Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1

Synaptic clustering of neurotransmitter receptors is crucial for efficient signal transduction and integration in neurons. PDZ domain-containing proteins such as PSD-95/SAP90 interact with the intracellular C termini of a variety of receptors and are thought to be important in the targeting and anchoring of receptors to specific synapses. Here, we show that PICK1 (protein interacting with C kinase), a PDZ domain-containing protein, interacts with the C termini of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in vitro and in vivo. In neurons, PICK1 specifically colocalizes with AMPA receptors at excitatory synapses. Furthermore, PICK1 induces clustering of AMPA receptors in heterologous expression systems. These results suggest that PICK1 may play an important role in the modulation of synaptic transmission by regulating the synaptic targeting of AMPA receptors.     

Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes

Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction.     

Protein kinase C stimulates the acid-sensing ion channel ASIC2a via the PDZ domain-containing protein PICK1

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. They are expressed in central and sensory neurons where they are involved in neuromodulation and in pain perception. Recently, the PDZ domain-containing protein PICK1 (protein interacting with C-kinase) has been shown to interact with ASIC1a and ASIC2a, raising the possibility that protein kinase C (PKC) could regulate ASICs. We now show that the amplitude of the ASIC2a current, which was only modestly increased ( approximately +30%) by the PKC activator 1-oleyl-2-acetyl-sn-glycerol (OAG, 50 microm) in the absence of PICK1, was strongly potentiated ( approximately +300%) in the presence of PICK1. This PICK1-dependent regulatory effect was inhibited in the presence of a PKC inhibitory peptide and required the PDZ domain of PICK1 as well as the PDZ-binding domain of ASIC2a. We have also shown the direct PICK1-dependent phosphorylation of ASIC2a by [(32)P]phosphate labeling and immunoprecipitation and identified a major phosphorylation site, (39)TIR, on the N terminus part of ASIC2a. The OAG-induced increase in ASIC2a current amplitude did not involve any change in the unitary conductance of the ASIC2a channel, whether co-expressed with PICK1 or not. These data provide the first demonstration of a regulation of ASICs by protein kinase phosphorylation and its potentiation by the partner protein PICK1.     

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and EC₅₀ for glutamate. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [³H]glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but may also affect neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotrophic lateral sclerosis and cerebral hypoxia, which are associated with neuronal GLT1b up-regulation.     

Parkin-mediated monoubiquitination of the PDZ protein PICK1 regulates the activity of acid-sensing ion channels

Mutations in the parkin gene result in an autosomal recessive juvenile-onset form of Parkinson's disease. As an E3 ubiquitin-ligase, parkin promotes the attachment of ubiquitin onto specific substrate proteins. Defects in the ubiquitination of parkin substrates are therefore believed to lead to neurodegeneration in Parkinson's disease. Here, we identify the PSD-95/Discs-large/Zona Occludens-1 (PDZ) protein PICK1 as a novel parkin substrate. We find that parkin binds PICK1 via a PDZ-mediated interaction, which predominantly promotes PICK1 monoubiquitination rather than polyubiquitination. Consistent with monoubiquitination and recent work implicating parkin in proteasome-independent pathways, parkin does not promote PICK1 degradation. However, parkin regulates the effects of PICK1 on one of its other PDZ partners, the acid-sensing ion channel (ASIC). Overexpression of wild-type, but not PDZ binding- or E3 ubiquitin-ligase-defective parkin abolishes the previously described, protein kinase C-induced, PICK1-dependent potentiation of ASIC2a currents in non-neuronal cells. Conversely, the loss of parkin in hippocampal neurons from parkin knockout mice unmasks prominent potentiation of native ASIC currents, which is normally suppressed by endogenous parkin in wild-type neurons. Given that ASIC channels contribute to excitotoxicity, our work provides a mechanism explaining how defects in parkin-mediated PICK1 monoubiquitination could enhance ASIC activity and thereby promote neurodegeneration in Parkinson's disease.     

Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters

Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20-fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5-h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10-20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase-deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co-transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.     

The PDZ domain of PICK1 differentially accepts protein kinase C-alpha and GluR2 as interacting ligands

The C terminus (ct) of protein kinase C-alpha (PKCalpha) has a type I PDZ binding motif, whereas GluR2 has a type II PDZ binding motif. Both motifs are recognized by the PDZ domain of protein interacting with protein kinase C (PICK1), and PICK1-PKCalpha-controlled phosphorylation regulates the synaptic expression and function of GluR2. Here, we show that a specific mutation within the carboxylate-binding loop of the PDZ domain of PICK1 (K27E; PICK1-KE) results in a loss of interaction with GluR2 but not with PKCalpha. In GST pull-down studies, PICK1-WT (wild type) but not PICK1-KE was retained by GST-ct-GluR2. Furthermore, PICK1-WT co-immunoprecipitated both PKCalpha and GluR2, whereas PICK1-KE only co-immunoprecipitated PKCalpha. In heterologous cells, PICK1-WT, but not PICK1-KE, clustered GluR2 and also clustered GluR1 in a GluR2-dependent manner. However, neither PICK1-WT nor PICK1-KE altered the distribution of PKCalpha, even after phorbol ester-induced redistribution of PKCalpha to the membrane. Finally, PICK1-KE showed no mislocalization when compared with PICK1-WT in neurons. Taken together, it appears that the PDZ domain of PICK1 is less sensitive to mutations for PKCalpha when compared with GluR2 binding. These results suggest that the PDZ domain of PICK1 has distinct PKCalpha and GluR2 binding subsite(s).     

Functional importance of the synaptic plasma membrane calcium pump and sodium-calcium exchanger

Two major Ca2+ transport mechanisms co-function in a preparation of synaptosomal plasma membrane vesicles: an (ATP + Mg2+)-dependent Ca2+ pump, and a reversible Na+-Ca2+ exchanger (Gill, D. L., Grollman, E.F., and Kohn, L. D. (1981) J. Biol. Chem. 256, 184-192). An accurate comparative analysis of the kinetics of the two Ca2+ transporters under free Ca2+ conditions precisely buffered with EGTA, reveals that both mechanisms have high affinity for Ca2+. The ATP-dependent Ca2+ pump displays simple saturation kinetics with a Km for Ca2+ of 0.11 microM and a Vmax of 2.2 nmol/min/mg of protein. In contrast, the Na+-Ca2+ exchanger has a complex dependence on free Ca2+, the activity continuing to saturate over a wide range of free Ca2+ concentrations from 0.03 microM to 3 mM. The curvilinear Eadie-Hofstee analysis reveals a distinct high affinity component for the exchanger with a Km for Ca2+ of approximately 0.5 microM, and a lower affinity component not accurately resolvable into a discrete Km value. 2 mM amiloride blocks Na+-Ca2+ exchange-mediated Ca2+ uptake by 90% over a wide range of free Ca2+ (0.3-3000 microM), suggesting a similar noncompetitive inhibition of both low and high affinity Ca2+ sites. Ca2+ accumulated in vesicles via either the Ca2+ pump or Na+-Ca2+ exchanger is rapidly (in less than 1 min) released by 0.1% saponin (w/v), although a minor component (8-10%) of Ca2+ pump activity is resistant to saponin addition. The IC50 for the effect of saponin is the same (0.01%, w/v) for both Ca2+ transport mechanisms. The ATP-dependent Ca2+ pump is shown to be highly sensitive to vanadate inhibition (Ki = 0.5 microM). The high saponin sensitivity of both Ca2+ transporters and the potent effect of vanadate on Ca2+ pumping, together with previous Na+ channel and Na+ pump flux studies in the same membrane vesicles (Gill, D. L. (1982) J. Biol. Chem. 257, 10986-10990), all strongly suggest that both of the high affinity Ca2+ transporters function in the plasma membrane where they are of major functional importance to the regulation of intrasynaptic free Ca2+ levels.     

