Cypin

166

155

Strong inward rectifier potassium (Kir2) channels are important in the control of cell excitability, and their functions are modulated by interactions with intracellular proteins. Here we identified a complex of scaffolding/ trafficking proteins in brain that associate with Kir2.1, Kir2.2, and Kir2.3 channels. By using a combination of affinity interaction pulldown assays and co-immunoprecipitations from brain and transfected cells, we demonstrated that a complex composed of SAP97, CASK, Veli, and Mint1 associates with Kir2 channels via the C-terminal PDZ-binding motif. We further demonstrated by using in vitro protein interaction assays that SAP97, Veli-1, or Veli-3 binds directly to the Kir2.2 C terminus and recruits CASK. Co-immunoprecipitations indicated that specific Veli isoforms participate in forming distinct protein complexes in brain, where Veli-1 stably associates with CASK and SAP97, Veli-2 associates with CASK and Mint1, and Veli-3 associates with CASK, SAP97, and Mint1. Additionally, immunocytochemistry of rat cerebellum revealed overlapping expression of Kir2.2, SAP97, CASK, Mint1, with Veli-1 in the granule cell layer and Veli-3 in the molecular layer. We propose a model whereby Kir2.2 associates with distinct SAP97-CASK-Veli-Mint1 complexes. In one complex, SAP97 interacts directly with the Kir2 channels and recruits CASK, Veli, and Mint1. Alternatively, Veli-1 or Veli-3 interacts directly with the Kir2 channels and recruits CASK and SAP97; association of Mint1 with the complex requires Veli-3. Expression of Kir2.2 in polarized epithelial cells resulted in targeting of the channels to the basolateral membrane and co-localization with SAP97 and CASK, whereas a dominant interfering form of CASK caused the channels to mislocalize. Therefore, CASK appears to be a central protein of a macromolecular complex that participates in trafficking and plasma membrane localization of Kir2 channels.Strong inward rectifier potassium (Kir2) 1 channels are a widely expressed family of ion channel proteins distinguished by their ability to pass K ϩ current in the inward direction more readily than outward. Kir2 channels are key components in control of neuronal excitability in brain, electrical activity in heart, vascular tone, and glial buffering of potassium (1, 2). Kir2 channels are important in the modulation of cell excitability, repolarization of the action potential, and determination of the cellular resting potential. Furthermore, Kir2 mutations are implicated in at least one genetic disease causing periodic paralysis and heart arrhythmias (Andersen's syndrome (3)).The function, localization, and trafficking behavior of many ion channels are regulated by interactions of their intracellular domains with members of the MAGUK protein family. MAGUK proteins have a characteristic domain structure containing up to three PDZ domains, a Src homology 3 (SH3) domain, and a catalytically inactive guanylate kinase-like domain (GK). These domains interact with numerous other proteins allowing MAGUK proteins to particip...

Section: Discussionmentioning

confidence: 99%

A Multiprotein Trafficking Complex Composed of SAP97, CASK, Veli, and Mint1 Is Associated with Inward Rectifier Kir2 Potassium Channels

Leonoudakis

Conti

Radeke

et al. 2004

166

155

“…PSD-93 (9), SAP-97 (15), SAP-102 (18) and monoclonal antibodies to PSD-95 (#046; Affinity Bioreagents) and GFP (Quantum, CLONTECH). GAD-65 antibody was a gift from Dr. Steinunn Baekkeskov (Dept.…”

Section: Methodsmentioning

confidence: 99%

“…Axonal staining for SAP-102 is likely specific as it is blocked by preadsorbing the antibody with the immunizing antigen (data not shown). Furthermore, the affinity-purified antibody used for staining recognizes only SAP-102 by Western blot analysis of crude brain extracts (18).…”

