brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Brain Slice Biotinylation: An <em>Ex Vivo</em> Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

The dopamine transporter (DAT) removes dopamine from the extracellular milieu and is potently inhibited by number of psychoactive drugs, including cocaine, amphetamines, and methylphenidate (Ritalin). Multiple lines of evidence demonstrate that protein kinase C (PKC) down-regulates dopamine transport, primarily by redistributing DAT from the plasma membrane to endosomal compartments, although the mechanisms facilitating transporter sequestration are not defined. Here, we demonstrate that DAT constitutively internalizes and recycles in rat pheochromocytoma (PC12) cells. Temperature blockades demonstrated basal internalization and reliance on recycling to maintain DAT cell surface levels. In contrast, recycling blockade with bafilomycin A1 significantly decreased transferrin receptor (TfR) surface expression but had no effect on DAT surface levels, suggesting that DAT and TfR traffic via distinct endosomal mechanisms. Kinetic analyses reveal robust constitutive DAT cycling to and from the plasma membrane, independent of transporter expression levels. In contrast, phorbol ester-mediated PKC activation accelerated DAT endocytosis and attenuated transporter recycling in a manner sensitive to DAT expression levels. These data demonstrate constitutive DAT trafficking and that PKC-mediated DAT sequestration is achieved by a combination of accelerated internalization and reduced recycling. Additionally, the differential sensitivity to expression level exhibited by constitutive and regulated DAT trafficking suggests that these two processes are mediated by independent cellular mechanisms.

The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.

Die Dopamin-Transporter konstitutiv internalisiert und Recycling in einem Proteinkinase-C-geregelten Art und Weise stabil transfizierten PC12-Zelllinien.

Neurotransmitter-Transporter des Menschenhandels: Endozytose, Recycling und Regulierung.

Sodium- and chloride-dependent transporters in the SLC6 gene family are key regulators of extracellular neurotransmitter levels and are required for normal neurotransmission. Copious evidence supports the premise that membrane trafficking dynamically modulates transporter surface expression in response to psychostimulant exposure, receptor activation, and neuronal activity. Recent work from our group and others demonstrates that many SLC6 transporters not only traffic in response to exogenous stimuli, but also constitutively traffic, with exogenous signaling modulating intrinsic transporter trafficking kinetics. This review focuses on what is currently understood about constitutive and regulated transporter trafficking, and poses a model wherein endocytic trafficking dynamically primes transporters for multi-faceted regulatory events.

Neurotransmitter transporter trafficking: endocytosis, recycling, and regulation.

Neurotransmitter transporters are critical for synaptic neurotransmitter inactivation. Transporter inhibitors markedly increase the duration and magnitude of synaptic transmission, underscoring the importance of transporter activity in neurotransmission. Recent studies indicate that membrane trafficking dynamically governs neuronal transporter cell-surface presentation in a protein kinase C-regulated manner, suggesting that transporter trafficking profoundly affects synaptic signaling. However, the molecular architecture coupling neurotransmitter transporters to the endocytic machinery is not defined. Here, we identify nonclassical, distinct endocytic signals in the dopamine transporter (DAT) that are necessary and sufficient to drive constitutive and protein kinase C-regulated DAT internalization. The DAT internalization signal is conserved across SLC6 neurotransmitter carriers and is functional in the homologous norepinephrine transporter, suggesting that this region is likely to be the endocytic signal for all SLC6 neurotransmitter transporters. The DAT endocytic signal does not conform to classic internalization motifs, suggesting that SLC6 neurotransmitter transporters may have evolved unique endocytic mechanisms.

Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.

Nichtklassische, deutliche Signale zu diktieren konstitutive Endozytose und PKC-regulierten Neurotransmitter-Transporter Verinnerlichung.

Amphetamine (AMPH) is a potent dopamine (DA) transporter (DAT) inhibitor that markedly increases extracellular DA levels. In addition to its actions as a DAT antagonist, acute AMPH exposure induces DAT losses from the plasma membrane, implicating transporter-specific membrane trafficking in amphetamine's actions. Despite reports that AMPH modulates DAT surface expression, the trafficking mechanisms leading to this effect are currently not defined. We recently reported that DAT residues 587-596 play an integral role in constitutive and protein kinase C (PKC)-accelerated DAT internalization. In the current study, we tested whether the structural determinants required for PKC-stimulated DAT internalization are necessary for AMPH-induced DAT sequestration. Acute amphetamine exposure increased DAT endocytic rates, but DAT carboxy terminal residues 587-590, which are required for PKC-stimulated internalization, were not required for AMPH-accelerated DAT endocytosis. AMPH decreased DAT endocytic recycling, but did not modulate transferrin receptor recycling, suggesting that AMPH does not globally diminish endocytic recycling. Finally, treatment with a PKC inhibitor demonstrated that AMPH-induced DAT losses from the plasma membrane were not dependent upon PKC activity. These results suggest that the mechanisms responsible for AMPH-mediated DAT internalization are independent from those governing PKC-sensitive DAT endocytosis.

Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent.

Amphetamine (Ladeeffekt) ist ein potenter Dopamin (DA)-Transporter (DAT)-Hemmer, der extrazellul&auml;re DA Ebenen deutlich erh&ouml;ht. Zus&auml;tzlich zu seinen Aktionen als ein DAT-Antagonist induziert akute Verbraucherexposition bei Ladeeffekt DAT Verluste aus der Plasmamembran, zielbewu&szlig;t Transporter-spezifischen Membran Menschenhandel Amphetamin Aktionen. Trotz Berichte, dass Ladeeffekt DAT Oberfl&auml;che Ausdruck moduliert sind die Menschenhandel Mechanismen dazu, derzeit nicht definiert. Vor kurzem berichteten wir, dass DAT R&uuml;ckst&auml;nde 587-596 integral in eine konstitutive Rolle und Proteinkinase C PKC-beschleunigte DAT Internalisierung. In der aktuellen Studie haben wir getestet, ob die strukturellen Determinanten f&uuml;r die PKC-stimuliert DAT Internalisierung Ladeeffekt induziert DAT-Sequestrierung notwendig sind. Akuter Amphetamin Exposition erh&ouml;ht DAT endocytic Preise, aber DAT Carboxy terminal R&uuml;ckst&auml;nde 587-590, die f&uuml;r PKC-stimuliert Internalisierung erforderlich sind, sind nicht zur Ladeeffekt-beschleunigte DAT Endozytose erforderlich. Ladeeffekt verringert, DAT endocytic Verwertung, aber nicht modulieren Transferrin-Rezeptor-recycling, darauf hindeutet, dass Ladeeffekt nicht global endocytic Wiederverwertung vermindert wird. Behandlung mit einer PKC-Hemmer nachweislich schlie&szlig;lich Ladeeffekt induziert DAT Verluste von der Plasmamembran nicht PKC Aktivit&auml;t abh&auml;ngig waren. Diese Ergebnisse legen nahe, dass die Mechanismen, DAT Ladeeffekt-vermittelte Internalisierung von regierenden PKC-Sensitive DAT Endozytose unabh&auml;ngig sind.

Amphetamin-induzierten R&uuml;ckgang der Dopamin-Transporter-Oberfl&auml;che-Ausdruck sind Proteinkinase C-unabh&auml;ngig.

Dopamine (DA) reuptake terminates dopaminergic neurotransmission and is mediated by DA transporters (DATs). Acute protein kinase C (PKC) activation accelerates DAT internalization rates, thereby reducing DAT surface expression. Basal DAT endocytosis and PKC-stimulated DAT functional downregulation rely on residues within the 587-596 region, although whether PKC-induced DAT downregulation reflects transporter endocytosis mechanisms linked to those controlling basal endocytosis rates is unknown. Here, we define residues governing basal and PKC-stimulated DAT endocytosis. Alanine substituting DAT residues 587-590 1) abolished PKC stimulation of DAT endocytosis, and 2) markedly accelerated basal DAT internalization, comparable to that of wildtype DAT during PKC activation. Accelerated basal DAT internalization relied specifically on residues 588-590, which are highly conserved among SLC6 neurotransmitter transporters. Our results support a model whereby residues within the 587-590 stretch may serve as a locus for a PKC-sensitive braking mechanism that tempers basal DAT internalization rates.

Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization.

