Ligand-Gated Ion Channels Molecular Biophysics 28 September 2007

LGIC mediate fast synaptic transmission.

LGICs are responsible for changing a chemical signal in the synapse (neurotransmitter) to either an inhibitory or excitatory post synaptic potential in the post synaptic cell.

The EPSPs and IPSPs are summed from all of the dendrites, changing the membrane potential at the axon hillock. If the depolarization is high enough, an AP will be initiated.

Fast exchange of bath is needed to study ligand-gated ion channels FSU Neuroscience Website Paul Trombley

Families of Ligand-Gated Ion Channels Cys-loop receptors –Nicotinic Acetylcholine receptor –GABA A and GABA C Receptors –Glycine Receptor –5-HT 3 Receptor Ionotrophic Glutamate Receptors –NMDA –AMPA –Kainate P2X Receptors Kandel, Schwartz & Jessel, Principles of Neural Science 4th Ed. (2000)

Cystine-Loop Superfamily of Ligand-Gated Ion Channels Heteromeric or homomeric pentamers Characterized by a large N-terminal loop cross-linked by cystine bridges Each subunit is made up of 4 membrane spanning helices The large intracellular M3-M4 linker is the site for many cytoskeletal protein- protein interactions. M2 lines the pore Keramidas et al., Cys Ashcroft 2000

Cystine-Loop Superfamily of Ligand-Gated Ion Channels nAChR

Activated by Acetylcholine and Nicotine –µs activation times Blocked by curare and some general anesthetics. Non-selective cation channel including sodium, potassium and calcium. Isolated from Torpedo marmorata and visualized by N. Unwin and colleagues in the mid 1980’s

It’s pentameric structure consists of 2  subunits and a mixture of , and  subunits. Unwin’s  resolution electron microscopy structure. Ligand binding domain Pore lined by TM2 Intracellular M3-M4 linker

Open and closed state of the channel at the gate is different by 3  Unwin 2003 Van der Waal’s surface representation at the gate.

nAChR kinetics are dependent on subunit composition. Giniatullin et al 2005 Desensitization

Cystine-Loop Superfamily of Ligand-Gated Ion Channels 5-HT 3 Receptor

5-HT3 is a non-selective cation channel and is sensitive to curarie Yan et al. 1999

Homomultimers and heteromultimers of 5- HT3A and 5-HT3B Receptor Subunits produce channels with different characteristics. Peters et al 2005

Arginine residues with in the cytoplasmic domain strongly influence conductance of the 5-HT3 receptor Peters et al Peters et al Electrostatic potential surface representation

Cystine-Loop Superfamily of Ligand-Gated Ion Channels Glycine and GABA Receptors

Ion Selection: Chloride Channels Basic Residues Gate Region Modified from Keramidas et al., Prog. Biophys. Mol. Biol. 86: 161 (2004) Slide from Blitzer, Teaching Resource, Science’s STKE 2005

GABA Receptor Subunit Composition Two GABA Binding Sites at  Interfaces Benzodiazepine Site at  Interface Katzung (Ed.) Basic & Clinical Pharmacology, Lange (2004) Slide from Blitzer, Teaching Resource, Science’s STKE 2005 GABA = Gamma-aminobutyric Acid

Benzodiazepines and Barbiturates Enhance GABA A Currents Through Different Mechanisms Open Time Probability of Opening Twyman et al (1989) Ann. Neurol. 25: (1989) Slide from Blitzer, Teaching Resource, Science’s STKE 2005

GlyR Betz and Laube 2006

PNAS

Cys

Characterization of the Chimeric channel Grutter et al 2005 Expressed in Hek 293 cells Whole-cell patch recording of macroscopic chloride currents Functional Ca2+ potentiation site of the ECD ACh gates the channel Reversal potential shifted closer to Na when Cl cdriving force is removed

Activation is slowed in the  7/Cly chimeria possibly due to mismatched interactions of the poorly conserved Cys-loop of nAChR and the M2-M3 linker of the Glycine receptor.

