Ligand-Gated Ion Channels Genevieve Bell, Erminia Fardone, Kirill Korshunov Membrane Biophysics – Fall 2014

Primary Secondary Tertiary Quaternary

L + R Na+

Na+ LRNa+

Na+ LR O Na+Na+

Na+ LR O D Na+Na+ Na+

LR O D

100 µM Zinc 50 µM AMPA 100 µM Zinc 50 µM AMPA 20 pA 50 pA 5 s

Control

Control Zinc

Threshold for action potential Control Zinc

Control Zinc

Introduction Phosphorylated Ligand Gated Ion Channels (pLGICs) include: – nAChRs – GABA A Rs – 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 GABA A Rs PKC inhibits GABA A Rs 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 GABA A R 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 GABA A Rs.

Conformational changes in pLGICs can be targeted by drugs to treat several diseases.

GABA A 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 GABA A 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 -GABA A 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 GABA A 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 GABA A R containing γ2 subunits Acts indiscriminately on GABA A 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 GABA A 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 Zn 2+ 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 ( L247T α7) exhibits spontaneous inward currents in the absence of ACh. Zn 2+ is also largely found in the hippocampus and retina. – How does Zn 2+ affect α7 nAChRs? – Influence on the spontaneous currents? α7-nicotinic acetylcholine receptor

Methods and Materials Model organism = Xenopus oocytes. cDNA (for either WT α7 or L247T α7) was injected into the nuclei of stage 6 oocyte. Electrophysiology – Two-four days after cDNA injection – Voltage-clamp – ACh applied at 3 min intervals – Zn 2+ from ZnCl 2 and Zn 2+ acetate – nAChR blockers methyllycaconitine (MLA) and α- bungarotoxin (Bgt)

Zinc’s effect on WT α7 ACh current (I ACh )

Zn 2+ Blocks WT α7 I ACh [Zn 2+ ] = 10 nM to 10 mM [ACh] = 150 μM

Zn 2+ Blocks WT α7 I ACh Voltage- Independently [Zn 2+ ] = 30 μM [ACh] = 150 μM [Zn 2+ ] = 20 μM

L247T α7 and the zinc current (I Zn )

Zn 2+ Induces Currents in L247T α7 [Zn 2+ ] = 1 mM [ACh] = 0.2 μM [MLA] = 1 μM [αBuTx ] = 100 nM

The Dual Role of Zn 2+ on L247T α7 [Zn 2+ ] = 10 fM to 10 mM

I Zn on L247T α7 Mimics I ACh on WT α7 [Zn 2+ ] = mV I ACh on WT α7

L247T α7 has one Zn 2+ -Gated Channel Pore [Zn 2+ ] = mV

Zn 2+ -induced Modulation of I ACh in L247T α7

Zn 2+ Modulation of I ACh in L247T α7 For A + C: [Zn 2+ ] = 1 mM For B: [Zn 2+ ] = 10 mM

Conclusion WT α7 – Pretreatment (20-30 s) of Zn 2+ blocks I ACh – Blockage increases with [Zn 2+ ] – Blockage is voltage-independent L247T α7 – Zn 2+ produces its own current (I Zn ) – Zn 2+ 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. – Zn 2+ activates one open state

The tetrameric structure of a glutamate receptor channel Rosenmund C, Stern-Bach Y, Stevens CF (1998). Science 280:

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: GluR3 flip 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 state Staircase fashion: S= Small, 5 pS* M= Medium, 15 pS L = 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