Section: Differential Localization Of Psd-95/93 and Sap-102 In Neu-mentioning

confidence: 99%

Ion Channel Clustering by Membrane-associated Guanylate Kinases

El‐Husseini

Topinka

Lehrer‐Graiwer

et al. 2000

Self Cite

Neurotransmission requires appropriate assembly of signal transduction machinery at synaptic sites. Although mechanisms for organization of these synaptic signaling complexes are unclear, recent studies suggest a general role for PDZ domain-containing membrane-associated guanylate kinases (MAGUK) 1 in receptor clustering at pre-and postsynaptic sites (1-4).Molecular cloning has identified four neuronal MAGUK proteins in mammals: PSD-95 (SAP-90), PSD-93 (Chapsyn-110), SAP-97 (hDLG), and SAP-102 (5-10). Immunohistochemical studies indicate that PSD-95 and PSD-93 occur primarily in a somatodendritic distribution and are specifically enriched at the PSD (11). Consistent with this postsynaptic localization, targeted disruption of PSD-95 in mouse disrupts N-methyl-Daspartate receptor-dependent synaptic plasticity (12). Recent studies show that SAP-102 can be found at postsynaptic sites (7, 13). On the other hand, SAP-97 occurs in presynaptic terminals in forebrain neurons (8). Although SAP-97 knockout mice have not yet been reported, SAP-97 is the mammalian homologue of Drosophila discs large (DLG), and flies lacking DLG show prominent presynaptic defects (14).Mechanisms for differential localization and function of MAGUK proteins remain uncertain. MAGUK proteins share extensive sequence homology in their PDZ, SH3, and guanylate kinase domains. Significant differences between these proteins occur primarily at their N termini, suggesting roles for these regions in differential functions of these proteins. Importantly, PSD-95 contains a pair of cysteine residues that are sites for protein palmitoylation (15), a post-translational modification that is essential for postsynaptic sorting (16). In contrast, SAP-97 lacks N-terminal cysteines, and it is not palmitoylated. PSD-93 is alternatively spliced at the N terminus to yield two major isoforms: the first, which we now term PSD-93␣, has cysteines at positions 5, 7, and 12; and the second, which we term PSD-93␤, has cysteines at positions 3 and 5, like PSD-95 (9). Finally SAP-102 has cysteines at positions 7, 8, 10, and 13 (7). It is uncertain whether PSD-93 or SAP-102 are palmitoylated and whether this regulates cellular trafficking and ion channel clustering by these proteins.Here, we report that both PSD-95 and PSD-93 occur prominently at postsynaptic sites in hippocampal neurons, whereas SAP-102 localizes to both dendrites and axons in these cells. Similarly, we find that PSD-95 and both isoforms of PSD-93 mediate surface ion channel clustering, although SAP-97 and SAP-102 do not. Metabolic labeling studies show that PSD-95 and both isoforms of PSD-93 are robustly palmitoylated and that SAP-97 and SAP-102 are not. Mutants of PSD-95 and PSD-93 that disrupt palmitoylation also block surface receptor clustering, indicating a critical role for this lipid modification in ion channel aggregation at the plasma membrane. Rather than being palmitoylated, we find that N-terminal cysteines in SAP-102 tightly bind to zinc and that this unique N terminus of SAP-102 confers axonal t...

“…Thus, INAD is required for anchoring the signaling complex in the rhabdomeres, but in turn the INAD target-interacting protein TRP is required to localize INAD to the rhabdomeres. It has also been shown in transfected neuron cultures that the PDZ domain protein PSD-95 could be localized in axons as a result of interactions with the membrane protein shaker-type K ϩ channel Kv1.4 (32) and that transfected Kv1.4 also depended on the interaction with PSD-95 to be selectively localized in axons (32) and in postsynaptic sites (33). Our data suggest the existence of a similar dual dependence to properly localize the PDZ protein PICK1 and its target binding proteins.…”

Section: Recombinant Myc-pick1 Is Selectively Clustered At Excitatorymentioning

confidence: 98%

Molecular Determinants for PICK1 Synaptic Aggregation and mGluR7a Receptor Coclustering

Boudin

Craig

2001

PSD-95/Disc-large/ZO-1 (PDZ) domain-containing proteins play a central role in synaptic organization by their involvement in neurotransmitter receptor clustering and signaling complex assembly. The protein interacting with protein kinase C (PICK1), a synaptic PDZ domain protein that also contains a coiled-coil and acidic domain, binds to several synaptic components including the metabotropic glutamate receptor mGluR7a. Coexpression of PICK1 and mGluR7a in heterologous cells induces coclustering of these two proteins. To examine the role of the different structural motifs of PICK1 in synaptic aggregation of PICK1 and mGluR7a coclustering, several PICK1 mutants were generated to analyze their distribution in transfected hippocampal cultured neurons and to test their ability to induce coclusters with mGluR7a when coexpressed in fibroblast cells. The PDZ and coiled-coil domains are both required, whereas the acidic region plays an inhibitory role in these processes. Our data suggest that synaptic aggregation and receptor coclustering depend on PICK1 binding to a target membrane receptor, e.g. mGluR7a, by a PDZ-mediated interaction and on PICK1 oligomerization through the coiled-coil domain. This study defined three structural signals within PICK1 regulating its synaptic localization and receptor coclustering activity, which could represent molecular substrates involved in synaptic development and plasticity.A characteristic and important feature of central nervous system synapses is the clustering of receptors and signaling molecules at both postsynaptic and presynaptic sites, which underlies an efficient synaptic transmission in the brain. For instance, at glutamate excitatory synapses of the hippocampus, the ionotropic glutamate N-methyl-D-aspartate and ␣-amino-3-hydroxy-5-methylsoxazole-4-propionate (AMPA) 1 receptors are both concentrated at postsynaptic sites (1), whereas the G protein-coupled metabotropic glutamate receptor mGluR7a is specifically targeted to the presynaptic active zone of nerve terminals (2). Recent studies showed the importance of PDZ domain-containing proteins in mediating the specific synaptic aggregation of neurotransmitter receptors and in regulating receptor signaling at synapses (3-5). Among them, protein interacting with protein kinase C (PICK1) is a PDZ domain protein originally cloned on the basis of its interaction with protein kinase C (PKC) by a yeast two-hybrid screen (6). PICK1 has subsequently been shown to interact with the Eph receptor tyrosine kinases and ephrin-B ligands (7), GluR2/3/4c subunits of the AMPA receptor (8, 9), mGluR7a receptor (10, 11), and dopamine and norepinephrine transporters (12).Functionally, PICK1 can induce coclustering of the AMPA and mGluR7a receptors in heterologous cells, suggesting a role of PICK1 in the synaptic clustering of these receptors (8 -11). Indeed, disruption of the interaction between PICK1 and mGluR7a by deletion of the PICK1 binding domain in mGluR7a abolished the presynaptic clustering of recombinant mGluR7a in hippocampal neurons...