Post on 30-Jan-2016
description
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 WebsitePaul Trombley
Families of Ligand-Gated Ion Channels
• Cys-loop receptors– Nicotinic Acetylcholine receptor– GABAA and GABAC Receptors– Glycine Receptor– 5-HT3 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., 2004.
Cys
Cys
Ashcroft 2000
Cystine-Loop Superfamily of Ligand-Gated Ion Channels
nAChR
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 2005 4 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-HT3 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. 2005
Peters et al. 2004
Electrostatic potential surface representation
Cystine-Loop Superfamily of Ligand-Gated Ion Channels
Glycine and GABA Receptors
Ion Selection: Chloride Channels
BasicResidues
GateRegion
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 EnhanceGABAA Currents Through Different
Mechanisms
Open Time
Probabilityof Opening
Twyman et al (1989) Ann. Neurol. 25: 213-220 (1989)Slide from Blitzer, Teaching Resource, Science’s STKE 2005
GlyR
Betz and Laube 2006
PNAS
Cys
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
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 affinityAMPA Receptor antagonist
MNQX = lowerAffinity 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 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 Mg2+
Zigmond et al. 1999
• Blocks channel at rest• Depolarization --> Mg2+
ion leaves the pore• Glu + depolarization =
Coincidence Detector• Other channel blockers:
PCP, ketamine, MK801
Ionotrophic Glutamate ReceptorsAMPA 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 Ca2+ and have a wide variety
of kineticsP2X6 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 32P 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 2007
- + +
-+
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