Ligand-Gated Ion Channels

Genevieve Bell, Erminia Fardone, Kirill KorshunovMembrane Biophysics – Fall 2014

Primary

Secondary

Tertiary

Quaternary

L + RL + R

Na+Na+

L + RL + R

Na+Na+

LRLR

Na+Na+

L + RL + R

Na+Na+

LRLR

OO

Na+Na+

Na+Na+

L + RL + R

Na+Na+

LRLR

OO

DD

Na+Na+

Na+Na+

Na+Na+

L + RL + R LRLR

OO

DD

100 µM Zinc100 µM Zinc

50 µM AMPA50 µM AMPA

100 µM Zinc100 µM Zinc

100 µM Zinc100 µM Zinc

50 µM AMPA50 µM AMPA

50 µM AMPA50 µM AMPA

20 pA20 pA

50 pA50 pA

50 pA50 pA

5 s5 s

5 s5 s

5 s5 s

ControlControl

ControlControl

ZincZinc

Threshold for action potentialThreshold for action potential

ControlControl

ZincZinc

Threshold for action potentialThreshold for action potential

ControlControl

ZincZinc

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/de-phosphorylation 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

Neurological disorders

Global allosteric conformational change

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 γ2-containing 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 MechanismsChristine 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 anxiety-

related disorders• Disorders are typically managed via

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

Benzodiazepines

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. EusebiPNAS 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 α-

bungarotoxin (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 Voltage-Independently

[Zn2+] = 30 μM [ACh] = 150 μM [Zn2+] = 20 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

[Zn2+] = 10 nM

@ +45 mV

@ -100 mV

IACh on WTα7

L247Tα7 has one Zn2+-Gated Channel Pore

[Zn2+] = 10 nM

@ - 63 mV

Zn2+-induced Modulation of IACh in L247Tα7

Zn2+ Modulation of IACh in L247Tα7

For A + C: [Zn2+] = 1 mMFor B: [Zn2+] = 10 mM

ConclusionWTα7

– 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 fM-

10 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.

• 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 glutamate-gated ion channel comprised of 4 pore-forming subunits GluA1–4.

Backgroud

cDNA Expression and Cell Culture: HEK (Human embryonic kidney)cell line transfected with a-amino-3-hydroxy-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-6-

nitro-7-sulfamoyl-benzoquinoxaline (NBQX)

Methods used

Saturating agonist concentrations (1 mM) consistently caused a noninactivating channels to open state

Foundings (1)

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 stateStaircase fashion:S= Small, 5 pS*M= Medium, 15 pSL = Large, 23 pS

*pS=picoSiemens (electrical conductance)

Foundings (2)

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 (3)

Activation of single receptor/channels proceeds through the staircase of openings to three different conductance levels of increasing amplitude

Foundings (4)

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