Biology subjects

Luethi, D.

Publications and source records attributed to Luethi, D..

3 recordsLinked to original sources

bioRxiv2022

Structural basis of organic cation transporter-3 inhibition

Organic cation transporters (OCTs) facilitate the translocation of catecholamines and xenobiotics across the plasma membrane in various tissues throughout the human body. OCT3 plays a key role in low-affinity, high-capacity uptake of monoamines in most tissues including heart, brain and liver. Its deregulation plays a role in diseases. Despite its importance, the structural basis of OCT3 function and its inhibition has remained enigmatic. Here we describe the cryo-EM structure of human OCT3 at 3.2 [A] resolution. Structures of OCT3 bound to two inhibitors, corticosterone and decynium-22, define the ligand binding pocket and reveal common features of major facilitator transporter inhibitors. In addition, we relate the functional characteristics of an extensive collection of previously uncharacterized human genetic variants to structural features, thereby providing a basis for understanding the impact of OCT3 polymorphisms.

pharmacology and toxicology↗

bioRxiv2021

Dopaminergic Ric GTPase activity impacts amphetamine sensitivity and sleep quality in a dopamine transporter-dependent manner in Drosophila melanogaster

Dopamine (DA) is required for movement, sleep, and reward, and DA signaling is tightly controlled by the presynaptic DA transporter (DAT). Therapeutic and addictive psychostimulants, including methylphenidate (Ritalin; MPH), cocaine, and amphetamine (AMPH), markedly elevate extracellular DA via their actions as competitive DAT inhibitors (MPH, cocaine) and substrates (AMPH). DAT silencing in mice and invertebrates results in hyperactivity, reduced sleep, and blunted psychostimulant responses, highlighting DATs essential role in DA-dependent behaviors. DAT surface expression is not static; rather it is dynamically regulated by endocytic trafficking. PKC-stimulated DAT endocytosis requires the neuronal GTPase, Rit2, and Rit2 silencing in mouse DA neurons impacts psychostimulant sensitivity. However, it is unknown whether or not Rit2-mediated changes in psychostimulant sensitivity are DAT-dependent. Here, we leveraged Drosophila melanogaster to test whether the Drosophila Rit2 ortholog, Ric, impacts dDAT function, trafficking, and DA-dependent behaviors. Orthologous to hDAT and Rit2, dDAT and Ric directly interact, and the constitutively active Ric mutant Q117L increased dDAT surface levels and function in cell lines and ex vivo Drosophila brains. Moreover, DAergic RicQ117L expression caused sleep fragmentation in a DAT-dependent manner but had no effect on total sleep and daily locomotor activity. Importantly, we found that Rit2 is required for AMPH-stimulated DAT internalization in mouse striatum, and that DAergic RicQ117L expression significantly increased Drosophila AMPH sensitivity in a DAT-dependent manner, suggesting a conserved impact of Ric-dependent DAT trafficking on AMPH sensitivity. These studies support that the DAT/Rit2 interaction impacts both baseline behaviors and AMPH sensitivity, potentially by regulating DAT trafficking.

neuroscience↗

bioRxiv2019

Dopamine transporter trafficking and Rit2 GTPase:In vivo impact and mechanism of action

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 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.

neuroscience↗