus opens it.
• Changes in membrane potential.
• Membrane deformation (e.g., mechanical
pressure).
• Selectivity by charge (i.e., positively lined
pore allows anions through; negatively lined
pore allows cations through).
Extracellular ligand-gated
• nicotinic ACh (muscle): 2 (embryonic), 2
(adult)
• nicotinic ACh (neuronal): (2-10), (2-4)
• glutamate: NMDA, kainate, AMPA
• P2X (ATP)
• 5-HT3
• GABAA: (1-6), (1-4),  (1-4), , , (1-3)
• Glycine
Intracellular ligand-gated
• leukotriene C4-gated
Ca2+
• ryanodine receptor
Ca2+
• IP3-gated Ca2+
• IP4-gated Ca2+
• Ca2+-gated K+
• Ca2+-gated nonselective cation
Ca2+-gated Cl–
cAMP cation
cGMP cation
cAMP chloride
ATP Cl–
volume-regulated Cl–
arachidonic acidactivated K+
• Na+-gated K+
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G-protein linked receptors coupled to
ion channels
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Acetylcholine (muscarinic)
Adenosine & adenine nucleotides
Adrenaline & noradrenaline
Angiotensin
Bombesin
Bradykinin
Calcitonin
Cannabinoid
Chemokine
Cholecystokinin & gastrin
Dopamine
Endothelin
Galinin
GABA (GABAB)
Glutamate (quisqualate)
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Histamine
5-Hydroxytryptamine (1,2)
Leukotriene
Melatonin
Neuropeptide Y
Neurotensin
Odorant peptides
Opioid peptides
Platelet-activating factor
Prostanoid
Protease-activated
Tachykinins
Taste receptors
VIP
Vasopressin and oxytocin
Gated Ion Channels
• Another type of membrane transport
• Pores in the membrane that open and close in a
regulated manner and allow passage of ions
-“Dispose” of the gradients
• Passive transporters
-Ions flow from high to low concentration
-No energy is used
-If there is no gradient ions will not flow
Gated Ion Channels
• Small highly selective pores in the cell
membrane
• Move ions or H2O
• Fast rate of transport 107 ions x s-1
• Transport is always down the gradient
• Cannot be coupled to an energy source
Ion channels are everywhere
• Channels are present in almost every cell
• Functions
-Transport of ions and H2O
-Regulation of electrical
potential across the
membrane
-Signaling
Gating mechanisms
• Two discrete states ;open (conducting) closed
(nonconducting)
• Some channels have also inactivated state
(open but nonconducting)
• Part of the channel structure or external
particle blocks otherwise open channel
What gates ion channels?
• Non gated - always open
• Gated
􀁺 Voltage across the cell membrane
􀁺 Ligand
􀁺 Mechanical stimulus, heat (thermal
fluctuations)
Gating mechanisms
• Conformational changes in channel protein
are responsible for opening and closing of the
pore
-3D conformational shape is determined by
atomic, electric, and hydrophobic forces
• Energy to switch the channel protein from one
conformational shape to another comes from
the gating source
Ligand gated channels
• Glutamate receptors
• Nicotinic acetylcholine receptor
• Vanilloid receptor family (TRPV)
= Neurotransmitter
Ion Flow = Current
Ligand gated ion channels
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Gated by ligands present outside of the cell
In fact they are receptors
All of them are nonselective cation channels
Mediate effects of neurotransmitters
Acetylcholine Receptor
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ACh
 (or )
ACh
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consists of a pentamer of
protein subunits, with two
binding sites for
acetylcholine, which,
when bound, alter the
receptor's configuration
and cause an internal
pore to open.
This pore allows Na+ ions
to flow down their
electrochemical gradient
into the cell.
Structure of the AChR at 4.6Å
Miyazawa et al, (1999)
(left figure was modified)
filled site in ACh binding protein
Brejc, 2001
Channel Gating Mechanisms
AChR: Proposed gating mechanism
(Unwin, 1995)
Closed
Open
The ACh receptor also responds to
nicotine, and so is called the “nicotinic”
acetylcholine receptor -nAChR
Acetylcholine Receptor
Nicotinic Acetylcholine Receptor
A ligand gated ion channel
the resting (closed) ion channel to acetylcholine (ACh)
produces the excited (open) state. Longer exposure leads to desensitization and
channel closure
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Acetylcholine
binding sites
Na+, Ca2+
ACh
Continued
excitation
Outside
Inside
Resting
(gate closed)
Desensitized
(gate closed)
Excited
(gate open)
ACh
•Synaptic transmission throughout the
nervous system is predominantly
Chemical
•At the chemical level, the key players
include integral membrane
proteins that control signaling
Neurotransmission is fast and precise
Action Potential opens voltage gated Ca2+
channels
Ca2+ enters the terminal.
