Molecular Determinants for Subcellular Localization of PSD-95 with an Interacting K+ Channel

Arnold, Donald B.; Clapham, David E.

doi:10.1016/s0896-6273(00)80761-8

Cited by 113 publications

(107 citation statements)

References 40 publications

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“…Lysates from tsA201 cells that had been transfected with the different constructs were immunoblotted with the Kv4.2 antibody to verify that the different proteins were well expressed. The antibody recognized one major band for each construct: at about 96, 68, and 94 kDa for C-Kv4.2, Kv4.2⌬VSAL, and C-Kv4.2⌬VSAL, respectively ( 3A, lanes [5][6][7][8]. Again, the product sizes are close to those expected and observed for an ECFP-tagged Kv4.2 fusion protein (38) and the slightly reduced size of the VSAL deletion mutant.…”

Section: Methodssupporting

confidence: 65%

Cell Surface Targeting and Clustering Interactions between Heterologously Expressed PSD-95 and the Shal Voltage-gated Potassium Channel, Kv4.2

Wong¹,

Newell²,

Jugloff³

et al. 2002

Journal of Biological Chemistry

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Kv4.2 is a voltage-gated potassium channel that is critical in controlling the excitability of myocytes and neurons. Processes that influence trafficking and surface distribution patterns of Kv4.2 will affect its ability to contribute to cellular functions. The scaffolding/clustering protein PSD-95 regulates trafficking and distribution of several receptors and Shaker family Kv channels. We therefore investigated whether the C-terminal valine-serine-alanine-leucine (VSAL) of Kv4.2 is a novel binding motif for PSD-95. By using co-immunoprecipitation assays, we determined that full-length Kv4.2 and PSD-95 interact when co-expressed in mammalian cell lines. Mutation analysis in this heterologous expression system showed that the VSAL motif of Kv4.2 is necessary for PSD-95 binding. PSD-95 increased the surface expression of Kv4.2 protein and caused it to cluster, as shown by deconvolution microscopy and biotinylation assays. Deleting the C-terminal VSAL motif of Kv4.2 eliminated these effects, as did substituting a palmitoylation-deficient PSD-95 mutant. In addition to these effects of PSD-95 on Kv4.2 distribution, the channel itself promoted redistribution of PSD-95 to the cell surface in the heterologous expression system. This work represents the first evidence that a member of the Shal subfamily of Kv channels can bind to PSD-95, with functional consequences.The influence that voltage-gated ion channels exert on the integrative physiology of excitable cells is determined by the inherent biophysical properties of the channels and their cell surface distribution. Kv4.2 is a fast transient (A-type) voltagegated potassium (Kv) 1 channel of the Shal subfamily that is found in heart and neurons (1). In myocytes, Kv4.2 is a major component of the I to current, which is crucial for recovery from the QT interval and for repolarization (2). In neurons of the central nervous system, Kv4.2 is expressed primarily somatodendritically (3, 4), where its concentration at postsynaptic sites may affect the back propagation of action potentials (5) and may therefore modulate long term potentiation at synapses (6). In contrast, Kv1.4, a Kv channel of the Shaker subfamily, is primarily expressed on axons (3, 4). Its positioning along the axon and at presynaptic sites (7-9) is thought to regulate the efficiency of action potential propagation (10) and to control neurotransmitter release (11). It is therefore crucial to understand the basis for the specialized distributions of Kv channels in order to fully understand their roles in neuronal excitability. The mechanisms that specify ion channel distributions are not well known and likely differ between those that determine localization in sub-domains of cells (e.g. axon, soma, and dendrite) versus lateral organization at the membrane, such as within channel clusters. Post-synaptic density 95 (PSD-95) is a membrane-associated guanylate kinase that helps localize and cluster numerous proteins. Kv1.4 and other Kv1 members are among the best known examples of in vitro ion channel clusteri...

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Section: Methodssupporting

confidence: 65%

Cell Surface Targeting and Clustering Interactions between Heterologously Expressed PSD-95 and the Shal Voltage-gated Potassium Channel, Kv4.2

Wong¹,

Newell²,

Jugloff³

et al. 2002

Journal of Biological Chemistry

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“…The precise mechanism by which the intramolecular SH3-GK interaction is involved in channel clustering by PSD-95 remains to be resolved. Our observation that L460P and ⌬C mutants often form perinuclear clusters with Kv1.4 raises the additional possibility that an intact SH3-GKr interaction is required for proper intracellular trafficking of PSD-95 (Arnold and Clapham, 1999;Craven et al, 1999;ElHusseini et al, 2000;Tiffany et al, 2000). Whatever the precise mechanism, this study raises the interesting idea that regulation of the intramolecular SH3-GK interaction (e.g., by binding of GK or SH3 ligands, or by phosphorylation) could control the targeting and clustering activity of PSD-95 at postsynaptic sites.…”

