dissertation talk 2009
TRANSCRIPT
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Trafficking of Syntaxins and Kir2 Potassium Channels in Neurons
Linda Soo Hoo
University of California, Santa Barbara
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The Meaning of Life
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Overview
• Introduction– Neuronal polarity and polarized trafficking pathways
• Parts 1 & 2:– Polarized protein trafficking: Are SNARE proteins
polarized in neurons, and are they involved in polarized trafficking of neuronal membrane proteins?
– Ion channel localization: Are trafficking proteins associated with ion channels, and do they play a role in their localization and clustering?
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Neurons are polar and need to traffic cargoesasymmetrically Communication
• Directional transmission of information
Compartments • Axons
(e.g. Kv1.4, Nav1.2, GAP-43, etc.)
• Dendrites
(e.g. GluR1-4, mGluRα, transferrin receptors, Kv4.2, etc.)
Dendrites
CellBody
Axon
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AXON
DENDRITE
Neuronal signaling occurs via dendritic spines
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Synaptic Transmission
• Communication between neurons at synapses• Changes to strength of this communication
defines synaptic plasticity• Mechanisms underlying synaptic plasticity may
include:– Formation or loss of synaptic contacts
(developmental)
– Physiological changes:
Changes in quantity of neurotransmitters release and response by receptors
Changes in electrical signaling driven by ionotropic receptors or ion channels
Changes in cell surface localization of ion channels and receptors
Science 2006
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Cultured Hippocampal Neurons (Banker)
One process becomes the axon and grows dramatically, while the other processes become dendrites.
Craig and Banker (1994) Annu. Rev. Neurosci.
Question: How are the axonal and dendritic compartments maintained—vesicle targeting specificity?
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Polarized Vesicle Targeting in Neurons
• Polarized delivery
• Selective membrane fusion
• Selective endocytosis
Potential Players Involved:Motor ProteinsSNARE ComplexEndocytic Machinery
Horton and Ehlers (2003) Neuron
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Making Fusion Possible• SNARE protein superfamily
– (Soluble-N-ethylmaleimide-sensitive factor Attachment protein REceptor)
– Characteristic 60-70 aa cytoplasmic coiled-coil domain (SNARE motif)
• Vesicle membrane fusion– V-SNARE targets vesicle to its correct membrane fusion partner T-SNARE (target)
– SNARE core complex, tight and stable 4 helical bundle
– Membranes merge
Target membrane
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SNARE Core Complex
• SNAPs• VAMPs• Syntaxins
Cargo Vesicle
Target Membrane
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MDCK polarized epithelial cells:
• Syntaxin 3: apical membrane.
• Syntaxin 3 F31A mutant: non-polarized.
• Syntaxin 4: basolateral membrane.
(Low et al., 1998; Low et al., 2006; Sharma et al.,2006)
Golgi
Nucleus
MT
Apical
Question: Are these syntaxins involved in specifying the targeting of selective proteins to specific neuronal domains?
Different syntaxin distribution suggests that these syntaxins may participate in specifying fusion of vesicles to specific compartments.
Plasma membrane syntaxins
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Syntaxins 3 and 4 are expressed in hippocampal neurons
Question: Are the plasma membrane syntaxinsStx3 and Stx4 polarized in neurons?
Syntaxin 3
NRK = Normal rat kidney cells
Syntaxin 4
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Total Syntaxin 3Total Syntaxin 3GFP
Syntaxin 3 Localizes to Axon and Neurite Tips
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Total Syntaxin 4Total Syntaxin 4GFP
Syntaxin 4 Localizes to Dendrites
Axon
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• N-terminal trafficking motif:
Syntaxin 3 mutant constructs
FMDE
Cytoplasmic Extracellular
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N-terminal targeting motif is required for polarized syntaxin 3 trafficking
axon
dendrites
axonaxon
Stx3Δ38GFP
Stx3AAA
GFP
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Is syntaxin 3 responsible for polarized trafficking of vesicles carrying cargo to the
axons?
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• NgCAM (L1) is a neural cell adhesion molecule, a member of the immunoglobulin superfamily, that is expressed in axons and growth cones.
• Intracellular NgCAM is expressed in a non-polarized manner in hippocampal neurons.
• NgCAM surface expression is highly polarized to the axon.
• This suggests that selective fusion is the mechanism involved in axonal polarity of NgCAM.
C
N
FN IIIrepeats
Ig-likedomains
Extracellular
Intracellular
NgCAM (L1)—an axonal targeted cargo
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Syntaxin 3 localizes NgCAM to axon
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Syntaxin 3 mutants mislocalize NgCAM
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Syntaxin 3 does not affect trafficking of a dendritic cargo transferrin receptor
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Mislocalization of syntaxin 3 leads to loss of NgCAM polarity
• Axonal proteins have high Polarity Index Ratios
• Dendritic proteins have low Polarity Index Ratios
N =25-30 cells
Polarity Index: Ratio of surface NgCAM (axon) surface NgCAM (dendrite)
Conclusion: Syntaxin 3 is required for mediating targeting of axonal cargo protein and not dendritic cargo protein
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Is syntaxin 3 necessary for localizing NgCAM to axon?
