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Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function.

Kim G, Luján R, Schwenk J, Kelley MH, Aguado C, Watanabe M, Fakler B, Maylie J, Adelman JP - Elife (2016)

Bottom Line: We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels.Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP.Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.

View Article: PubMed Central - PubMed

Affiliation: Vollum Institute, Oregon Health and Science University, Portland, United States.

ABSTRACT
Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca(2+) influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.

No MeSH data available.


GST-fusion protein expression.(A) Coomassie stained gel showing input bacterial lysates (left lanes) for the indicated GST-fusion proteins prior to being bound to glutathione agarose beads, and after binding to beads (right lanes). (B) Western blot of the gel in panel A, probed with anti-GST antibody.DOI:http://dx.doi.org/10.7554/eLife.12637.008
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fig2s2: GST-fusion protein expression.(A) Coomassie stained gel showing input bacterial lysates (left lanes) for the indicated GST-fusion proteins prior to being bound to glutathione agarose beads, and after binding to beads (right lanes). (B) Western blot of the gel in panel A, probed with anti-GST antibody.DOI:http://dx.doi.org/10.7554/eLife.12637.008

Mentions: To test for direct interaction with SK2 channels, SK2-L and SK2-S, the SK2 isoforms that contribute to synaptic SK2-containing channels (Allen et al., 2011), and either C8-MPP2 or PSD-95 were co-expressed in HEK293 cells. Immunoprecipitations were performed using an anti-SK2 antibody raised in guinea pig that is directed to the common C-terminal domain of the two SK2 isoforms, or using IgG as a control. Precipitated proteins were prepared as Western blots. Probing with anti-SK2 antibody that was raised in rabbits and directed against the same C-terminal sequence, demonstrated equivalent SK2 expression for input and after immunoprecipitation in each sample (Figure 2—figure supplement 1). Probing with anti-C8 antibody detected a band of the appropriate apparent molecular weight for C8-MPP2 in the sample co-expressed with SK2-S plus SK2-L but not in the IgG control sample (Figure 2A). Probing with anti-PSD-95 antibody did not detect co-immunoprecipitation with SK2 (Figure 2B). The results demonstrated that C8-MPP2 but not PSD-95 was specifically co-precipitated with SK2. To examine the possibility that SAP97 interacts with SK2-S, C8-SAP97 was co-expressed in HEK293 cells together with either myc-SK2-S or GluA1, a known SAP97 interaction partner (Leonard et al., 1998). Immunoprecipitations were performed using anti-SK2 antibody, anti-GluA1 antibody, or IgG as a control. Western blotting with anti-C8 antibody revealed that C8-SAP97 only co-immunoprecipitated when co-expressed with GluA1, despite equivalent levels of input and immunoprecipitated proteins (Figure 2—figure supplement 1). To test if MPP2 interacts with the unique N-terminal domain of SK2-L that is required for synaptic localization GST pull-down experiments were performed. While PDZ interactions are important for many MAGUK-partner protein interactions, the unique N-terminal domain of SK2-L does not contain a PDZ ligand motif and using the PDZ domain from MPP2 failed to show an interaction with the N-terminal domain of SK2-L. Therefore, the SH3-HOOK-GK domain of MPP2 was employed. This domain of MPP2 as well the SH3-HOOK-GK domains from SAP97, a MAGUK scaffold of a distinct subfamily from MPP2 (Oliva et al., 2012), or the SH3-HOOK-GK domain from CaCNB4, a non-canonical MAGUK protein that is a beta subunit for voltage-gated Ca2+ channels (Van Petegem et al., 2004) were prepared as GST-fusion proteins, bound to glutathione-agarose beads, and used as baits for the prey, the His-tagged N-terminal domain of SK2-L. A GST-fusion protein of the C-terminal domain of Kv1.4, and a His-fusion protein of PSD-93 (Chapsyn) were also prepared as positive interaction controls (Lunn et al., 2007). Coomassie staining and Western blotting with anti-GST antibody verified equivalent amounts of the baits, either as input or bound to beads (Figure 2—figure supplement 2). After exposure to the baits, bound prey proteins were eluted and prepared as Western blots. Probing with an anti-His antibody showed that the SH3-HOOK-GK domain from MPP2, but not from SAP97 or CaCNB4, specifically pulled down the N-terminal domain of SK2-L. As expected, the C-terminal domain of Kv1.4 pulled down PSD-93 (Figure 2C). Taken together these results suggest that MPP2 interacts with the unique N-terminal domain of SK2-L that is required for synaptic localization (Allen et al., 2011).10.7554/eLife.12637.006Figure 2.MPP2 interacts with the N-terminal domain of SK2-L.


Membrane palmitoylated protein 2 is a synaptic scaffold protein required for synaptic SK2-containing channel function.

