Binding of L-[3H]Glutamate to Fresh or Frozen Synaptic Membrane and Postsynaptic Density Fractions Isolated from Cerebral Cortex and Cerebellum of Fresh or Frozen Canine Brain

Abstract: Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[³H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the B_max value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl^? increased the number of L-glutamate binding sites and the effect was enhanced by Ca²⁺; Ca²⁺ alone had no significant effect; (b) the Cl^?/Ca²⁺ -sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing: (c) the effect of Na⁺ ion was biphasic: low concentration of Na⁺ (< 5 mM) decreased Cl^?7Ca²⁺ -de-pendent L-glutamate binding sites, whereas at higher concentrations of Na⁺ the binding of glutamate was found to increase either in the presence or absence of Ca²⁺ and Cl^?. In addition, the K⁺ ion (50 mM) was found to decrease the Na⁺-independent and Cl^?/Ca^2--independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na^?-dependent and Cl^?/Ca²⁺-independent L-glutamate binding by 93%; in the presence of Cl, ^?/Ca²⁺, the K⁺ ion decreased the Na⁺-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na⁺-dependent L-glutamate binding sites assayed in the absence of Ca²⁺ and Cl^?. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na^?-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na⁺-dependent nor APB (or Cl^?/Ca²⁺)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin. Also, phosphorylation of CTX-SPM or CTX-PSD fraction, whether incubated simultaneously or after removal of the phosphorylating reagents, had no effect on binding of L-glutamate. Furthermore, binding of L-glutamate to CTX-SPM or CTX-PSD was found to have no significant effect on subsequent phosphorylation of the fractions. Treatment of the CTX-PSD fraction with 0.5% deoxycholate, 1.0% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, and 1.0 M KCl removed the L-glutamate receptors from the PSD by 25%, 44%, 40%, 8%, and 11%. respectively. The respective percentages of total protein solubilized by these reagents were similar, indicating no preferential dissociation of the receptors, and suggesting that the L-glutamate receptor is an intrinsic PSD component. The present findings, together with the earlier ones showing the presence of γ-aminobutyric acid and flunitrazepam binding sites, of the Ca²⁺-dependent K⁺ channel, and of the voltage-dependent Ca²⁺ channel proteins in the isolated PSD fraction, suggest that many, if not all, neurotransmitter receptor proteins and ion channel proteins are anchored in the PSD at the synapse, and thus the PSD may play an important role in neurotransmission at the postsynaptic site.     

Affinity purification of PSD-95-containing postsynaptic complexes

A widely used method for the preparation of postsynaptic density (PSD) fractions consists of treatment of synaptosomal membranes with Triton X-100 and further purification by density gradient centrifugation. In the present study, the purity of this preparation was assessed by electron microscopic analysis. Thin-section and rotary shadow immuno-electron microscopy of the Triton X-100-derived PSD fraction shows many PSD-95-positive structures that resemble in situ PSDs in shape and size. However, the fraction also includes contaminants such as CaMKII clusters, spectrin filaments and neurofilaments. We used magnetic beads coated with an antibody against PSD-95 to further purify PSD-95-containing complexes from the Triton-derived PSD fraction. Biochemical analysis of the affinity-purified material shows a substantial reduction in the astrocytic marker glial fibrillary acidic protein and electron microscopic analysis shows mostly individual PSDs attached to magnetic beads. This preparation was used to assess the association of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type glutamate receptors with the PSD-95-containing complex. AMPA receptors are demonstrated by immunoblotting to be present in the complex, although they do not co-purify exclusively with PSD-95, suggesting the existence of two pools of receptors, one associated with the PSD-95 scaffold and the other not. Of the AMPA receptor-anchoring proteins tested, SAP-97 is present in the affinity-purified preparation whereas GRIP is found only in trace amounts. These results imply that a subpopulation of AMPA receptors is anchored to the PSD-95-containing scaffold through interaction of GluR1 with SAP-97.     