Dopamin (DA)-Reuptake beendet die dopaminergen Neurotransmission und ist von DA-Transporter (DAT) vermittelt. Akute Proteinkinase C (PKC) Aktivierung beschleunigt DAT Internalisierung Preise, wodurch DAT Oberfl&auml;che Ausdruck. Basale DAT Endozytose und PKC-stimuliert DAT funktionale Heraufregulation Vertrauen auf R&uuml;ckst&auml;nde innerhalb der 587-596-Region, obwohl ob PKC-induzierte DAT Heraufregulation Transporter Endozytose Mechanismen verbunden spiegelt diese steuernde basale Endozytose-Raten ist unbekannt. Hier definieren wir R&uuml;ckst&auml;nde f&uuml;r basale und PKC-stimuliert DAT Endozytose. Alanin ersetzen DAT R&uuml;ckst&auml;nde 587-590 1) abgeschafft PKC Stimulation der DAT Endozytose und 2) deutlich beschleunigten basale DAT Internalisierung, vergleichbar mit der Wildtyp DAT w&auml;hrend der PKC-Aktivierung. Beschleunigte basale DAT Internalisierung st&uuml;tzte sich speziell auf R&uuml;ckst&auml;nde 588-590, die stark unter SLC6 Neurotransmitter-Transporter konserviert werden. Unsere Ergebnisse unterst&uuml;tzen ein Modell, wobei R&uuml;ckst&auml;nde innerhalb der 587-590-Strecke als ein Ort f&uuml;r ein PKC-Sensitive-Bremsen-Mechanismus dienen k&ouml;nnen, die basale DAT Internalisierung Preise m&auml;&szlig;igt.

Dopamin Transporter endocytic Determinanten: Carboxy-terminal-R&uuml;ckst&auml;nde f&uuml;r basale und PKC-stimuliert Internalisierung von entscheidender Bedeutung.

The neuronal dopamine transporter (DAT) is a major determinant of extracellular dopamine (DA) levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC) activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC) to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly) dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [(3)H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.

Insertion of tetracysteine motifs into dopamine transporter extracellular domains.

Der neuronale Dopamin-Transporter (DAT) ist ein ausschlaggebender Faktor f&uuml;r extrazellul&auml;re Dopamin (DA) Ebenen und ist das prim&auml;re Ziel f&uuml;r eine Vielzahl von s&uuml;chtig und therapeutische Psychopharmaka. DAT wird akut durch Proteinkinase C (PKC) Aktivierung und Amphetamin-Exposition, reguliert beide DAT Oberfl&auml;che Ausdruck von endocytic Menschenhandel modulieren. Um verwenden sind live imaging Ans&auml;tze um DAT Endozytose, studieren Methoden notwendig, ausschlie&szlig;lich den DAT-Oberfl&auml;chen-Pool zu kennzeichnen. Die Verwendung der Membran impermeant, sulfonierte Biarsenic Farbstoffe enth&auml;lt potential wie einen solchen Ansatz und erfordert die Einf&uuml;hrung einer extrazellul&auml;ren Tetracysteine Motiv (TetraCys; CCPGCC) Farbstoff Bindung zu erleichtern. In der aktuellen Studie nutzten wir systeminterne Prolin-Glycin (Pro-Gly) Dipeptide in vorhergesagten DAT extrazellul&auml;re Dom&auml;nen zur Einf&uuml;hrung von TetraCys Motiven in DAT extrazellul&auml;re Schleifen, 2, 3 und 4 codiert. [(3) H]DA Aufnahme Studien, Oberfl&auml;che Biotinylierung und Fluoreszenz-Mikroskopie in PC12 Zellen zeigen, dass TetraCys Einf&uuml;gung in die DAT zweite extrazellul&auml;re Schleife ergibt eine funktionelle Transporter, der PKC-vermittelte Heraufregulation verwaltet. Einf&uuml;hrung des TetraCys in extrazellul&auml;ren Schleifen, 3 und 4 hat mit stark beeintr&auml;chtigte Funktion, die nicht Reifen und Verkehr an der Zelloberfl&auml;che DATs ergeben. Dies ist die erste Demonstration der erfolgreichen Einf&uuml;hrung eines Tetracysteine-Motivs in eine DAT extrazellul&auml;ren Dom&auml;ne und Versprechen f&uuml;r die Verwendung von Biarsenic Farbstoffe in live DAT imaging Studien halten kann.

Einf&uuml;gen von Tetracysteine Motiven in Dopamin Transporter extrazellul&auml;re Dom&auml;nen.

Dopaminergic signaling and plasticity are essential to numerous CNS functions and pathologies, including movement, cognition, and addiction. The amphetamine- and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT C terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (for Ras-like in neurons) (Rit2), as a protein that interacts with the DAT C-terminal endocytic signal. Yeast two-hybrid, GST pull down and FRET studies establish that DAT and Rin directly interact, and colocalization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Coimmunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis.

The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking.

