Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant

Abstract

The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in attentiondeficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKCsensitive negative endocytic mechanism, or "endocytic brake," controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation.is a modulatory neurotransmitter critical for locomotion and reward (1), and dopaminergic (DAergic) dysregulation is linked to multiple neuropsychiatric disorders, including Parkinson's disease, schizophrenia, attention-deficit/hyperactivity disorder (ADHD), and autism spectrum disorder (ASD) (2, 3). Presynaptic recapture, facilitated by the high-affinity DA transporter (DAT), spatially and temporally restricts extracellular DA availability (4-6). Addictive psychostimulants that target DAT and its monoamine transporter homologs for 5HT (SERT) and NE (NET) are either competitive ligands, such as cocaine, or competitive substrates, such as amphetamine (7). Although these drugs interact with DAT, SERT, and NET with equimolar affinity, their binding to DAT is requisite for reward (8, 9). Transporter inhibitors with differential DAT, SERT, and NET specificity are widely used to treat neuropsychiatric disorders (10, 11). However, their therapeutic efficacy differs significantly among patients, consistent with the model that monoamines may differentially contribute to the pathogenesis of these disorders (10, 12). Thus, regulatory mechanisms specific to DAT, SERT, or NET may provide a novel route to develop transporter-specific therapeutics.DAT plasma membrane expression is req...

Scaffold Repurposing of Nucleosides (Adenosine Receptor Agonists): Enhanced Activity at the Human Dopamine and Norepinephrine Sodium Symporters

“…Chemical shifts are expressed in δ values (ppm) with tetramethylsilane (δ 0.00) for CDCl 3 and water (δ 3.30) for CD 3 OD. NMR spectra were collected with a Bruker AV spectrometer equipped with a z-gradient [ 1 H, 13 C, 15 N]-cryoprobe. TLC analysis was carried out on glass sheets precoated with silica gel F254 (0.2 mm) from Sigma-Aldrich.…”

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…A recent report by Wu et al (2015) found that the increased rate of constitutive endocytosis of the Cys615 variant could be rescued by overexpression of a constitutively active version of the tyrosine kinase Ack1, which functions downstream of the Rho-family GTPase Cdc42. The actions of this kinase appear to interact with PKC regulation of DAT because Ack1 or Cdc42 inhibition increased clathrin-dependent DAT internalization and reduced DAT surface levels and activity in a manner that was nonadditive with PMA.…”

Extracellular α‐synuclein enters dopaminergic cells by modulating flotillin‐1–assisted dopamine transporter endocytosis

“…Because DAT functions in the neuronal plasma membrane, the regulation of subcellular targeting and the surface levels of DAT determine the overall activity of dopamine systems (39). The regulatory effect of PKC activation is among the most thoroughly studied mechanism of DAT trafficking (40)(41)(42)(43)(44), and importantly, FLOT1 is involved in PKC-triggered endocytosis and membrane microdomain DAT localization (20). On the basis of the predilection site of FLOT1 expression in the mammalian brain (22), FLOT1 likely coexists with DAT in dopaminergic neurons in situ.…”