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Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy.

Blom H, Rönnlund D, Scott L, Spicarova Z, Widengren J, Bondar A, Aperia A, Brismar H - BMC Neurosci (2011)

Bottom Line: Despite this, there is as yet little known about the isoform specific distribution in neurons.The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine.A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Physics, Royal Institute of Technology, Stockholm, Sweden.

ABSTRACT

Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons.

Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine.

Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

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Co-immunoprecipitation and GST pull down assay of PSD-95 and α3 NKA. Co-immunoprecipitation (CoIP) shows interaction between PSD-95 and α3 NKA. GST pull down assays shows interaction of PSD-95 with the α3 NKA N-terminus and a specific PDZ3 interaction.
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Figure 1: Co-immunoprecipitation and GST pull down assay of PSD-95 and α3 NKA. Co-immunoprecipitation (CoIP) shows interaction between PSD-95 and α3 NKA. GST pull down assays shows interaction of PSD-95 with the α3 NKA N-terminus and a specific PDZ3 interaction.

Mentions: We first tested the possibility that the neuron specific α3 NKA is expressed in spines, using different biochemical methods. We found that the α3 isoform coimmunoprecipitated (CoIP) with the synaptic scaffolding protein PSD-95, a wellknown synaptic marker, typically located in the head of the spines in excitatory synapses [10]. Figure 1 shows Western blot images displaying this interaction, where the co-immunoprecipitation of the α3 NKA/PSD-95 complex was performed in five separate experiments using the α3 antibody and in three separate experiments using the PSD-95 antibody. To further confirm this interaction, we used glutathione-S-transferase (GST) fused peptides and the GST pull down technique (cf. Figure 1). It is well known that the N-terminus of the α-subunit of NKA can bind and interact with other proteins [11]. We thus generated a GST fused peptide corresponding to the Ntail of α3 NKA. This GST-fused N-tail of α3 NKA was found to pull down PSD-95. The PSD-95 protein contains several domains capable to bind with other proteins including three PDZ domains (UniProtKB/Swiss-Prot database entry P31016) also known as PDZ1, PDZ2 and PDZ3. By scanning the primary structure of rat α3 NKA against known protein motifs http://elm.eu.org we predicted five PDZ3 binding domains (LD23DL, VE39EV, TD48CV, QE60IL and PE78WV) in the N-tail of α3 in concordance with the consensus motif x[DE]x[IVL]. To find whether one or more of the three PDZ domains of PSD-95 will binds with rat α3 NKA, we produced soluble GST fused peptides corresponding to each of the three PDZ domains of rat PSD-95. It was found that the GST-fused PDZ3 domain effectively pulls down α3 NKA from the rat striatal lysate (cf. Figure 1). GST-pull down of PSD-95 using α3 NKA N-terminus was performed in five separate experiments. The pull down of α3 NKA with PSD-95 PDZ domains was performed in three separate experiments.


Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy.

Blom H, Rönnlund D, Scott L, Spicarova Z, Widengren J, Bondar A, Aperia A, Brismar H - BMC Neurosci (2011)

Co-immunoprecipitation and GST pull down assay of PSD-95 and α3 NKA. Co-immunoprecipitation (CoIP) shows interaction between PSD-95 and α3 NKA. GST pull down assays shows interaction of PSD-95 with the α3 NKA N-terminus and a specific PDZ3 interaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040715&req=5

Figure 1: Co-immunoprecipitation and GST pull down assay of PSD-95 and α3 NKA. Co-immunoprecipitation (CoIP) shows interaction between PSD-95 and α3 NKA. GST pull down assays shows interaction of PSD-95 with the α3 NKA N-terminus and a specific PDZ3 interaction.
Mentions: We first tested the possibility that the neuron specific α3 NKA is expressed in spines, using different biochemical methods. We found that the α3 isoform coimmunoprecipitated (CoIP) with the synaptic scaffolding protein PSD-95, a wellknown synaptic marker, typically located in the head of the spines in excitatory synapses [10]. Figure 1 shows Western blot images displaying this interaction, where the co-immunoprecipitation of the α3 NKA/PSD-95 complex was performed in five separate experiments using the α3 antibody and in three separate experiments using the PSD-95 antibody. To further confirm this interaction, we used glutathione-S-transferase (GST) fused peptides and the GST pull down technique (cf. Figure 1). It is well known that the N-terminus of the α-subunit of NKA can bind and interact with other proteins [11]. We thus generated a GST fused peptide corresponding to the Ntail of α3 NKA. This GST-fused N-tail of α3 NKA was found to pull down PSD-95. The PSD-95 protein contains several domains capable to bind with other proteins including three PDZ domains (UniProtKB/Swiss-Prot database entry P31016) also known as PDZ1, PDZ2 and PDZ3. By scanning the primary structure of rat α3 NKA against known protein motifs http://elm.eu.org we predicted five PDZ3 binding domains (LD23DL, VE39EV, TD48CV, QE60IL and PE78WV) in the N-tail of α3 in concordance with the consensus motif x[DE]x[IVL]. To find whether one or more of the three PDZ domains of PSD-95 will binds with rat α3 NKA, we produced soluble GST fused peptides corresponding to each of the three PDZ domains of rat PSD-95. It was found that the GST-fused PDZ3 domain effectively pulls down α3 NKA from the rat striatal lysate (cf. Figure 1). GST-pull down of PSD-95 using α3 NKA N-terminus was performed in five separate experiments. The pull down of α3 NKA with PSD-95 PDZ domains was performed in three separate experiments.

Bottom Line: Despite this, there is as yet little known about the isoform specific distribution in neurons.The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine.A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Physics, Royal Institute of Technology, Stockholm, Sweden.

ABSTRACT

Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons.

Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine.

Conclusions: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.

Show MeSH