Trafficking of Syntaxins and Kir2 Potassium Channels in Neurons

Linda Soo Hoo

University of California, Santa Barbara

The Meaning of Life

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?

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

AXON

DENDRITE

Neuronal signaling occurs via dendritic spines

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

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?

Polarized Vesicle Targeting in Neurons

• Polarized delivery

• Selective membrane fusion

• Selective endocytosis

Potential Players Involved:Motor ProteinsSNARE ComplexEndocytic Machinery

Horton and Ehlers (2003) Neuron

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

SNARE Core Complex

• SNAPs• VAMPs• Syntaxins

Cargo Vesicle

Target Membrane

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

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

Total Syntaxin 3Total Syntaxin 3GFP

Syntaxin 3 Localizes to Axon and Neurite Tips

Total Syntaxin 4Total Syntaxin 4GFP

Syntaxin 4 Localizes to Dendrites

Axon

• N-terminal trafficking motif:

Syntaxin 3 mutant constructs

FMDE

Cytoplasmic Extracellular

N-terminal targeting motif is required for polarized syntaxin 3 trafficking

axon

dendrites

axonaxon

Stx3Δ38GFP

Stx3AAA

GFP

Is syntaxin 3 responsible for polarized trafficking of vesicles carrying cargo to the

axons?

• 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

Syntaxin 3 localizes NgCAM to axon

Syntaxin 3 mutants mislocalize NgCAM

Syntaxin 3 does not affect trafficking of a dendritic cargo transferrin receptor

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

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

Surface NgCAM GFP/Surface NgCAM

shNontarget

shSyntaxin 3

axon

Syntaxin 3 is necessary for NgCAM axonal

polarization • Surface NgCAM polarity measurements

**

p ≤ 0.001

Stx 4

Control

Stx 3Syntaxin 3 involved in axonal outgrowth

• 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

Neuronal Polarity & Precise Localization of Ion Channels are Key to

Neuronal Function

Potassium Channels

Sodium Channels

Calcium Channels

Neurotransmitter Channels

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

• 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

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

Are Kir2 channels associated with trafficking complexes?

• Proteomic identification of interacting proteins

How do these complexes affect:• Channel targeting & clustering in neurons

Questions

PDZ Domain

RRESEI

Purification of Channel-interacting Complexes

(Leonoudakis et al., JBC, 2004)

Kir2-Associated Proteins identified by Mass Spectrometry

Brain: Interacting scaffolding/adaptor proteins include:

• SAP102• Chapsyn-110/PSD-93• PSD-95 • SAP97

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.

Kir2.3 is clustered in dendritic spines of hippocampal neurons

HA-Kir2.3 (red) and GFP (green) transfected into neurons

Kir2.3 clustering in spines depends on PDZ interaction

HA-Kir2.3 is clustered HA-Kir2.3D3 is not clustered

Kir2.3 clustering in spines depends on PDZ interaction

n = 14-15 cells

Colocalization with PSD-95

shPSD95 effectively knocks down PSD-95

H1 si-PSD-95 CMV GFP

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

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

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.

SummaryWhat enables proteins to be expressed correctly at specific subcellular locations?

• Syntaxin 3-dependent membrane fusion of NgCAM to axons.

Axon Dendrite

X X

• Potassium channel clustering to dendritic spines requires PDZ-mediated dependent interactions by PSD-95.

NH3

Kir2

RRESAI

Postsynaptic density

Dendritic shaft

Spine

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