Molecular mechanisms contributing to TARP regulation of channel conductance and polyamine block of calcium-permeable AMPA receptors.

Many properties of fast synaptic transmission in the brain are influenced by transmembrane AMPAR regulatory proteins (TARPs) that modulate the pharmacology and gating of AMPA-type glutamate receptors (AMPARs). Although much is known about TARP influence on AMPAR pharmacology and kinetics through their modulation of the extracellular ligand-binding domain (LBD), less is known about their regulation of the ion channel region. TARP-induced modifications in AMPAR channel behavior include increased single-channel conductance and weakened block of calcium-permeable AMPARs (CP-AMPARs) by endogenous intracellular polyamines. To investigate how TARPs modify ion flux and channel block, we examined the action of γ-2 (stargazin) on GluA1 and GluA4 CP-AMPARs. First, we compared the permeation of organic cations of different sizes. We found that γ-2 increased the permeability of several cations but not the estimated AMPAR pore size, suggesting that TARP-induced relief of polyamine block does not reflect altered pore diameter. Second, to determine whether residues in the TARP intracellular C-tail regulate polyamine block and channel conductance, we examined various γ-2 C-tail mutants. We identified the membrane proximal region of the C terminus as crucial for full TARP-attenuation of polyamine block, whereas complete deletion of the C-tail markedly enhanced the TARP-induced increase in channel conductance; thus, the TARP C-tail influences ion permeation. Third, we identified a site in the pore-lining region of the AMPAR, close to its Q/R site, that is crucial in determining the TARP-induced changes in single-channel conductance. This conserved residue represents a site of TARP action, independent of the AMPAR LBD

AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

AMPARs mediate the vast majority of fast excitatory synaptic transmission in the brain and their biophysical and trafficking properties depend on their subunit composition and on several posttranscriptional and posttranslational modifications. Additionally, in the brain AMPARs associate with auxiliary subunits, which modify the properties of the receptors. Despite the abundance of AMPAR partners, recent proteomic studies have revealed even more interacting proteins that could potentially be involved in AMPAR regulation. Amongst these, carnitine palmitoyltransferase 1C (CPT1C) has been demonstrated to form an integral part of native AMPAR complexes in brain tissue extracts. Thus, we aimed to investigate whether CPT1C might be able to modulate AMPAR function. Firstly, we confirmed that CPT1C is an interacting protein of AMPARs in heterologous expression systems. Secondly, CPT1C enhanced whole-cell currents of GluA1 homomeric and GluA1/GluA2 heteromeric receptors. However, CPT1C does not alter the biophysical properties of AMPARs and co-localization experiments revealed that AMPARs and CPT1C are not associated at the plasma membrane despite a strong level of co-localization at the intracellular level. We established that increased surface GluA1 receptor number was responsible for the enhanced AMPAR mediated currents in the presence of CPT1C. Additionally, we revealed that the palmitoylable residue C585 of GluA1 is important in the enhancement of AMPAR trafficking to the cell surface by CPT1C. Nevertheless, despite its potential as a depalmitoylating enzyme, CPT1C does not affect the palmitoylation state of GluA1. To sum up, this work suggests that CPT1C plays a role as a novel regulator of AMPAR surface expression in neurons. Fine modulation of AMPAR membrane trafficking is fundamental in normal synaptic activity and in plasticity processes and CPT1C is therefore a putative candidate to regulate neuronal AMPAR physiology

AMPAR/TARP stoichiometry differentially modulates channel properties

AMPARs control fast synaptic communication between neurons and their function relies on auxiliary subunits, which importantly modulate channel properties. Although it has been suggested that AMPARs can bind to TARPs with variable stoichiometry, little is known about the effect that this stoichiometry exerts on certain AMPAR properties. Here we have found that AMPARs show a clear stoichiometry-dependent modulation by the prototypical TARP γ2 although the receptor still needs to be fully saturated with γ2 to show some typical TARP-induced characteristics (i.e. an increase in channel conductance). We also uncovered important differences in the stoichiometric modulation between calcium-permeable and calcium-impermeable AMPARs. Moreover, in heteromeric AMPARs, γ2 positioning in the complex is important to exert certain TARP-dependent features. Finally, by comparing data from recombinant receptors with endogenous AMPAR currents from mouse cerebellar granule cells, we have determined a likely presence of two γ2 molecules at somatic receptors in this cell type

Metazoan evolution of glutamate receptors reveals unreported phylogenetic groups and divergent lineage-specific events

Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings

Mechanisms of CPT1C-Dependent AMPAR trafficking enhancement

In neurons, AMPA receptor (AMPAR) function depends essentially on their constituent components:the ion channel forming subunits and ion channel associated proteins. On the other hand, AMPAR trafficking is tightly regulated by a vast number of intracellular neuronal proteins that bind to AMPAR subunits. It has been recently shown that the interaction between the GluA1 subunit of AMPARs and carnitine palmitoyltransferase 1C (CPT1C), a novel protein partner of AMPARs, is important in modulating surface expression of these ionotropic glutamate receptors. Indeed, synaptic transmission in CPT1C knockout (KO) mice is diminished supporting a positive trafficking role for that protein. However, the molecular mechanisms of such modulation remain unknown although a putative role of CPT1C in depalmitoylating GluA1 has been hypothesized. Here, we explore that possibility and show that CPT1C effect on AMPARs is likely due to changes in the palmitoylation state of GluA1. Based on in silico analysis, Ser 252, His 470 and Asp 474 are predicted to be the catalytic triad responsible for CPT1C palmitoyl thioesterase (PTE) activity. When these residues are mutated or when PTE activity is inhibited, the CPT1C effect on AMPAR trafficking is abolished, validating the CPT1C catalytic triad as being responsible for PTE activity on AMPAR. Moreover, the histidine residue (His 470) of CPT1C is crucial for the increase in GluA1 surface expression in neurons and the H470A mutation impairs the depalmitoylating catalytic activity of CPT1C. Finally, we show that CPT1C effect seems to be specific for this CPT1 isoform and it takes place solely at endoplasmic reticulum (ER). This work adds another facet to the impressive degree of molecular mechanisms regulating AMPAR physiology

Molecular characterization of carnitine palmitoyltransferase 1C

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the conversion of long chain fatty acyl-CoAs into acylcarnitines, the first step in the transport of long chain fatty acids from the cytoplasm to the mitochondrial matrix, where they undergo β-oxidation. This reaction is not only central to the control of fatty acid oxidation, but it also determines the availability of long chain acyl-CoA for other processes. There are three different CPT1 isozymes: CPT1A (expressed in liver, pancreas, kidney, brain, blood, and embryonic tissues), CPT1B (expressed only in brown adipose tissue, muscle, and heart) and the recently described CPT1C. CPT1C protein sequence is highly similar to that of the other two isozymes. Expression studies indicate that CPT1C is localized exclusively in the central nervous system, with homogeneous distribution in all areas (hippocampus, cortex, hypothalamus, and others). It has also been reported that CPT1c is localized in neurons but not in astrocytes of adult brain. 1. CPT1C strucutral model A 3-D structural model of the isozyme has been constructed by homology modeling. Residues contacting both substrates have been determined and compared to the same amino acid positions in CPT1A. The results obtained from the analysis show that the residues involved in the catalysis of the reaction in CPT1A and residues contacting both substrates are conserved mainly conserved in CPT1C or show semi-conservative substitutions. 2. CPT1 enzymatic activity Expression of rat CPT1C in Saccharomyces cerevisiae yields no catalytic activity when testing different conditions (longer periods of time, increased temperature, increased substrate concentration, testing of microsomal fraction or chimeric protein CPT1·ACA). Thus, the yeast expression system is not suitable for studying CPT1C enzymatic activity. 3. Subcellular localization Endogenous and overexpressed CPT1C is basically localized in the endoplasmic reticulum of mammalian cells (HEK293T, PC12, SH-SY5Y, primary cultures of fibroblasts and neurons). Some evidences indicated that CPT1C could also be found, in lower amounts, in mitochondrial associated membranes (MAMs). The specific sequence of CPT1C N-terminal domain (first 150 amino acids) drives the protein to the endoplasmic reticulum. 4. CPT1C N-terminus processing The N-terminal end of endogenous CPT1C in wild type mouse brain is processed (at least until Val27) and is not detected in mouse brain cortex lysates. 5. CPT1C membrane topology The N- and C-terminal domains of CPT1C are facing the cytosolic side of the endoplasmic reticulum membrane, whereas the loop domain is facing the endoplasmic reticulum lumen. 6. CPT1C interacting partners The data provided by the yeast two-hybrid assay do not indicate a unique binding partner of CPT1C. Instead the assay retrieved proteins involved in different functions: protein degradation, membrane trafficking, cell structure, signal transduction and metabolism. KEYWORDS: Carnitine palmitoyltransferase, Endoplasmatic reticulum, Subcelular localization, CPT1 activity, Structural model, Membrane topologyLa carnitina palmitoiltransferasa 1 (CPT1) es una enzima que cataliza la conversión de aciles-CoA de cadena larga en acil-carnitinas, reacción crucial para el control de la oxidación de ácidos grasos. Existen tres isoformas diferentes de CPT1: CPT1A (isoforma más ubicua), CPT1B (expresada en tejido adiposo, músculo y corazón) y CPT1C que es la isoforma más recientemente descrita. La secuencia de la proteína CPT1C es muy parecida a la de las otras dos isoformas. Estudios de expresión indican que CPT1C se localiza exclusivamente en el sistema nervioso central. También se ha descrito que CPT1C se localiza en neuronas de cerebro adulto pero no en astrocitos. Las conclusiones obtenidas de los resultados presentados en esta tesis son (por apartados): 1. Modelo structural A través de técnicas de modelaje por homología se ha construido un modelo tridimensional teórico de la proteína. De su estudio se concluye que los residuos implicados en la catálisis de la reacción y los residuos en contacto con los sustratos están bien conservados en la secuencia de CPT1C. 2. Actividad enzimática La expresión de CPT1C en la levadura Saccharomyces cerevisiae no muestra actividad CPT1 aunque se testen diferentes condiciones (tiempos de reacción más largos, incrementos en la concentración de sustratos, pruebas en fracciones microsomales o pruebas con proteínas quiméricas como la CPT1·ACA) 3. Localización subcelular La proteína CPT1C se localiza básicamente en el retículo endoplasmático de células de mamífero (HEK293T, PC12, SH-SY5Y y en cultivos primarios de fibroblastos y de neuronas). La secuencia concreta de los 150 primeros aminoácidos dirige la porteína al retículo endoplasmático. 4. Procesamiento del extremo N-terminal de CPT1C El extremo N-terminal de la proteína CPT1C endógena sufre un procesamiento. 5. Topología en la membrana de CPT1C Los dominios N- y C-terminal (centro catalítico) están orientados hacia la cara citosólica de la membrana del retículo endoplasmático. 6. Proteínas de unión Los resultados obtenidos del ensayo de dobles híbridos no indican que CPT1C interaccione con una sola proteína de unión. Del ensayo se obtuvieron proteínas implicadas en diferentes funciones: degradación de proteínas, tráfico de membranas, estructura celular, vías de transducción de la señal y metabolismo

