Stargazin Stargazin Regulates Synaptic Regulates Synaptic Targeting of AMPA Targeting of AMPA

ReceptorsReceptors

A Presentation By:A Presentation By:GROUP 6GROUP 6

Jennifer MillerJennifer Miller Stewart McIlvenaStewart McIlvena Udai ModyUdai Mody Ozzie MurrayOzzie Murray Lou MurdochLou Murdoch Joshua MichaelJoshua Michael Marissa MiyaoMarissa Miyao Bethany MorganBethany Morgan

BackgroundBackground In the year 2000, a mouse was found exhibiting In the year 2000, a mouse was found exhibiting

epileptic seizures and ataxia (which results in epileptic seizures and ataxia (which results in wobbliness). Through a number of experiments, wobbliness). Through a number of experiments, two defects, (among others), were located in the two defects, (among others), were located in the cerebellar granule cells of the mouse :cerebellar granule cells of the mouse :

1) Lack of functional AMPARs1) Lack of functional AMPARs

2) Mutated stargazin 2) Mutated stargazin

* For an AMPAR to be “functional,” it needs to be * For an AMPAR to be “functional,” it needs to be localizedlocalized at a synapse. at a synapse.

* In the rest of the presentation, “mutated granule cells” refers to those * In the rest of the presentation, “mutated granule cells” refers to those that have the defective stargazin protein, and “normal granule cells” that have the defective stargazin protein, and “normal granule cells”

refers to those that had the normal stargazin.refers to those that had the normal stargazin.

In the first half of this presentation, In the first half of this presentation,

I am going to present the experiments performed that I am going to present the experiments performed that provedproved

AMPARsAMPARs were non-functionalwere non-functional in the mutant granule in the mutant granule cellscells…..…..

And in the second half of the presentation,And in the second half of the presentation,

I am going to present the experiments that showed… I am going to present the experiments that showed…

1) 1) WhyWhy Mutant stargazin caused the AMPARs to be Mutant stargazin caused the AMPARs to be non-functionalnon-functional

2) How stargazin interacts with AMPAR subunits to 2) How stargazin interacts with AMPAR subunits to make it functional.make it functional.

Figuring out AMPARs were not Figuring out AMPARs were not Functional in the Mutant Functional in the Mutant

Granule CellsGranule Cells

Normal granule cells Normal granule cells exhibited spontaneous exhibited spontaneous inward current that inward current that was abolished by was abolished by CNQX. CNQX.

Mutant granule cells Mutant granule cells exhibited essentially exhibited essentially no spontaneous no spontaneous current.current.

*Spontaneous currents were mediated by *Spontaneous currents were mediated by the release of glutamate from a the release of glutamate from a presynaptic granule cell and recorded presynaptic granule cell and recorded via whole-cell patch clamp recordingvia whole-cell patch clamp recording

Normal – 1.7 currents/sec

Mutant – 1 current/10 sec

Figuring out AMPARs were not Figuring out AMPARs were not Functional in the Mutant Functional in the Mutant

Granule CellsGranule Cells

To eliminate the possibility To eliminate the possibility that the current was that the current was abolished due to defective abolished due to defective NMDARs, the granule cell NMDARs, the granule cell cultures were depolarized cultures were depolarized in the presence of CNQX.in the presence of CNQX.

Frequency:

Insignificantly different for the two cell types

As stated before, AMPARS need to As stated before, AMPARS need to be localized at the synapse to be be localized at the synapse to be functional because they need the functional because they need the glutamate that is released there in glutamate that is released there in order to activate.order to activate.

Thus, the next experiment compares Thus, the next experiment compares the localization of AMPARS in the localization of AMPARS in mutant and normal cell cultures via mutant and normal cell cultures via immunogold-labeling.immunogold-labeling.

Figuring out AMPARs were not Figuring out AMPARs were not Functional in the Mutant Functional in the Mutant

Granule CellsGranule Cells Immunogold-labeling Immunogold-labeling

experiments labeled experiments labeled AMPAR subunits. AMPAR subunits.

- In mutant mice, little - In mutant mice, little labeling was found in labeling was found in PSD.PSD.

-In normal mice, abundant -In normal mice, abundant labeling was found in labeling was found in PSD.PSD.

What If the Mutant Granule What If the Mutant Granule Cells Just Couldn’t Produce Cells Just Couldn’t Produce

AMPARs?AMPARs?

- A Cytoplasmic AMPAR subunit labeling was - A Cytoplasmic AMPAR subunit labeling was performed basically the same in both normal and performed basically the same in both normal and

mutant granule cell cultures.mutant granule cell cultures.

This indicates the problem was not This indicates the problem was not due to the due to the productionproduction of AMPAR of AMPAR

subunits, but rather their subunits, but rather their transport to the synapsetransport to the synapse..

Now, that we have shown that there Now, that we have shown that there is a lack of functional AMPARs in the is a lack of functional AMPARs in the granule cells, we are going to talk granule cells, we are going to talk about the mutant protein about the mutant protein stargazinstargazin, , and what its role in the cell could and what its role in the cell could possibly be.possibly be.

What Could Stargazin’s What Could Stargazin’s Role Be in Granule Cells?Role Be in Granule Cells?

1) A calcium channel subunit1) A calcium channel subunit

*Stargazin has a weak homology to a *Stargazin has a weak homology to a γ-1 Ca channel subunit

OROR

2) Regulate AMPARs in the synapse2) Regulate AMPARs in the synapse

*When stargazin is mutated, there is a lack of functional AMPARs in granule *When stargazin is mutated, there is a lack of functional AMPARs in granule cellscells

Which is It???Which is It???