Ion channels of glutamate receptors: structural modeling

Ionotropic glutamate receptors belong to the superfamily of P-loop channels as well as K⁺, Na⁺, and Ca²⁺ channels. However, the structural similarity between ion channels of the glutamate receptors and K⁺ channels is a matter of discussion. The aim of this study was to analyze differences between the structures of K⁺ channels and glutamate receptor channels. For this purpose, homology models of NMDA and AMPA receptor channels (M2 and M3 segments) were built using X-ray structures of K⁺ channels as templates. The models were optimized and used to reproduce specific data on the structure of glutamate receptor channels. Particular attention was paid to the data of the binding of channel blockers and to the results of scanning mutagenesis. The modeling demonstrates that properties of glutamate receptor channel can be reproduced assuming only local structural deformations of the K⁺ channel templates. The most valuable differences were found in the selectivity-filter region, whereas helical parts of M2 and M3 segments could have similar spatial organization with homologous segments in K⁺ channels. It is concluded that the current experimental data on glutamate receptor channels does not reveal global structural differences with K⁺ channels.     

Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins.

Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.     

Molecular targets underlying SUMO‐mediated neuroprotection in brain ischemia

SUMOylation (small ubiquitin‐like modifier conjugation) is an important post‐translational modification which is becoming increasingly implicated in the altered protein dynamics associated with brain ischemia. The function of SUMOylation in cells undergoing ischemic stress and the identity of small ubiquitin‐like modifier (SUMO) targets remain in most cases unknown. However, the emerging consensus is that SUMOylation of certain proteins might be part of an endogenous neuroprotective response. This review brings together the current understanding of the underlying mechanisms and downstream effects of SUMOylation in brain ischemia, including processes such as autophagy, mitophagy and oxidative stress. We focus on recent advances and controversies regarding key central nervous system proteins, including those associated with the nucleus, cytoplasm and plasma membrane, such as glucose transporters (GLUT1, GLUT4), excitatory amino acid transporter 2 glutamate transporters, K⁺ channels (K2P1, K_v1.5, K_v2.1), GluK2 kainate receptors, mGluR8 glutamate receptors and CB1 cannabinoid receptors, which are reported to be SUMO‐modified. A discussion of the roles of these molecular targets for SUMOylation could play following an ischemic event, particularly with respect to their potential neuroprotective impact in brain ischemia, is proposed.     

Distinct serum proteome profiles associated with collagen‐induced arthritis and complete Freund's adjuvant‐induced inflammation in CD38−/− mice: The discriminative power of protein species or proteoforms

Collagen‐type‐II‐induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38^?/? than in wild‐type (WT) mice. ProteoMiner‐equalized serum samples were subjected to 2D‐DiGE and MS‐MALDI‐TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38^?/? versus WT mice either with arthritis (CIA⁺), with no arthritis (CIA^?), or with inflammation (complete Freund's adjuvant (CFA)‐treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA⁺ from CIA^? mice, and WT from CD38^?/? mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA⁺ CD38^?/? mice from CIA⁺ WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38^?/? and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low‐abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 ( http://proteomecentral.proteomexchange.org/dataset/PXD001788 , http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071 ).     

Co-localization of Shaker A-type K+ channel (Kv1.4) and AMPA-glutamate receptor (GluR4) immunoreactivities to dendrites of OFF-bipolar cells of goldfish retina

Immunocytochemical methods were used to determine the comparative distribution of Shaker Kv1.4 and Shal Kv4.2 A-type voltage-gated K⁺ channels and AMPA-type GluR4 glutamate receptors in the goldfish retina. Kv1.4-immunoreactivity (IR) was restricted to a very narrow band of bright puncta and filamentous processes in the outer plexiform layer (OPL), whereas GluR4-IR was found in radial processes of Müller cells in addition to a narrow band in the OPL. Kv4.2-IR was most prominent over cell bodies of horizontal cell, amacrine cells and ganglion cells, with very weak labeling over the synaptic terminal of cone photoreceptors. Double label experiments revealed complete co-localization of Kv1.4-IR and GluR4-IR in the OPL and showed that the Kv1.4 puncta in the OPL appeared enclosed by the Kv4.2-IR cone terminals. Electron microscopical immunocytochemistry showed that Kv1.4-IR and GluR4-IR were restricted to the dendrites of OFF-bipolar cells that innervated cone photoreceptor terminals and thin processes that coursed between the rod and cone terminals in the OPL. These data are consistent with other studies demonstrating the selective clustering of A-type voltage-gated K⁺ channels and ionotropic glutamate receptors. However, they differ from mammalian preparations in which Shal-like Kv4.2 rather than Shaker-like Kv1.4 co-localize postsynaptically with glutamate receptors.     