Replacement of the nAChR Cys-loop with the Glycine R Cys-loop speeds activation Grutter et al 2005

Cys-loop / M2-M3 linler interactions are important for activation Kinetics and they are receptor specific. Grutter et al 2005 WT Glycine R activation Activation of Glycine R w/cys-loop point mutations to nAChR specific residues Chimeric channel w/2-3 linker of nAChR Slow activation of Chimeric

Receptor gating

Ionotrophic Glutamate Receptors

How many subunits make up an ionotrophic glutamate receptor?

Determination of binding sites by single channel electrophysiology AMPA receptor composed of GluR6/GluR3 chimeric channel expressed in HEK293 cells –Form homomultimers –No desensitization The assumptions –# of binding sites = # of subunits –Binding sites must be equivalent

These channels have 3 conductances and a closed state. Rosenmund et al 1998 Quisqualate = AMPA Receptor agonist NBQX = high affinity AMPA Receptor antagonist MNQX = lower Affinity AMPA Receptor antagonist Cyclothiazide = blocks inactivation

The relative frequency current amplitude histogram should shift in a predictable manner with increasing concentration of agonist if the states observed are due to different #’s of bound ligand.

Dwell time analysis for each transition state indicates 4 subunits 2 components

Ionotrophic Glutamate Receptors NMDA Receptors

NMDA = N-methyl-D-aspartic acid Made up of at least 1 NR1subunit and a combination of NR2A-D and NR3A-B Permeable to K+, Na+, Ca2+ High conductance Activate slowly Desensitize slowly & incompletely – Prolonged Ca2+ influx in the face of sustained glutamate release

Different combinations of NMDA subunits produce channels with an array of kinetics Cull-Candy et al 2001

NMDA receptor and Mg 2+ Zigmond et al Blocks channel at rest Depolarization --> Mg 2+ ion leaves the pore Glu + depolarization = Coincidence Detector Other channel blockers: PCP, ketamine, MK801

Ionotrophic Glutamate Receptors AMPA and Kainate Receptors Activate rapidly Desensitize within a few milliseconds Kainate – GluR5-7, KA1-2 AMPA – GluR1-4 –With GluR2 subunit: permeable only to K+ and Na+ –Without GluR2 subunit: Ca2+-permeable –AMPA = alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid

AMPA receptor

Isolating AMPA-R and NMDA-R Currents With Selective Blockers Nestler, Hyman, & Malenka, Molecular Neuropharmacology McGraw-Hill (2001) Slide from Blitzer, Teaching Resource, Science’s STKE 2005

P2X Receptors

Gated by extracellular ATP Trimeric arrangement determined by crosslinking and agonist binding studies 7 subtypes, heteromultimers produce a variety of kinetic outcomes Do not contain common ATP consensus motifs (ie. Walker motif) M1 is involved in gating, M2 lines the pore Intracellular N and C termini are important for protein-protein interactions P2X Receptors

P2X receptors are permeable to both Na + and Ca 2+ and have a wide variety of kinetics P2X6 is silent but can be expressed with other subunits to modulate their kinetics Egan et al. 2006

hP2X1 Cystine point mutations of S286-I329 Oocyte expression, two-electrode voltage-clamp Hek293 expression, whole-cell patch-clamp

Potency shift due to agonist binding and/or channel gating changes. Roberts and Evans 2007 oocytes

Some mutations that do affect ATP potency, have decreased binding efficiency but not all Roberts and Evans 2007 Protein expression is not different across mutants ATP binding is decreased in 4 of the mutants in 32 P 2-azido ATP /UV cross linking studies

Some mutations slow activation and desensitization in conjunction with or regardless of ATP potency changes. Roberts and Evans 2007 Increased EC50 Decreased EC50

Addition of a charge to some mutated residues modulate peak current magnitude. MTS compounds forms disulfide bonds with the side chain of cystine when exposed MTSES adds a negative charge MTSEA adds a positive charge WT P2X1 does not have an exposed cystine in the region in question

Modulation of peak current magnitude in some mutants is due to changes in ATP potency but not all Roberts and Evans

These residues are accessible to the outside of the cell, some of which is ATP binding dependent. Roberts and Evans 2007 MTSEA Biotin forms disulfide bridges with aqueously exposed cystines, here only from the outside of the cell

Proposed binding site of P2X receptor Roberts and Evans 2007