CaCa2+2+ currents are normal in currents are normal in mutant granule cellsmutant granule cells

Because the stargazin protein was Because the stargazin protein was similar in sequence to the similar in sequence to the γγ-1 calcium -1 calcium channel subunit, it was possible the channel subunit, it was possible the primary defect in mutant granule cells primary defect in mutant granule cells was due to altered calcium channel was due to altered calcium channel function.function.

Whole-cell patch clamping showed no Whole-cell patch clamping showed no difference in calcium currents.difference in calcium currents.

CaCa2+2+ currents are normal in currents are normal in mutant granule cellsmutant granule cells

Activation of whole-cell calcium currents

Steady-state inactivation of calcium currents

CaCa2+2+ currents are normal in currents are normal in mutant granule cellsmutant granule cells

Result: Result:

Even though stargazin shows weak Even though stargazin shows weak homology to the calcium channel homology to the calcium channel

subunit, this data makes it unlikely that subunit, this data makes it unlikely that the primary defect is altered calcium the primary defect is altered calcium

channel function.channel function.

As of now, we know the following As of now, we know the following things: things: 1) Mutant granule cells lack functional 1) Mutant granule cells lack functional AMPARsAMPARs

2) Stargazin is mutant in granule cells2) Stargazin is mutant in granule cells

3) Stargazin is probably not a Ca3) Stargazin is probably not a Ca2+2+ channel channel subunitsubunit

Thus, the next reasonable step was to Thus, the next reasonable step was to perform an experiment to see if stargazin perform an experiment to see if stargazin interacted with AMPAR subunits.interacted with AMPAR subunits.

Stargazin interacts with Stargazin interacts with AMPARs and PDZ ProteinsAMPARs and PDZ ProteinsExperiment 1Experiment 1

Stargazin, upon being Stargazin, upon being transfected into cells and co-transfected into cells and co-immunoprecipitated, was immunoprecipitated, was found to bind with GluR1, 2 found to bind with GluR1, 2 and 4. and 4.

Experiment 2Experiment 2

Stargazin interacts with Stargazin interacts with PSD-95 via Stargazin’s PDZ – PSD-95 via Stargazin’s PDZ – binding sitebinding site

* * When binding site is deleted (known When binding site is deleted (known now as stargazinnow as stargazinΔΔC), AMPAR subunits C), AMPAR subunits can still bind, but PSD-95 cannot.can still bind, but PSD-95 cannot.

Is Stargazin the Connector Is Stargazin the Connector Between AMPARs and PSD-Between AMPARs and PSD-

95?95?

Because PSD-95 can mediate Because PSD-95 can mediate clustering of ion channels, it was clustering of ion channels, it was

hypothesized that maybe this was the hypothesized that maybe this was the connection between stargazin and the connection between stargazin and the clustering of AMPAR subunits at the clustering of AMPAR subunits at the

synapse. synapse.

Stargazin Interaction with Stargazin Interaction with PSD-95 and GluR4PSD-95 and GluR4

Co-expression of GluR4 with PSD-95 or with Stargazin results in diffuse distributions of these proteins at the cell surface.

However, transfecting the three together causes patch-like clusters at the cell surface.

Stargazin Rescues AMPAR Stargazin Rescues AMPAR Responses in Mutant CellsResponses in Mutant Cells

Transfecting stargazin in Transfecting stargazin in mutant cells restores mutant cells restores synaptic AMPAR synaptic AMPAR functionfunction

When stargazin-GFP When stargazin-GFP expressing cells were expressing cells were transfected into mutant transfected into mutant cells spontaneous currents cells spontaneous currents increased in amplitude and increased in amplitude and frequencyfrequency

When stargazinWhen stargazinΔΔC was C was transfected in mutant transfected in mutant cells, the spontaneous cells, the spontaneous currents do not increase in currents do not increase in amplitude or frequency.amplitude or frequency.

StargazinStargazinΔΔC Cannot Rescue C Cannot Rescue AMPAR EPSCsAMPAR EPSCs

Transfecting Transfecting stargazinstargazinΔΔC in mutant C in mutant

cells cannot rescue cells cannot rescue AMPAR EPSCsAMPAR EPSCs

Transfecting stargazin Transfecting stargazin in mutant cells in mutant cells

restores AMPAR restores AMPAR EPSCsEPSCs

Interaction Between Interaction Between AMPAR, Stargazin, and AMPAR, Stargazin, and

PSD-95PSD-95

Stargazin interacts with AMPA receptors in an intracellular compartment in the cell and promotes their delivery to the cell surface. The carboxyl terminus of stargazin binds specifically to the anchor protein PSD-95 and mediates recruitment of the stargazin-AMPA receptor complex to postsynaptic sites.

ConclusionsConclusions

Mutant cerebellar granule cells lacked Mutant cerebellar granule cells lacked AMPAR synaptic currents because they AMPAR synaptic currents because they contained defective stargazin proteinscontained defective stargazin proteins

Normal stargazin proteins are necessary Normal stargazin proteins are necessary to localize AMPAR subunits in the to localize AMPAR subunits in the synaptic membranesynaptic membrane The association of the PSD-95 anchor The association of the PSD-95 anchor

protein with stargazin allows the stargazin-protein with stargazin allows the stargazin-AMPAR complex to be anchored in the AMPAR complex to be anchored in the synaptic membrane.synaptic membrane.