Predominant role of plasma membrane monoamine transporters in monoamine transport in 1321N1, a human astrocytoma‐derived cell line

Monoamine neurotransmitters should be immediately removed from the synaptic cleft to avoid excessive neuronal activity. Recent studies have shown that astrocytes and neurons are involved in monoamine removal. However, the mechanism of monoamine transport by astrocytes is not entirely clear. We aimed to elucidate the transporters responsible for monoamine transport in 1321N1, a human astrocytoma‐derived cell line. First, we confirmed that 1321N1 cells transported dopamine, serotonin, norepinephrine, and histamine in a time‐ and dose‐dependent manner. Kinetics analysis suggested the involvement of low‐affinity monoamine transporters, such as organic cation transporter (OCT) 2 and 3 and plasma membrane monoamine transporter (PMAT). Monoamine transport in 1321N1 cells was not Na⁺/Cl^? dependent but was inhibited by decynium‐22, an inhibitor of low‐affinity monoamine transporters, which supported the importance of low‐affinity transporters. RT‐PCR assays revealed that 1321N1 cells expressed OCT3 and PMAT but no other neurotransmitter transporters. Another human astrocytoma‐derived cell line, U251MG, and primary human astrocytes also exhibited the same gene expression pattern. Gene‐knockdown assays revealed that 1321N1 and primary human astrocytes could transport monoamines predominantly through PMAT and partly through OCT3. These results might indicate that PMAT and OCT3 in human astrocytes are involved in monoamine clearance.

     

Inhibition of endosomal trafficking by brefeldin A interferes with long-distance interaction between chloroplasts and plasma membrane transporters

The huge internodal cells of the characean green algae are a convenient model to study long-range interactions between organelles via cytoplasmic streaming. It has been shown previously that photometabolites and reactive oxygen species released by illuminated chloroplasts are transmitted to remote shaded regions where they interfere with photosynthetic electron transport and the differential activity of plasma membrane transporters, and recent findings indicated the involvement of organelle trafficking pathways. In the present study, we applied pulse amplitude-modulated microscopy and pH-sensitive electrodes to study the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on long-distance interactions in Chara australis internodal cells. These data were compared with BFA-induced changes in organelle number, size and distribution using fluorescent dyes and confocal laser scanning microscopy. We found that BFA completely and immediately inhibited endocytosis in internodal cells and induced the aggregation of organelles into BFA compartments within 30–120 min of treatment. The comparison with the physiological data suggests that the early response, the arrest of endocytosis, is related to the attenuation of differences in surface pH, whereas the longer lasting formation of BFA compartments is probably responsible for the acceleration of the cyclosis-mediated interaction between chloroplasts. These data indicate that intracellular turnover of membrane material might be important for the circulation of electric currents between functionally distinct regions in illuminated characean internodes and that translational movement of metabolites is delayed by transient binding of the transported substances to organelles.     

Influence of pentobarbital on synaptic plasma membrane protein synthesis

The effects of acute and chronic pentobarbital treatment on the incorporation of ³H-lysine into rat brain synaptic plasma membranes (SPM) were examined by means of SDS polyacrylamide gel electrophoresis. The data suggest the major effects of both drug treatments are found in a unique population of SPM derived from a light population of nerve ending particles (NEP). These light NEP preferentially accumulate labelled GABA, suggesting this fraction is enriched in GABA containing nerve terminals.     

Relations between plasma membrane and lysosomal membrane. 1. Fate of covalently labelled plasma membrane protein

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4 degrees C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37 degrees C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell-associated labels) was rapidly released (half-life less than 1 h), and most label (65%) was released slowly (half-life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37 degrees C, the patterns of 125I-polypeptides quantified by autoradiography after SDS-PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides. The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2-4% of cell-associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H-labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell-associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.     