AMPAR interacting protein CPT1C enhances surface expression of GluA1-containing receptors

AMPARs mediate the vast majority of fast excitatory synaptic transmission in the brain and their biophysical and trafficking properties depend on their subunit composition and on several posttranscriptional and posttranslational modifications. Additionally, in the brain AMPARs associate with auxiliary subunits, which modify the properties of the receptors. Despite the abundance of AMPAR partners, recent proteomic studies have revealed even more interacting proteins that could potentially be involved in AMPAR regulation. Amongst these, carnitine palmitoyltransferase 1C (CPT1C) has been demonstrated to form an integral part of native AMPAR complexes in brain tissue extracts. Thus, we aimed to investigate whether CPT1C might be able to modulate AMPAR function. Firstly, we confirmed that CPT1C is an interacting protein of AMPARs in heterologous expression systems. Secondly, CPT1C enhanced whole-cell currents of GluA1 homomeric and GluA1/GluA2 heteromeric receptors. However, CPT1C does not alter the biophysical properties of AMPARs and co-localization experiments revealed that AMPARs and CPT1C are not associated at the plasma membrane despite a strong level of co-localization at the intracellular level. We established that increased surface GluA1 receptor number was responsible for the enhanced AMPAR mediated currents in the presence of CPT1C. Additionally, we revealed that the palmitoylable residue C585 of GluA1 is important in the enhancement of AMPAR trafficking to the cell surface by CPT1C. Nevertheless, despite its potential as a depalmitoylating enzyme, CPT1C does not affect the palmitoylation state of GluA1. To sum up, this work suggests that CPT1C plays a role as a novel regulator of AMPAR surface expression in neurons. Fine modulation of AMPAR membrane trafficking is fundamental in normal synaptic activity and in plasticity processes and CPT1C is therefore a putative candidate to regulate neuronal AMPAR physiology

Molecular mechanisms contributing to TARP regulation of channel conductance and polyamine block of calcium-permeable AMPA receptors.