Ca2+ initiates vesicular release of
neurotransmitter
Note that nerve transmission of the
AP involves BOTH VG ion channels
AND LG ion channels.
Mechanism of Transmitter release
Reserve vesicles are outside the active zone. Synapsins tethers vesicles to the
cytoskeleton
Ca+2 activates Ca2+/calmodulin dependent protein kinase
which phosphorylates synapsin I and frees the vesicles.
Molecular Diversity and its Control
Genes and Gene Expression:
• Sets of gene families encoding an ion channel
receptor correspond to the sets of protein
subunits of the same functional class:
e.g., There are gene families each for the
major α, β, and γ subunits for GABAA
receptors, and for the GluR1-4 (AMPA), Glur57 (kainate), KA-1 and KA-2 (kainate), and
NR2A-D (NMDA) glu receptor subunits.
Molecular Diversity and its Control
Genes and Gene Expression:
• Generally, the genes for each receptor class
are scattered over many chromosomes, with
occasional clusters;
e.g., human GABAA receptor α1, α6, β2, and γ2
genes are all close together at q31-35 on
chromosome 5.
What is this called…?
Molecular Diversity and its Control
Genes and Gene Expression:
• Introns – There does not seem to be any
consistency in the lengths of the introns and
exons and, therefore, the genes, of various
receptor genes both between and within a
species.
e.g., the gene encoding the mouse GABAA
receptor δ subunit is ~ 14 kb long, but the β1
subunit gene, which has the same intron-exon
structure, is 65 kb long.
Molecular Diversity and its Control
Genes and Gene Expression:
• Transcriptional Control
Most attention, to date, given to:
1. Timing of gene expression during development.
2. Mechanism of expression of gene expression to
neurons and types of neurons.
3. Regulation of receptor gene expression during
synapse formation.
However, relatively little is known about the
mechanism by which gene expression responds to
environ signals impinging on the individual neuron.
Molecular Diversity and its Control
Genes and Gene Expression:
• Multiprotein transcriptional complex consisting of
RNA Pol II and a plethora of ancillary factors.
• TATA box? Not all genes have them!
• Some use other initiator elements.
• Simple binding to TATA box insufficient to transcribe
a gene to satisfy physiol levels of txn.
• Need additional seq-specific interactions of various
txn factors with cis-acting enhancer and silencer
elements.
Molecular Diversity and its Control
Genes and Gene Expression:
• Much evidence for the role of silencing in neurons
acting at 2 levels:
1. Global silencing of neuronal genes in non-neuronal
cell types.
2. Silencing at the fine-tuning level to restrict
expression of neuronal genes to a subset of neurons.
What is the most important example of #2?
Molecular Diversity and its Control
Genes and Gene Expression:
• The GluR subunit genes have been the most heavily
investigated.
• Have no TATA nor CAAT start sites.
• GC-rich with multiple txn start sites within a CpG
island.
• Promotors contain overlapping Sp1 and GSG recog
sites near the major txn start sites and an NSR
silencer.
• NSR sequence in the NR1 and GluR2 genes has a
small modulatory effect with respect to neuronal
specificity of expression.
Molecular Diversity and its Control
Genes and Gene Expression:
• The GluR2 NSR sequence is a site of mediation of the
stimulatory effects on gene expression of the signaling
pathways initiated by the neurotrophic factor, GDNF and
BDNF.
• Ligand-switching-induced changes in gene expression: occurs
a lot during development;
e.g., replacement of the nAchR fetal γ subunits by functionally
homologous ε subunits in myocytes.
e.g., fetal expression of only GlyR α2 subunits to adult expression
of only GlyR α1 and α3 subunits in the s.c.  underlies the
differential sensitivities to strychnine affinities for the GlyR
subtypes.
e.g., NR2B to NR2C NMDAR subunits in cereb gran cells ~ 2 wks
after birth.