H1 Stx3 CMV GFP
• Cos cell expression
Syntaxin 3
GAPDH
shSyn
taxin
3
shNon
targ
et
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Surface NgCAM GFP/Surface NgCAM
shNontarget
shSyntaxin 3
axon
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Syntaxin 3 is necessary for NgCAM axonal
polarization • Surface NgCAM polarity measurements
**
p ≤ 0.001
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Stx 4
Control
Stx 3Syntaxin 3 involved in axonal outgrowth
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• Syntaxin 3 localizes to axons, whereas syntaxin 4 localizes to dendrites
• Mislocalization of syntaxin 3 leads to mislocalization of axonal protein NgCAM and not dendritic protein transferrin receptor
• Necessity: Knockdown of endogenous syntaxin 3 leads to misloclalization of NgCAM.
• Syntaxin 3 is involved in axonal outgrowth in early neurons.
• Results demonstrate that selective fusion contributes to specifying polarized protein trafficking in neurons.
Summary: Syntaxin 3 & NgCAM
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Neuronal Polarity & Precise Localization of Ion Channels are Key to
Neuronal Function
Potassium Channels
Sodium Channels
Calcium Channels
Neurotransmitter Channels
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Potassium Channels
Kir2 subclass of K+ channels
• Involved in action potential repolarization
• Set and maintain the resting potential
Adapted from Lily Jan’s lab, UCSF
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• Restore resting membrane potential in heart
• Regulate neuronal excitability
• Maintain skeletal muscle excitability
• Mediate K+ buffering by glia
• Involved in dilation of blood vessels
• Salt reabsorption in kidney
Kir 2 Channel Physiology
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Kir2 Channel Diseases
• Cardiac Arrhythmias• Periodic Paralysis• Developmental Alterations
Mutations in Kir2.1 Cause Andersen-Tawil syndrome
Short QT-Syndrome
• Cardiac Arrhythmias• Ventricular Fibrillation• Sudden Death
ir2.1
Plaster et al., 2001; Andelfinger et al., 2002
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Are Kir2 channels associated with trafficking complexes?
• Proteomic identification of interacting proteins
How do these complexes affect:• Channel targeting & clustering in neurons
Questions
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PDZ Domain
RRESEI
Purification of Channel-interacting Complexes
(Leonoudakis et al., JBC, 2004)
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Kir2-Associated Proteins identified by Mass Spectrometry
Brain: Interacting scaffolding/adaptor proteins include:
• SAP102• Chapsyn-110/PSD-93• PSD-95 • SAP97
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How are Kir2 channels localized in neurons??
?• Regulating neuronal excitability
• Involved in action potential repolarization
• Setting and restoring the cell’s resting membrane potential
• Kir2 channels are critical regulators of dendritic excitability and synaptic integration in the forebrain and striatum.
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Kir2.3 is clustered in dendritic spines of hippocampal neurons
HA-Kir2.3 (red) and GFP (green) transfected into neurons
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Kir2.3 clustering in spines depends on PDZ interaction
HA-Kir2.3 is clustered HA-Kir2.3D3 is not clustered
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Kir2.3 clustering in spines depends on PDZ interaction
n = 14-15 cells
Colocalization with PSD-95
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shPSD95 effectively knocks down PSD-95
H1 si-PSD-95 CMV GFP
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GFP
si-PSD-95 Eliminates PSD-95 Clustering in Spines
Infected with Adeno-si-PSD-95 GFP
Uninfected
PSD-95 (endogenous)PSD-95 (endogenous)GFP
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GFP
PSD-95
HA-Kir2.3
HA-Kir2.3PSD-95
PSD-95 is Required for Channel Clustering in Dendritic Spines
Transfected with si-PSD-95 GFP and HA-Kir2.3 Untransfected
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Conclusions
• PDZ-binding motif is required for potassium channel clustering to dendritic spines.
• PSD-95 is required for targeting and clustering Kir2 channels to dendritic spines
• These studies demonstrate that Kir2 channel localization to spines depend on its association with PSD-95 via the PDZ-binding motif.
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SummaryWhat enables proteins to be expressed correctly at specific subcellular locations?
• Syntaxin 3-dependent membrane fusion of NgCAM to axons.
Axon Dendrite
X X
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• Potassium channel clustering to dendritic spines requires PDZ-mediated dependent interactions by PSD-95.
NH3
Kir2
RRESAI
Postsynaptic density
Dendritic shaft
Spine
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Acknowledgements
Lab Members:Chris D. Banna Dmitri LeonoudakisCarolyn M. Radeke Melanie WilliamsLior Dessau Lisa Conti
Advisor: Dr. Carol Vandenberg
Funding:Cottage Hospital GrantMuscular DystrophyAmerican Heart Association
Collaborators:Thomas WeimbsSeng Wei LowNikunj Sharma(UCSB)
Neuroscience Research Institute