Dopaminergen Signalisierung und Plastizit&auml;t sind wesentlich f&uuml;r zahlreiche CNS-Funktionen und Krankheiten, einschlie&szlig;lich Bewegung, Kognition und sucht. Der Amphetamine und Kokain-Sensitive-Dopamin (DA)-Transporter (DAT) steuert eng extrazellul&auml;ren Konzentrationen in DA und Half-Life. DAT-Funktion und Oberfl&auml;che Ausdruck sind nicht statisch, sondern werden dynamisch durch Membranen moduliert. Wir vor kurzem gezeigt, dass das DAT Carboxyl-Terminus ein PKC-Sensitive Internalisierung Signal codiert, die auch basale DAT Endozytose unterdr&uuml;ckt. Die zellul&auml;re Maschinerie, die EZB geregelten DAT Menschenhandel ist jedoch nicht klar definiert. In der hier vorgestellten Arbeit, wir identifiziert die Ras-&auml;hnlichen GTPase, Rin (f&uuml;r Ras-wie in Neuronen) (Rit2), als ein Protein, das mit dem DAT-C-terminalen endocytic Signal interagiert. Hefe-zwei-Hybrid, GST herunterziehen und FRET Studien nachweisen, dass DAT und Rin direkt interagieren und Kolokalisation Studien zeigen, dass DAT/Rin Verb&auml;nde in erster Linie in Lipid Raft Microdomains auftreten. Coimmunoprecipitations zeigen, dass PKC Aktivierung Rin Verbindung mit DAT. St&ouml;rung von Rin-Funktion mit GTPase Mutanten reguliert und ShRNA-vermittelte Rin Niederschlag zeigt, dass Rin f&uuml;r PKC-vermittelte DAT Internalisierung und funktionale Heraufregulation wichtig ist. Diese Ergebnisse belegen, dass Rin ein DAT-Interaktion-Protein, das ist f&uuml;r DAT PKC geregelten Handels erforderlich ist. Dar&uuml;ber hinaus empfiehlt diese Arbeit Rin an geregelten Endozytose beteiligt ist.

Die Plasmamembran-assoziierten GTPase Rin interagiert mit den Dopamin-Transporter und ist erforderlich f&uuml;r Protein Kinase C geregelten Dopamin-Transporter-Handel.

The acid-sensitive neuronal potassium leak channel, KCNK3, is vital for setting the resting membrane potential and is the primary target for volatile anesthetics. Recent reports demonstrate that KCNK3 activity is down-regulated by PKC; however, the mechanisms responsible for PKC-induced KCNK3 down-regulation are undefined. Here, we report that endocytic trafficking dynamically regulates KCNK3 activity. Phorbol esters and Group I metabotropic glutamate receptor (mGluR) activation acutely decreased both native and recombinant KCNK3 currents with concomitant KCNK3 surface losses in cerebellar granule neurons and cell lines. PKC-mediated KCNK3 internalization required the presence of both 14-3-3β and a novel potassium channel endocytic motif, because depleting either 14-3-3β protein levels or ablating the endocytic motif completely abrogated PKC-regulated KCNK3 trafficking. These results demonstrate that neuronal potassium leak channels are not static membrane residents but are subject to 14-3-3β-dependent regulated trafficking, providing a straightforward mechanism to modulate neuronal excitability and synaptic plasticity by Group I mGluRs.

The acid-sensitive, anesthetic-activated potassium leak channel, KCNK3, is regulated by 14-3-3β-dependent, protein kinase C (PKC)-mediated endocytic trafficking.

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.

Article	Year
The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines. The Journal of biological chemistry\| PubMed ID: 12682063	2003
Neurotransmitter transporter trafficking: endocytosis, recycling, and regulation. Pharmacology & therapeutics\| PubMed ID: 15500906	2004
Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization. Nature neuroscience\| PubMed ID: 15924135	2005
Amphetamine-induced decreases in dopamine transporter surface expression are protein kinase C-independent. Neuropharmacology\| PubMed ID: 18164041	2008
Dopamine transporter endocytic determinants: carboxy terminal residues critical for basal and PKC-stimulated internalization. Molecular and cellular neurosciences\| PubMed ID: 18638559	2008
Insertion of tetracysteine motifs into dopamine transporter extracellular domains. PloS one\| PubMed ID: 20161733	2010
The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 21957239	2011
The acid-sensitive, anesthetic-activated potassium leak channel, KCNK3, is regulated by 14-3-3β-dependent, protein kinase C (PKC)-mediated endocytic trafficking. The Journal of biological chemistry\| PubMed ID: 22846993	2012
Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 24198373	2013

Haley E. Melikian

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