Molecular Diversity and its Control
Alternative Splicing:
• Not very widespread among the RNA
transcripts of LG ion channel receptors.
• Where it does occur, often entails little more
than a difference in a short aa sequence that
may regulate postranslational modification
processes, such as phosphorylation and
glycosylation sites.
• Alt Splicing also produces variants of the same
receptor (e.g., AMPA) subunits.
Molecular Diversity and its Control
Alternative Splicing:
e.g., Each of the 4 AMPAR subunits occur in 2 alternatively
spliced variants, called flip and flop.
Correspond to the alternative inclusion of either of 2 adjacent
exons (exons 14 and 15 in the GluR2 gene).
Functional difference: the flip forms of most subunits desensitize
more slowly and to a lesser degree than the flop forms distinction that is easily displayed probed with various agents.
• NR1 subunit of the NMDARs has 3 alt spliced exons  8 splice
variants  variations of the C-term exons  different
receptor clustering properties in the membrane (e.g., only
NR1 variants with the C2” cassette interact with the PSD) and
Zn2+ and H+ sensitivities.
Molecular Diversity and its Control
RNA Editing:
• As ---- oxidative deaminated  inosine
• Inosine bp like G  Δ the codon  different aa in
the translated protein.
• Carried out by 2 dsRNA A deaminases, ADAR1 and
ADAR2.
• ADAR1 and ADAR2 depend on the formation of ds
structures involving intronic sequences, which bp
with the exonic sequences to be edited.
• Other protein factors prob participate as well,
because some cells express ADARs, but cannot edit.
Molecular Diversity and its Control
RNA Editing
GluR Q/R Editing
GluR R/G Editing
• $ RNA editing sites: GluR2,
5, 6:  replacement of a gln
codon (CAG) by an arg
codon (CIG=CGG) and
insertion of arg (R) into the
TM domain
• Occurs in M2 has low Ca2+
permeability, low singlechannel conductance and
linear rectifying properties.
• GluR2, 3, 4 undergo editing
at the R/G site in exon 13,
just N-term to the flip flop
region of alt splicing.
• Reduces sensitization and
accel recovery.
Molecular Diversity and its Control
Translational Control:
• Not exactly known how widespread this mech is
among LG ion channel subunits.
• Thus, should not make inferences about the levels of
protein subunits from the mRNAs.
• In many GluR subunits and NR subunits, removal of
(some e.g., 15 bp) the 5’UTR that is involved in the
putative stem-loop structure results in signif
disinihibition of translation.
• Translational suppression of gluR2 mRNA largely due
to broad region containing a repeat sequence near
the 5’ end of the mRNA, which may affect various txn
start sites differentially.
Molecular Diversity and its Control
Post-translational Modification:
• Extensively phosphorylated and glycosylated.
• Kinases known to phosphorylate LCICs:
PKA, CaMKII, PKC.
• Phosphorylation by these kinases affect the function
of LGICs:
i. PKA phos AMPARs  incr channel open time or the
P(O) state.
ii. CaMKII phos AMPARs corr with incr synaptic
responses (synaptic plasticity).
iii. Phosphorylation of receptors is corr with activity.
Molecular Diversity and its Control
Post-translational Modification:
• Phosphorylation (and dephosphorylation) efficiency
dependent on proximity to of LGICs to kinases and
phosphatases in the high-[protein] milieu.
E.g., For gluRs, PSD-95/SAP97 required for clustering.
E.g., For AKAPs (A-kinase anchoring protein) anchor
kinases and phosphatases to the complex.
E.g., RACK-1 stimulates PKC to phosphorylate GABAAR
at the β subunits by binding these subunits to at a
site different from PKC.
Molecular Diversity and its Control
Post-translational Modification:
• ~ 5-10% of a subunit MW can be glycosylation.
• Oligomannosidic glycans + complex oligosaccharides.
• Incr the efficiency of receptor assembly and cellsurface density of GABAARs , but not essential for
these processes.
• But is strongly required for ER exit of assembled GlyR
and nAchR.
• Appears that glycosylation has a similar quality
control over all receptors.
• Apart from this, there does not seem to be a clear
general role for glycosylation of LGICs.
Molecular Diversity and its Control
Receptor Assembly and Trafficking:
• Peptide synthesis.
