Ligand-Gated Ion Channels
Genevieve Bell, Erminia Fardone, Kirill Korshunov
Membrane Biophysics – Fall 2014
Tertiary
Primary
Secondary
Quaternary
Na+
L+R
Na+
Na+
L+R
LR
Na+
O
Na+
Na+
L+R
LR
Na+
O
Na+
Na+
L+R
LR
Na+
D
O
L+R
LR
D
100 µM Zinc
50 µM AMPA
20 pA
5s
100 µM Zinc
50 µM AMPA
50 pA
5s
50 µM AMPA
100 µM Zinc
50 pA
5s
Control
Zinc
Control
Threshold for action potential
Zinc
Control
Threshold for action potential
Zinc
Control
Introduction
• Phosphorylated Ligand Gated Ion Channels
(pLGICs) include:
– nAChRs
– GABAARs
– GlyRs
– 5-HT3Rs
• Best known for mediating fast
neurotansmission in nervous system
Introduction
• Phosphorylation is well known to influence
synaptic function by directly modulating
pLGICs
– Implicated in various disorders and can elicit a
wide variety of effects
– Understanding the structural basis of these effects
 design of specifically targeted drugs to treat
pathological receptor modification
pLGIC Architecture
• Pentameric assemblies of identical
or different subunits
• 5 subunits together form a central
water filled pore
• Each subunit can be divided into
three domains
• Transmembrane α-helices form
concentric rings around a central
pore, directly lined by 5 M2 helices
M3-M4 Cytoplasmic Domain
• M3-M4 domain is poorly conserved in length
and AA sequence and therefore exhibits
structural variation
• Interactions between M3-M4 loops and other
proteins or ions are known to modulate pLGIC
activity, assembly, and trafficking
• M3-M4 domain is the only region known to
house phosphorylation sites
Receptor Phosphorylation
Biological Function
Chronic Inflammatory Pain
• The α3-glycine receptor (α3 GlyR) is
prominent in the spinal cord
– Lamina I and II nociceptive neurons
– Phosphorylation at serine346 attributed to chronic
inflammation
α3-Glycine receptor
Mechanism of Inflammation
Sensitization
• Prosteglandin 2 (PGE2) activates PGE2 receptor
• PGE2 stimulates adenylyl cyclase to produce
more cAMP
• cAMP activate cAMP-dependent protein kinase
A (PKA)
• PKA phosphorylates ser346 residue, causing a
block in the IPSCs produced by glycine
• Ultimately, this leads to a sensitization in
nociception in the spinal cord lamina I and II
neurons
Zeilofer HU., 2005
Other Disorders Implicated in
Phosphorylation of pLGICs
• Alcoholism
– Implicated in GABAARs
• PKC inhibits GABAARs IPSCs
• Nicotine addiction
– Implicated in α4β2 nAChRs
• Phosphorylation/dephosphorylation lead to
receptor desensitization at the
Ser368 residue
• Continuous nicotinic exposure
leads to permanent receptor
desensitization
• Myasthenia gravis
– Implicated in muscle AChRs
• PKA phosphorylates γ and δ subunits
• PKC phosphorylates α and δ subunits
• PTK phosphorylates β, γ, and δ subunits
Summary
• Nociception sensitization occurs in α3 GlyRs,
caused by phosphorylation of the ser346 residue
– This is a possible mechanism for chronic inflammation
• Alcoholism is attributed to GABAAR
phosphorylation
• Nicotine addiction is attributed to desensitization
of α4β2 nAChRs
– Continuous nicotinic exposure leads to permanent
channel desensitization
• Phosphorylation of different muscle AChRs
subunits could lead to myasthenia gravis
Possible medical application
Inappropriate phosphorilation
Global allosteric conformational change
Neurological disorders
Chronic pain:
PKA-mediated phosphorilation α3 GlyRs inhibit current and causes a
conformational change of the gly-binding site.
Alcholism:
Protein phosphorilation can casue an increase in ethanol sensitivity of γ2containing GABAARs.
Conformational changes in
pLGICs can be targeted by drugs
to treat several diseases.
GABAA Receptor α and γ Subunits
Shape Synaptic Currents via
Different Mechanisms
Christine Dixon, Pankaj Sah, Joseph
W. Lynch, and Angelo Keramidas
Queensland Brain Institute, University of Queensland, Australia
Introduction
GABAA Receptors
- Mediate the majority of inhibitory
neurotransmission in the mammalian
brain
- Pentamers : Consist of two α, two β,
and a γ subunit
- 6 different α subunits
- 4 different β subunits
- 3 different γ subunits
- GABAA R that contain a variety of
subunits are expressed throughout the
brain
Inhibitory Postsynaptic Currents
- IPSCs at GABA-ergic are determined by:
• The biophysical properties of postsynaptic receptors
• How receptors are clustered at the postsynaptic membrane
- α subunit = Key determinant of the functional properties of GABAA R
The Amygdala
• Plays a key role in processing fear
• Dysfunction associated with anxietyrelated disorders
• Disorders are typically managed via
benzodiazepines
Benzodiazepines
• Enhances the action of GABA at GABAA R
containing γ2 subunits
• Acts indiscriminately on GABAA R
throughout the brain, producing side
effects such as tolerance and sedation
α1 and γ2 subunits are expressed throughout the CNS, while the
α2 and γ1 subunits have restricted distribution :
•
•
•
•
•
•
Amygdala
Forebrain
Cerebellum
Hypothalamus
Pallidum
Substantia Nigra
Properties of receptors containing α1 and γ2 subunits and their
impact on synaptic currents are very well known, in contrast
nothing is known regarding the impact of γ1 containing GABAA R
on inhibitory synaptic current
Experimental Procedures
• Cell Culture and Molecular Biology
• Subunits were transfected into HEK293 cells
• Primary neuronal cell culture
• Immunofluorescent Labeling
• Electrophysiology
• Patch-clamp: Outside-out and macropatch
Effects of Zn2+ on wild and mutant
neuronal α7 nicotinic receptors
E. Palma, L. Maggi, and F. Eusebi
PNAS 1998
Introduction
• α7 nAChR is a ligand-gated ion channel
largely present in the hippocampus and
the retina.
