Clustered Kv2.1 decreases dopamine transporter activity and internalization

Lebowitz, Joseph J.; Pino, José A.; Mackie, Phillip; Lin, Min; Hurst, Cheyenne; Divita, Keeley; Collins, Anthony T.; Koutzoumis, Dimitri N.; Torres, Gonzalo E.; Khoshbouei, Habibeh

doi:10.1074/jbc.ra119.007441

Cited by 15 publications

(15 citation statements)

References 56 publications

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“…Rahbek -Clemmensen et al 2017;Thal et al 2019;Lebowitz et al 2019). Moreover, we previously reported that, 1) PKC activation preferentially depletes DAT from cholera toxin-positive (Ctx+) microdomains (Gabriel et al 2013), and 2) there is significantly more DAT-Rit2 co-localization in Ctx+ microdomains (Navaroli et al 2011).…”

Section: Discussionmentioning

confidence: 97%

“…6), AMPH-stimulated DAT internalization increased the surface DAT-Rit2 population. Surface DAT is distributed among several membrane microdomains (38,(61)(62)(63)(64)(65)(66)(67). Moreover, we previously reported that, 1) PKC activation preferentially depletes DAT from cholera toxin-positive (Ctx+) microdomains (26), and 2) there is significantly more DAT-Rit2 colocalization in Ctx+ microdomains (38).…”

Section: Discussionmentioning

confidence: 98%

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Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

Fagan

Kearney

Sweeney

et al. 2019

Preprint

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Following its evoked release, DA signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DAT. DAT surface availability is dynamically regulated by endocytic trafficking, and direct PKC activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals, or whether there are region-and/or sexdependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKCstimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation, and that the DAT N-terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization, and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals. _______________________________________ DA neurotransmission is required for motor control, learning, memory, motivation, and reward (1,2). DAergic dysregulation is evidenced in numerous neuropsychiatric disorders, including ADHD, ASD, schizophrenia, bipolar disorder, addiction, and PD (3-8). DA signaling is tightly controlled by the presynaptic DAT, which rapidly clears synaptically released DA. DAT is also the primary target for addictive and therapeutic

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 98%

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

Fagan

Kearney

Sweeney

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

Section: Discussionmentioning

confidence: 98%

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

Fagan

Kearney

Sweeney

et al. 2020

Journal of Biological Chemistry

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Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

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“…The Kv2 channel proteins Kv2.1 and Kv2.2 are highly expressed in neuronal tissue and they colocalize with VAP proteins at ER-PM contacts in the cell bodies and axon initial segments of cultured and in vivo neurons (Kirmiz et al, 2018; Lebowitz et al, 2019). The interaction between Kv2 channels and VAPs seems to be an important determinant in the localization of these proteins in neurons with overexpression of Kv2.1 causing VAPs to redistribute to large ER-PM clusters while loss of VAPs reduce Kv2 clustering in the PM (Johnson et al, 2018; Kirmiz et al, 2018).…”

Section: Er-plasma Membrane Contacts In Neuronsmentioning

confidence: 99%

NeurodegenERation: The Central Role for ER Contacts in Neuronal Function and Axonopathy, Lessons From Hereditary Spastic Paraplegias and Related Diseases

et al. 2019

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The hereditary spastic paraplegias (HSPs) are a group of inherited neurodegenerative conditions whose characteristic feature is degeneration of the longest axons within the corticospinal tract which leads to progressive spasticity and weakness of the lower limbs. Though highly genetically heterogeneous, the majority of HSP cases are caused by mutations in genes encoding proteins that are responsible for generating and organizing the tubular endoplasmic reticulum (ER). Despite this, the role of the ER within neurons, particularly the long axons affected in HSP, is not well understood. Throughout axons, ER tubules make extensive contacts with other organelles, the cytoskeleton and the plasma membrane. At these ER contacts, protein complexes work in concert to perform specialized functions including organelle shaping, calcium homeostasis and lipid biogenesis, all of which are vital for neuronal survival and may be disrupted by HSP-causing mutations. In this article we summarize the proteins which mediate ER contacts, review the functions these contacts are known to carry out within neurons, and discuss the potential contribution of disruption of ER contacts to axonopathy in HSP.

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Clustered Kv2.1 decreases dopamine transporter activity and internalization

Cited by 15 publications

References 56 publications

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact

NeurodegenERation: The Central Role for ER Contacts in Neuronal Function and Axonopathy, Lessons From Hereditary Spastic Paraplegias and Related Diseases

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