• Folding.
• Post-translational modifications.
• Insertion into ER.
• Strict selectivity is required so that only
certain combinations of subunits (and the
corrects ones!) are oligomerized and targeted
to the plasma membrane via the Golgi.
Molecular Diversity and its Control
Receptor Assembly and Trafficking:
• See earlier slide for the assembly of the nAchR
subunits.
• Stoichiometry: ααβγδ:
α + γ or α + δ = αγ or αδ heterodimers  assoc
with 1 other and the β subunit to form α2βγδ
complexes.
αβγ trimers form and recruit β and then the 2nd
α subunit.
For the GlyRs, αααββ are assembled via 2(αβ) +
α.
Molecular Diversity and its Control
Receptor Assembly and Trafficking:
• Sorting between the ER and plasma membrane:
• Driven by interactions between sequences on the
subunits and accessory proteins.
• Exit from ER subject to q.c. involving recog of
glycosylation of certain sites at the N-term.
• E.g., GlyRα1 mutant subunits that are not
glycosylated are retained in ER and rapidly degraded.
• Not all subunits require glycosylation.
• E.g., AMPA GluR2 subunit, where Q/R editing intro
arg at 607 creates an ER retention signal.
Molecular Diversity and its Control
Receptor Assembly and Trafficking:
• Moving receptors from ER to plasma membrane
involves targeting the correct sites on the menbrane.
• E.g., GABARAP found primarily in transport sites in
Golgi, interacts with NSF (N-ethylamide fusion prot).
GABARAP assoc spec with the γ2 subunit proteins of
GABAA Rs and also binds tubulin.
GABARAP selects vesicles with γ2 subunit-containing
GABAARs and enables transport along microtubules
to the synapse.
GABAARs lacking a γ subunit are localized
extrasynaptically and must use a diff mol mech for
transport.
Molecular Diversity and its Control
Receptor Assembly and Trafficking:
• At the synapse, LGICs are held in a dynamic
relationship ( lateral diffusion) with a protein
complex by multiple interactions with certain
proteins playing key roles in receptor clustering and
retention.
• E.g., gephyrin clusters the GlyR.
• E.g., rapsyn clusters nAchR. Rapsyn assoc with the
intracellular M3-M4 loop of the nAchR and mediates
the action of agrin-stimulated signaling pathway that
drives nAchR into synapses.
• Receptors have been shown to alternate between
clustering sites and assoc with the clustering
Molecular Diversity and its Control
Receptor Assembly and Trafficking:
• Once at the synapse, the receptors become
extensively involved with scaffolding proteins,
cytoskeletal anchoring proteins, and signal
transduction proteins.
• C.f., PSD complex.
• Receptors are also removed from the cell surface and
from synapses in response to environmental signals,
as well as for receptor production.
• Signal adaptor proteins, e.g., AP-2, arrestin,
ubiquitin.
Molecular Diversity and its Control
Receptor Assembly and Trafficking:
• Clathrin recruitment -> membrane invagination,
endocytosis  early endosomes  recycled to
plasma membrane or delivered to late endosomes
for sorting  recycled via trans-Golgi to plasma
membrane or to lysosomes for degradation.
• All these events involve protein-protein interactions,
most of which remain to be discovered.
E.g., Plic-1 binds only the α and β subunits of GABAAR;
responsible for GABAAR level: Achieved by protecting
receptors from degradation either by
i. Blocking ubiquitinization or
ii. Shuttlling them into recycling.
Toxins Target Ion Channels
• Neurotoxins produced by many organisms
attack neuronal ion channels,
• fast-acting
• deadly
• The Voltage-Gated Na+ Channel
– In genetic disease – channelopathies
• E.g., Generalized epilepsy with febrile seizures
– Toxins as experimental tools
• Toshio Narahashi – ion channel pharmacology
• Puffer fish: Tetrodotoxin (TTX)- Clogs Na+ permeable
pore
• Red Tide: Saxitoxin- Na+ Channel-blocking toxin
• The Voltage-Gated Na+ Channel (Cont’d)
– Varieties of toxins
• Batrachotoxin (frog): Blocks inactivation so channels
remain open
• Veratridine (lilies): Inactivates channels
• Aconitine (buttercups): Inactivates channels
– Differential toxin binding sites: Clues about 3-D
structure of channels