The binding affinities of proteins interacting with the PDZ domain of PICK1

Protein interacting with C kinase (PICK1) is well conserved throughout evolution and plays a critical role in synaptic plasticity by regulating the trafficking and posttranslational modification of its interacting proteins. PICK1 contains a single PSD95/DlgA/Zo-1 (PDZ) protein-protein interaction domain, which is promiscuous and shown to interact with over 60 proteins, most of which play roles in neuronal function. Several reports have suggested the role of PICK1 in disorders such as epilepsy, pain, brain trauma and stroke, drug abuse and dependence, schizophrenia and psychosis. Importantly, lead compounds that block PICK1 interactions are also now becoming available. Here, a new modeling approach was developed to investigate binding affinities of PDZ interactions. Using these methods, the binding affinities of all major PICK1 interacting proteins are reported and the effects of PICK1 mutations on these interactions are described. These modeling methods have important implications in defining the binding properties of proteins interacting with PICK1 as well as the general structural requirements of PDZ interactions. The study also provides modeling methods to support in the drug design of ligands for PDZ domains, which may further aid in development of the family of PDZ domains as a drug target.     

Molecular determinants for the complex binding specificity of the PDZ domain in PICK1

PICK1 (protein interacting with C kinase 1) contains a single PDZ domain known to mediate interaction with the C termini of several receptors, transporters, ion channels, and kinases. In contrast to most PDZ domains, the PICK1 PDZ domain interacts with binding sequences classifiable as type I (terminating in (S/T)XPhi; X, any residue) as well as type II (PhiXPhi; Phi, any hydrophobic residue). To enable direct assessment of the affinity of the PICK1 PDZ domain for its binding partners we developed a purification scheme for PICK1 and a novel quantitative binding assay based on fluorescence polarization. Our results showed that the PICK1 PDZ domain binds the type II sequence presented by the human dopamine transporter (-WLKV) with an almost 15-fold and >100-fold higher affinity than the type I sequences presented by protein kinase Calpha (-QSAV) and the beta(2)-adrenergic receptor (-DSLL), respectively. Mutational analysis of Lys(83) in the alphaB1 position of the PDZ domain suggested that this residue mimics the function of hydrophobic residues present in this position in regular type II PDZ domains. The PICK1 PDZ domain was moreover found to prefer small hydrophobic residues in the C-terminal P(0) position of the ligand. Molecular modeling predicted a rank order of (Val > Ile > Leu) that was verified experimentally with up to a approximately 16-fold difference in binding affinity between a valine and a leucine in P(0). The results define the structural basis for the unusual binding pattern of the PICK1 PDZ domain by substantiating the critical role of the alphaB1 position (Lys(83)) and of discrete side chain differences in position P(0) of the ligands.     

Molecular determinants for PICK1 synaptic aggregation and mGluR7a receptor coclustering: role of the PDZ, coiled-coil, and acidic domains

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.     

PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes

PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes. Am J Physiol Cell Physiol 288: C20–C29, 2005; doi:10.1152/ajpcell.00368.2004.—The plasma membrane of epithelial cells is subdivided into two physically separated compartments known as the apical and basolateral membranes. To obtain directional transepithelial solute transport, membrane transporters (i.e., ion channels, cotransporters, exchangers, and ion pumps) need to be targeted selectively to either of these membrane domains. In addition, the transport properties of an epithelial cell will be maintained only if these membrane transporters are retained and properly regulated in their specific membrane compartments. Recent reports have indicated that PDZ domain-containing proteins play a dual role in these processes and, in addition, that different apical and basolateral PDZ proteins perform similar tasks in their respective membrane domains. First, although PDZ-based interactions are dispensable for the biosynthetic targeting to the proper membrane domain, the PDZ network ensures that the membrane proteins are efficiently retained at the cell surface. Second, the close spatial positioning of functionally related proteins (e.g., receptors, kinases, channels) into a signal transduction complex (transducisome) allows fast and efficient control of membrane transport processes. retention of apical and basolateral membrane proteins; transducisomes; protein complex formation     

Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.