Section: Potential Functions Of Intramolecular Sh3-gk Region Interactionmentioning

confidence: 85%

An Intramolecular Interaction between Src Homology 3 Domain and Guanylate Kinase-Like Domain Required for Channel Clustering by Postsynaptic Density-95/SAP90

et al. 2000

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Members of the postsynaptic density-95 (PSD-95)/SAP90 family of membrane-associated guanylate kinase (MAGUK) proteins function as multimodular scaffolds that organize proteinsignaling complexes at neuronal synapses. MAGUK proteins contain PDZ, Src homology 3 (SH3), and guanylate kinase (GK)-like domains, all of which can function as sites for specific protein-protein interactions. We report here a direct proteinprotein interaction between the SH3 domain and the GK region in the PSD-95 family of MAGUKs. The SH3 domain of the PSD-95 family appears to have an atypical binding specificity, because the classical SH3 binding (-P-X-X-P-) motif is absent from the GK domain. Although SH3-GK binding can occur in either an intramolecular or intermolecular manner, the intramolecular mode is preferred, possibly because of additional tertiary interactions available when the SH3 and GK domains are adjacent in the same polypeptide. Mutations disrupting the intramolecular SH3-GK interaction do not interfere with PSD-95 association with the K ϩ channel Kv1.4 or with the GK domain-binding protein GKAP. The same mutations, however, inhibit the clustering of Kv1.4 by PSD-95, suggesting that the intramolecular SH3-GK interaction may modulate the clustering activity of PSD-95. Key words: PDZ domain; ion channel clustering; postsynaptic density; Src tyrosine kinase; polyproline helix; disks largeSynaptic transmission in neuronal synapses requires the proper organization of ion channels, neurotransmitter receptors, and signaling molecules in the synaptic junction. An emerging theme for the micro-organization of synapses is that functionally coupled proteins are placed in close proximity to each other via their interactions with scaffold proteins. The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) plays such a role at postsynaptic sites of glutamatergic synapses by interacting with a variety of ion channels, receptors, cytoskeletal components, and intracellular signaling proteins (Sheng and Wyszynski, 1997;Ziff, 1997;Craven and Bredt, 1998;Kennedy, 1998;O'Brien et al., 1998).The PSD-95 family of proteins (PSD-95/SAP90, SAP97/hdlg, chapsyn-110/PSD-93, and SAP102) share a common domain organization, consisting of three PDZ domains in their N-terminal half, a central Src homology 3 (SH3) domain, and a guanylate kinase (GK) domain at their C terminus. The PDZ domains of PSD-95 family proteins interact with the C terminal sequence motif (consensus -X-T/S-X-V) found in a variety of membrane and intracellular proteins, including Shaker K ϩ channels, NMDA receptors, and SynGAP (Kim et al., 1995Kornau et al., 1995;Doyle et al., 1996;Chen et al., 1998). In addition, PSD-95 binds to neuronal nitric oxide synthase (nNOS) through a PDZ-PDZ/␤-finger interaction (Brenman et al., 1996a;Hillier et al., 1999). In vitro and in vivo evidence suggests that these PDZmediated interactions are involved in the clustering of specific membrane proteins at synaptic sites (Kim et al., 1995Thomas et al., 1997).The GK domain of the PSD-95 family of MA...

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“…Based on the analogous membrane hypothesis (54), axonal Kv1.4 should assume an apical localization in Madin-Darby canine kidney cells, thus the predicted localization was not obtained. Another study using biolistic gene transfer to express recombinant Kv1.4 in high density postnatal hippocampal cultures also found an aberrant somatodendritic localization for Kv1.4 (55). It should be noted that both of these studies were performed in the absence of Kv␤ subunit coexpression.…”

Section: Discussionmentioning

confidence: 98%

Kvβ Subunit Oxidoreductase Activity and Kv1 Potassium Channel Trafficking

Campomanes¹,

Carroll²,

Manganas³

et al. 2002

Journal of Biological Chemistry

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Voltage-gated Kv1 potassium channels consist of poreforming ␣ subunits and cytoplasmic Kv␤ subunits. The latter play diverse roles in modulating the gating, stability, and trafficking of Kv1 channels. The crystallographic structure of the Kv␤2 subunit revealed surprising structural homology with aldo-keto reductases, including a triosephosphate isomerase barrel structure, conservation of key catalytic residues, and a bound NADP ؉ cofactor (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell 90, 943-952). Each Kv1-associated Kv␤ subunit (Kv␤1.1, Kv␤1.2, Kv␤2, and Kv␤3) shares striking amino acid conservation in key catalytic and cofactor binding residues. Here, by a combination of structural modeling and biochemical and cell biological analyses of structure-based mutations, we investigate the potential role for putative Kv␤ subunit enzymatic activity in the trafficking of Kv1 channels. We found that all Kv␤ subunits promote cell surface expression of coexpressed Kv1.2 ␣ subunits in transfected COS-1 cells. Kv␤1.1 and Kv␤2 point mutants lacking a key catalytic tyrosine residue found in the active site of all aldo-keto reductases have wild-type trafficking characteristics. However, mutations in residues within the NADP ؉ binding pocket eliminated effects on Kv1.2 trafficking. In cultured hippocampal neurons, Kv␤ subunit coexpression led to axonal targeting of Kv1.2, recapitulating the Kv1.2 localization observed in many brain neurons. Similar to the trafficking results in COS-1 cells, mutations within the cofactor binding pocket reduced axonal targeting of Kv1.2, whereas those in the catalytic tyrosine did not. Together, these data suggest that NADP ؉ binding and/or the integrity of the binding pocket structure, but not catalytic activity, of Kv␤ subunits is required for intracellular trafficking of Kv1 channel complexes in mammalian cells and for axonal targeting in neurons.

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Molecular Determinants for Subcellular Localization of PSD-95 with an Interacting K+ Channel

Cited by 113 publications

References 40 publications

Cell Surface Targeting and Clustering Interactions between Heterologously Expressed PSD-95 and the Shal Voltage-gated Potassium Channel, Kv4.2

Cell Surface Targeting and Clustering Interactions between Heterologously Expressed PSD-95 and the Shal Voltage-gated Potassium Channel, Kv4.2

An Intramolecular Interaction between Src Homology 3 Domain and Guanylate Kinase-Like Domain Required for Channel Clustering by Postsynaptic Density-95/SAP90

Kvβ Subunit Oxidoreductase Activity and Kv1 Potassium Channel Trafficking

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