ProSAP/Shank proteins - a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease

The postsynaptic density (PSD) is a specialized electron-dense structure underneath the postsynaptic plasmamembrane of excitatory synapses. It is thought to anchor and cluster glutamate receptors exactly opposite to the presynaptic neurotransmitter release site. Various efforts to study the molecular structure of the PSD identified several new proteins including membrane receptors, cell adhesion molecules, components of signalling cascades, cytoskeletal elements and adaptor proteins with scaffolding functions to interconnect these PSD components. The characterization of a novel adaptor protein family, the ProSAPs or Shanks, sheds new light on the basic structural organization of the PSD. ProSAPs/Shanks are multidomain proteins that interact directly or indirectly with receptors of the postsynaptic membrane including NMDA-type and metabotropic glutamate receptors, and the actin-based cytoskeleton. These interactions suggest that ProSAP/Shanks may be important scaffolding molecules of the PSD with a crucial role in the assembly of the PSD during synaptogenesis, in synaptic plasticity and in the regulation of dendritic spine morphology. Moreover the analysis of a patient with 22q13.3 distal deletion syndrome revealed a balanced translocation with a breakpoint in the human ProSAP2/Shank3 gene. This ProSAP2/Shank3 haploinsufficiency may cause a syndrome that is characterized by severe expressive language delay, mild mental retardation and minor facial dysmorphisms.     

Control of K+ channels by G proteins

Heterotrimeic G proteins are thought to couple receptors to ionic channels via cytoplasmic mediators such as cGMP in the case of retinal rods, cAMP in the case of olfactory cells, and the cAMP cascade in the case of cardiac myocytes. G protein-mediated second messenger effects on K⁺ channels are dealt with elsewhere in this series. Recently, membrane-delimited pathways have been uncovered and an hypothesis proposed in which the subunits of G proteins directly couple receptors to ionic channels, particularly K⁺ channels. While direct coupling has not been proven, the membrane-delimited nature has been established for specific G proteins and their specific K⁺ channel effectors.     

Effects of amyloid peptides on A‐type K+ currents of Drosophila larval cholinergic neurons

Accumulation of amyloid (Aβ) peptides has been suggested to be the primary event in Alzheimer's disease. In neurons, K⁺ channels regulate a number of processes, including setting the resting potential, keeping action potentials short, timing interspike intervals, synaptic plasticity, and cell death. In particular, A‐type K⁺ channels have been implicated in the onset of LTP in mammalian neurons, which is thought to underlie learning and memory. A number of studies have shown that Aβ peptides alter the properties of K⁺ currents in mammalian neurons. We set out to determine the effects of Aβ peptides on the neuronal A‐type K⁺ channels of Drosophila. Treatment of cells for 18 h with 1 μM Aβ1‐42 altered the kinetics of the A‐type K⁺ current, shifting steady‐state inactivation to more depolarized potentials and increasing the rate of recovery from inactivation. It also caused a decrease in neuronal viability. Thus it seems that alteration in the properties of the A‐type K⁺ current is a prelude to the amyloid‐induced death of neurons. This alteration in the properties of the A‐type K⁺ current may provide a basis for the early memory impairment that was observed prior to neurodegeneration in a recent study of a transgenic Drosophila melanogaster line over‐expressing the human Aβ1‐42 peptide. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Scaffold proteins (MAGUK, Shank and Homer) in postsynaptic density in the central nervous system

The postsynaptic density (PSD) is a dynamic multi-protein complex attached to the postsynaptic membrane composed of several hundred proteins such as receptors and channels, scaffolding and adaptor proteins, cell-adhesion proteins, cytoskeletal proteins, G-proteins and their modulators and signaling molecules including kinases and phosphtases. This review focuses on the prominent PSD scaffolds proteins such as members of the MAGUK (membrane-associated guanylyl kinase), Shank (SH3 domain and ankyrin repeat-containing protein) and Homer families. These molecules interact simultaneously with different kinds of receptors and modulate their function by linking the receptors to downstream signaling events. For example PSD 95, a main member of MAGUK family, interacts directly with carboxyl termini of NMDA receptor subunits and clusters them to the postsynaptic membrane. In addition, PSD 95 is involved in binding and organizing proteins connected with NMDAR signaling. Based on the modular character and ability to form multiproteins interactions, MAGUK, Shank and Homer are perfectly suited to act as a major scaffold in postsynaptic density.     

