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Insertion Of Tetracysteine Motifs Into Dopamine Transporter Extracellular Domains, Deanna M. Navaroli, Haley E. Melikian
Insertion Of Tetracysteine Motifs Into Dopamine Transporter Extracellular Domains, Deanna M. Navaroli, Haley E. Melikian
Haley Melikian
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. …
The Acid-Sensitive, Anesthetic-Activated Potassium Leak Channel, Kcnk3, Is Regulated By 14-3-3beta-Dependent, Pkc-Mediated Endocytic Trafficking, Luke Gabriel, Anatoli Lvov, Demetra Orthodoxou, Ann Rittenhouse, William Kobertz, Haley Melikian
The Acid-Sensitive, Anesthetic-Activated Potassium Leak Channel, Kcnk3, Is Regulated By 14-3-3beta-Dependent, Pkc-Mediated Endocytic Trafficking, Luke Gabriel, Anatoli Lvov, Demetra Orthodoxou, Ann Rittenhouse, William Kobertz, Haley Melikian
Haley Melikian
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 downregulated by PKC; however, the mechanisms responsible for PKC-induced KCNK3 downregulation are undefined. Here, we report that endocytic trafficking dynamically regulates KCNK3 activity. Phorbol esters and Group I 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 14-3-3beta and a novel potassium channel endocytic motif, as depleting either 14-3-3beta …
The Plasma Membrane-Associated Gtpase Rin Interacts With The Dopamine Transporter And Is Required For Protein Kinase C-Regulated Dopamine Transporter Trafficking, Deanna M. Navaroli, Zachary H. Stevens, Zeljko Uzelac, Luke Gabriel, Michael J. King, Lawrence M. Lifshitz, Harald H. Sitte, Haley E. Melikian
The Plasma Membrane-Associated Gtpase Rin Interacts With The Dopamine Transporter And Is Required For Protein Kinase C-Regulated Dopamine Transporter Trafficking, Deanna M. Navaroli, Zachary H. Stevens, Zeljko Uzelac, Luke Gabriel, Michael J. King, Lawrence M. Lifshitz, Harald H. Sitte, Haley E. Melikian
Haley Melikian
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 …
Dopamine Transporter Endocytic Trafficking In Striatal Dopaminergic Neurons: Differential Dependence On Dynamin And The Actin Cytoskeleton., Haley Melikian
Dopamine Transporter Endocytic Trafficking In Striatal Dopaminergic Neurons: Differential Dependence On Dynamin And The Actin Cytoskeleton., Haley Melikian
Haley Melikian
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 …
Nonclassical, Distinct Endocytic Signals Dictate Constitutive And Pkc-Regulated Neurotransmitter Transporter Internalization, Haley Melikian
Nonclassical, Distinct Endocytic Signals Dictate Constitutive And Pkc-Regulated Neurotransmitter Transporter Internalization, Haley Melikian
Haley Melikian
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 …