– Receptor dysfunction linked to epileptic
seizures and schizophrenia.
• A mutated form of α7 (L247Tα7) exhibits
spontaneous inward currents in the
absence of ACh.
• Zn2+ is also largely found in the
hippocampus and retina.
– How does Zn2+ affect α7 nAChRs?
– Influence on the spontaneous currents?
α7-nicotinic acetylcholine receptor
Methods and Materials
• Model organism = Xenopus oocytes.
• cDNA (for either WTα7 or L247Tα7) was injected
into the nuclei of stage 6 oocyte.
• Electrophysiology
–
–
–
–
–
Two-four days after cDNA injection
Voltage-clamp
ACh applied at 3 min intervals
Zn2+ from ZnCl2 and Zn2+ acetate
nAChR blockers methyllycaconitine (MLA) and αbungarotoxin (Bgt)
Zinc’s effect on WTα7 ACh current (IACh)
Zn2+ Blocks WTα7 IACh
[Zn2+] = 10 nM to 10 mM
[ACh] = 150 μM
Zn2+ Blocks WTα7 IACh VoltageIndependently
[Zn2+] = 20 μM
[Zn2+] = 30 μM
[ACh] = 150 μM
L247Tα7
and the zinc current (IZn)
Zn2+ Induces Currents in L247Tα7
[Zn2+] = 1 mM
[ACh] = 0.2 μM
[MLA] = 1 μM
[αBuTx ] = 100 nM
The Dual Role of Zn2+ on L247Tα7
[Zn2+] = 10 fM to 10 mM
IZn on L247Tα7 Mimics IACh on WTα7
IACh on WTα7
@ +45 mV
@ -100 mV
[Zn2+] = 10 nM
L247Tα7
has one Zn2+-Gated Channel
Pore
@ - 63 mV
[Zn2+] = 10 nM
Zn2+-induced Modulation of IACh in L247Tα7
Zn2+ Modulation of IACh in L247Tα7
For A + C: [Zn2+] = 1 mM
For B: [Zn2+] = 10 mM
Conclusion
WTα7
– Pretreatment (20-30 s) of Zn2+ blocks IACh
– Blockage increases with [Zn2+]
– Blockage is voltage-independent
L247Tα7
– Zn2+ produces its own current (IZn)
– Zn2+ acts as an agonist at low concentrations (10 fM10 nM)
– Acts as an antagonist at higher concentrations (10<).
• Voltage-dependent when co-applied with ACh.
– Zn2+ activates one open state
The tetrameric structure of a
glutamate receptor channel
Rosenmund C, Stern-Bach Y, Stevens CF (1998).
Science 280: 1596-1599.
Backgroud
• The AMPA-type glutamate receptor (AMPAR) is
widely expressed in the brain and mediates the
majority of fast excitatory neurotransmission.
• The AMPAR is a transmembrane glutamategated ion channel comprised of 4 pore-forming
subunits GluA1–4.
Methods used
cDNA Expression and Cell Culture: HEK (Human
embryonic kidney)cell line transfected with a-amino-3hydroxy-5-methyl-4-isoxazol propionate (AMPA)–
receptor.
- Receptor subunits: GluR6/GluR3
- Alternative splice variant: GluR3flip
Patch-clamp recording: outside-out patch
- AMPA receptor agonist: quisquillate (QUIS)
- AMPA receptor antagonist: 2,3-dihydroxy-6nitro-7-sulfamoyl-benzoquinoxaline (NBQX)
Foundings (1)
Saturating agonist concentrations (1 mM) consistently caused
a noninactivating channels to open state
Foundings (2)
Agonist-binding sites were presaturated with the competitive
antagonist, NBQX, before agonist (QUIS) application, so that
each agonist-binding site was only made available after an
antagonist molecule dissociated from the receptor.
NBQX: 10 to 30 µM
C= Closed state
Staircase fashion:
S= Small, 5 pS*
M= Medium, 15 pS
L = Large, 23 pS
*pS=picoSiemens
(electrical conductance)
Foundings (3)
No artifacts of GluR3/GluR6 following the sensitivity to
allosteric modulator (Cyclothiazide) with flop splicing variants of
GluR3 receptors
100 µm cyclothiazide to
remove the inactivation
Foundings (4)
Activation of single receptor/channels proceeds through the
staircase of openings to three different conductance levels of
increasing amplitude
Waiting times reveal 4 subunits
Conclusion
The authors proposed a model where each receptor
contains four functional antagonist/agonist-binding
sites, which is consistent with a tetrameric protein.
Outside-out patch