Calcium-Dependent Networks in Dopamine–Glutamate Interaction: The Role of Postsynaptic Scaffolding Proteins

Dopamine and glutamate systems are both involved in cognitive, behavioral, and motor processes. Dysfunction of dopamine–glutamate interplay has been suggested in several psychotic diseases, above all in schizophrenia, for which there exists a need for novel medications. Intracellular calcium-dependent transduction pathways are key determinants of dopamine–glutamate interactions, which take place mainly, albeit not exclusively, in the postsynaptic density (PSD), a highly specialized postsynaptic ultrastructure. Stimulation of dopamine and glutamate receptors modulates the gene expression and the function of specific PSD proteins, the “scaffolding” proteins (Homer, Shank, and PSD95), belonging to a complex Ca²⁺-regulated network that integrates and converges dopamine and glutamate signaling to appropriate nuclear targets. Dysfunction of scaffolding proteins leads to severe impairment of Ca²⁺-dependent signaling, which may underlie the dopamine–glutamate aberrations putatively implicated in the pathogenesis of psychotic disorders. Antipsychotic therapy has been demonstrated to directly and indirectly affect the neuronal Ca²⁺-dependent pathways through the modulation of PSD scaffolding proteins, such as Homer, therefore influencing both dopaminergic and glutamatergic functions and enforcing Ca²⁺-mediated long-term synaptic changes. In this review, we will discuss the role of PSD scaffolding proteins in routing Ca²⁺-dependent signals to the nucleus. In particular, we will address the implication of PSD scaffolding proteins in the intracellular connections between dopamine and glutamate pathways, which involve both Ca²⁺-dependent and Ca²⁺-independent mechanisms. Finally, we will discuss how new strategies for the treatment of psychosis aim at developing antipsychotics that may impact both glutamate and dopamine signaling, and what should be the possible role of PSD scaffolding proteins.     

Bicarbonate-Independent Sodium Conductance of Na/HCO3 Cotransporter NBCn1 Decreases NMDA Receptor Function

The sodium bicarbonate cotransporter NBCn1 is an electroneutral transporter with a channel activity that conducts Na⁺ in a HCO₃^–-independent manner. This channel activity was suggested to functionally affect other membrane proteins which permeate Na⁺ influx. We previously reported that NBCn1 is associated with the NMDA receptors (NMDARs) at the molecular and physiological levels. In this study, we examined whether NBCn1 channel activity affects NMDAR currents and whether this effect involves the interaction between the two proteins. NBCn1 and the NMDAR subunits GluN1A/GluN2A were expressed in Xenopus oocytes, and glutamate currents produced by the receptors were measured using two-electrode voltage clamp. In the absence of CO₂/HCO₃^–, NBCn1 channel activity decreased glutamate currents mediated by GluN1A/GluN2A. NBCn1 also decreased the slope of the current–voltage relationships for the glutamate current. Similar effects on the glutamate current were observed with and without PSD95, which can cluster NBCn1 and NMDARs. The channel activity was also observed in the presence of CO₂/HCO₃^–. We conclude that NBCn1 channel activity decreases NMDAR function. Given that NBCn1 knockout mice develop a downregulation of NMDARs, our results are unexpected and suggest that NBCn1 has dual effects on NMDARs. It stabilizes NMDAR expression but decreases receptor function by its Na⁺ channel activity. The dual effects may play an important role in fine-tuning the regulation of NMDARs in the brain.     

Characterization of zebrafish PSD‐95 gene family members

The PSD‐95 family of membrane‐ associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD‐95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD‐95, PSD‐93, and two isoforms of SAP‐97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan‐MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD‐95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Ca2+/calmodulin binding to PSD‐95 mediates homeostatic synaptic scaling down

Postsynaptic density protein‐95 (PSD‐95) localizes AMPA‐type glutamate receptors (AMPARs) to postsynaptic sites of glutamatergic synapses. Its postsynaptic displacement is necessary for loss of AMPARs during homeostatic scaling down of synapses. Here, we demonstrate that upon Ca²⁺ influx, Ca²⁺/calmodulin (Ca²⁺/CaM) binding to the N‐terminus of PSD‐95 mediates postsynaptic loss of PSD‐95 and AMPARs during homeostatic scaling down. Our NMR structural analysis identified E17 within the PSD‐95 N‐terminus as important for binding to Ca²⁺/CaM by interacting with R126 on CaM. Mutating E17 to R prevented homeostatic scaling down in primary hippocampal neurons, which is rescued via charge inversion by ectopic expression of CaM^R126E, as determined by analysis of miniature excitatory postsynaptic currents. Accordingly, increased binding of Ca²⁺/CaM to PSD‐95 induced by a chronic increase in Ca²⁺ influx is a critical molecular event in homeostatic downscaling of glutamatergic synaptic transmission.     