Many properties of fast synaptic transmission in the brain are influenced by transmembrane AMPAR regulatory proteins (TARPs) that modulate the pharmacology and gating of AMPA-type glutamate receptors (AMPARs). Although much is known about TARP influence on AMPAR pharmacology and kinetics through their modulation of the extracellular ligand-binding domain (LBD), less is known about their regulation of the ion channel region. TARP-induced modifications in AMPAR channel behavior include increased single-channel conductance and weakened block of calcium-permeable AMPARs (CP-AMPARs) by endogenous intracellular polyamines. To investigate how TARPs modify ion flux and channel block, we examined the action of γ-2 (stargazin) on GluA1 and GluA4 CP-AMPARs. First, we compared the permeation of organic cations of different sizes. We found that γ-2 increased the permeability of several cations but not the estimated AMPAR pore size, suggesting that TARP-induced relief of polyamine block does not reflect altered pore diameter. Second, to determine whether residues in the TARP intracellular C-tail regulate polyamine block and channel conductance, we examined various γ-2 C-tail mutants. We identified the membrane proximal region of the C terminus as crucial for full TARP-attenuation of polyamine block, whereas complete deletion of the C-tail markedly enhanced the TARP-induced increase in channel conductance; thus, the TARP C-tail influences ion permeation. Third, we identified a site in the pore-lining region of the AMPAR, close to its Q/R site, that is crucial in determining the TARP-induced changes in single-channel conductance. This conserved residue represents a site of TARP action, independent of the AMPAR LBD

Mechanisms of CPT1C-Dependent AMPAR trafficking enhancement

In neurons, AMPA receptor (AMPAR) function depends essentially on their constituent components:the ion channel forming subunits and ion channel associated proteins. On the other hand, AMPAR trafficking is tightly regulated by a vast number of intracellular neuronal proteins that bind to AMPAR subunits. It has been recently shown that the interaction between the GluA1 subunit of AMPARs and carnitine palmitoyltransferase 1C (CPT1C), a novel protein partner of AMPARs, is important in modulating surface expression of these ionotropic glutamate receptors. Indeed, synaptic transmission in CPT1C knockout (KO) mice is diminished supporting a positive trafficking role for that protein. However, the molecular mechanisms of such modulation remain unknown although a putative role of CPT1C in depalmitoylating GluA1 has been hypothesized. Here, we explore that possibility and show that CPT1C effect on AMPARs is likely due to changes in the palmitoylation state of GluA1. Based on in silico analysis, Ser 252, His 470 and Asp 474 are predicted to be the catalytic triad responsible for CPT1C palmitoyl thioesterase (PTE) activity. When these residues are mutated or when PTE activity is inhibited, the CPT1C effect on AMPAR trafficking is abolished, validating the CPT1C catalytic triad as being responsible for PTE activity on AMPAR. Moreover, the histidine residue (His 470) of CPT1C is crucial for the increase in GluA1 surface expression in neurons and the H470A mutation impairs the depalmitoylating catalytic activity of CPT1C. Finally, we show that CPT1C effect seems to be specific for this CPT1 isoform and it takes place solely at endoplasmic reticulum (ER). This work adds another facet to the impressive degree of molecular mechanisms regulating AMPAR physiology

Mechanisms of CPT1C-Dependent AMPAR trafficking enhancement

In neurons, AMPA receptor (AMPAR) function depends essentially on their constituent components:the ion channel forming subunits and ion channel associated proteins. On the other hand, AMPAR trafficking is tightly regulated by a vast number of intracellular neuronal proteins that bind to AMPAR subunits. It has been recently shown that the interaction between the GluA1 subunit of AMPARs and carnitine palmitoyltransferase 1C (CPT1C), a novel protein partner of AMPARs, is important in modulating surface expression of these ionotropic glutamate receptors. Indeed, synaptic transmission in CPT1C knockout (KO) mice is diminished supporting a positive trafficking role for that protein. However, the molecular mechanisms of such modulation remain unknown although a putative role of CPT1C in depalmitoylating GluA1 has been hypothesized. Here, we explore that possibility and show that CPT1C effect on AMPARs is likely due to changes in the palmitoylation state of GluA1. Based on in silico analysis, Ser 252, His 470 and Asp 474 are predicted to be the catalytic triad responsible for CPT1C palmitoyl thioesterase (PTE) activity. When these residues are mutated or when PTE activity is inhibited, the CPT1C effect on AMPAR trafficking is abolished, validating the CPT1C catalytic triad as being responsible for PTE activity on AMPAR. Moreover, the histidine residue (His 470) of CPT1C is crucial for the increase in GluA1 surface expression in neurons and the H470A mutation impairs the depalmitoylating catalytic activity of CPT1C. Finally, we show that CPT1C effect seems to be specific for this CPT1 isoform and it takes place solely at endoplasmic reticulum (ER). This work adds another facet to the impressive degree of molecular mechanisms regulating AMPAR physiology