Kim G, Luján R, Schwenk J, Kelley MH, Aguado C, Watanabe M, Fakler B, Maylie J, Adelman JP - Elife (2016)

GST-fusion protein expression.(A) Coomassie stained gel showing input bacterial lysates (left lanes) for the indicated GST-fusion proteins prior to being bound to glutathione agarose beads, and after binding to beads (right lanes). (B) Western blot of the gel in panel A, probed with anti-GST antibody.DOI:http://dx.doi.org/10.7554/eLife.12637.008
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4764564&req=5

fig2s2: GST-fusion protein expression.(A) Coomassie stained gel showing input bacterial lysates (left lanes) for the indicated GST-fusion proteins prior to being bound to glutathione agarose beads, and after binding to beads (right lanes). (B) Western blot of the gel in panel A, probed with anti-GST antibody.DOI:http://dx.doi.org/10.7554/eLife.12637.008
Mentions: To test for direct interaction with SK2 channels, SK2-L and SK2-S, the SK2 isoforms that contribute to synaptic SK2-containing channels (Allen et al., 2011), and either C8-MPP2 or PSD-95 were co-expressed in HEK293 cells. Immunoprecipitations were performed using an anti-SK2 antibody raised in guinea pig that is directed to the common C-terminal domain of the two SK2 isoforms, or using IgG as a control. Precipitated proteins were prepared as Western blots. Probing with anti-SK2 antibody that was raised in rabbits and directed against the same C-terminal sequence, demonstrated equivalent SK2 expression for input and after immunoprecipitation in each sample (Figure 2—figure supplement 1). Probing with anti-C8 antibody detected a band of the appropriate apparent molecular weight for C8-MPP2 in the sample co-expressed with SK2-S plus SK2-L but not in the IgG control sample (Figure 2A). Probing with anti-PSD-95 antibody did not detect co-immunoprecipitation with SK2 (Figure 2B). The results demonstrated that C8-MPP2 but not PSD-95 was specifically co-precipitated with SK2. To examine the possibility that SAP97 interacts with SK2-S, C8-SAP97 was co-expressed in HEK293 cells together with either myc-SK2-S or GluA1, a known SAP97 interaction partner (Leonard et al., 1998). Immunoprecipitations were performed using anti-SK2 antibody, anti-GluA1 antibody, or IgG as a control. Western blotting with anti-C8 antibody revealed that C8-SAP97 only co-immunoprecipitated when co-expressed with GluA1, despite equivalent levels of input and immunoprecipitated proteins (Figure 2—figure supplement 1). To test if MPP2 interacts with the unique N-terminal domain of SK2-L that is required for synaptic localization GST pull-down experiments were performed. While PDZ interactions are important for many MAGUK-partner protein interactions, the unique N-terminal domain of SK2-L does not contain a PDZ ligand motif and using the PDZ domain from MPP2 failed to show an interaction with the N-terminal domain of SK2-L. Therefore, the SH3-HOOK-GK domain of MPP2 was employed. This domain of MPP2 as well the SH3-HOOK-GK domains from SAP97, a MAGUK scaffold of a distinct subfamily from MPP2 (Oliva et al., 2012), or the SH3-HOOK-GK domain from CaCNB4, a non-canonical MAGUK protein that is a beta subunit for voltage-gated Ca2+ channels (Van Petegem et al., 2004) were prepared as GST-fusion proteins, bound to glutathione-agarose beads, and used as baits for the prey, the His-tagged N-terminal domain of SK2-L. A GST-fusion protein of the C-terminal domain of Kv1.4, and a His-fusion protein of PSD-93 (Chapsyn) were also prepared as positive interaction controls (Lunn et al., 2007). Coomassie staining and Western blotting with anti-GST antibody verified equivalent amounts of the baits, either as input or bound to beads (Figure 2—figure supplement 2). After exposure to the baits, bound prey proteins were eluted and prepared as Western blots. Probing with an anti-His antibody showed that the SH3-HOOK-GK domain from MPP2, but not from SAP97 or CaCNB4, specifically pulled down the N-terminal domain of SK2-L. As expected, the C-terminal domain of Kv1.4 pulled down PSD-93 (Figure 2C). Taken together these results suggest that MPP2 interacts with the unique N-terminal domain of SK2-L that is required for synaptic localization (Allen et al., 2011).10.7554/eLife.12637.006Figure 2.MPP2 interacts with the N-terminal domain of SK2-L.

Bottom Line: We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels.Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP.Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.

View Article: PubMed Central - PubMed

Affiliation: Vollum Institute, Oregon Health and Science University, Portland, United States.

ABSTRACT
Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca(2+) influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.

No MeSH data available.