Capping of the N‐terminus of PSD‐95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein‐95 (PSD‐95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD‐95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD‐95 is released from postsynaptic membranes in response to Ca²⁺ influx via NMDA receptors. Here, we show that Ca²⁺/calmodulin (CaM) binds at the N‐terminus of PSD‐95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD‐95 formed at its N‐terminus (residues 1–16). This N‐terminal capping of PSD‐95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD‐95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD‐95. The PSD‐95 mutant Y12E strongly impairs binding to CaM and Ca²⁺‐induced release of PSD‐95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD‐95 serves to block palmitoylation of PSD‐95, which in turn promotes Ca²⁺‐induced dissociation of PSD‐95 from the postsynaptic membrane.     

Mechanisms of Adenosine Release in the Developing and Adult Mouse Hippocampus

Adenosine is a neuromodulator known to inhibit the synaptic release of neurotransmitters, e.g., glutamate, and to hyperpolarize postsynaptic neurons. The release of adenosine is markedly enhanced under ischemic conditions. It may then act as an endogenous neuroprotectant against cerebral ischemia and excitotoxic neuronal damage. The mechanisms by which adenosine is released from nervous tissue are not fully known, particularly in the immature brain. We now characterized the release of [³H]adenosine from hippocampal slices from developing (7-day-old) and adult (3-month-old) mice using a superfusion system. The properties of the release differed only partially in the immature and mature hippocampus. The K⁺-evoked release was Ca²⁺ and Na⁺ dependent. Anion channels were also involved. Ionotropic glutamate receptor agonists potentiated the release in a receptor-mediated manner. Activation of metabotropic glutamate receptors enhanced the release in developing mice, with group II receptors alone being effective. The evoked adenosine release apparently provides neuroprotective effects against excitotoxicity under cell-damaging conditions. Taurine had no effect on adenosine release in adult mice, but depressed the release concentration dependently in the immature hippocampus.     

A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel

The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells. This purification procedure was also applied to study interactions between soluble proteins in mammalian cells. In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease. We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein. Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system. Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells.     

Zinc transporter‐1: a novel NMDA receptor‐binding protein at the postsynaptic density

Zinc (Zn²⁺) is believed to play a relevant role in the physiology and pathophysiology of the brain. Hence, Zn²⁺ homeostasis is critical and involves different classes of molecules, including Zn²⁺ transporters. The ubiquitous Zn²⁺ transporter‐1 (ZNT‐1) is a transmembrane protein that pumps cytosolic Zn²⁺ to the extracellular space, but its function in the central nervous system is not fully understood. Here, we show that ZNT‐1 interacts with GluN2A‐containing NMDA receptors, suggesting a role for this transporter at the excitatory glutamatergic synapse. First, we found that ZNT‐1 is highly expressed at the hippocampal postsynaptic density (PSD) where NMDA receptors are enriched. Two‐hybrid screening, coimmunoprecipitation experiments and clustering assay in COS‐7 cells demonstrated that ZNT‐1 specifically binds the GluN2A subunit of the NMDA receptor. GluN2A deletion mutants and pull‐down assays indicated GluN2A(1390–1464) domain as necessary for the binding to ZNT‐1. Most importantly, ZNT‐1/GluN2A complex was proved to be dynamic, since it was regulated by induction of synaptic plasticity. Finally, modulation of ZNT‐1 expression in hippocampal neurons determined a significant change in dendritic spine morphology, PSD‐95 clusters and GluN2A surface levels, supporting the involvement of ZNT‐1 in the dynamics of excitatory PSD.

     

Differential modulation of cardiac potassium channels by Grb adaptor proteins

Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K⁺ channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K⁺ channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K⁺ channels.