Sequence determinants of the Caenhorhabditis elegans dopamine transporter dictating in vivo axonal export and synaptic localization

Abstract

The monoamine neurotransmitter dopamine (DA) acts across phylogeny to modulate both simple and complex behaviors. The presynaptic DA transporter (DAT) is a major determinant of DA signaling capacity in ensuring efficient extracellular DA clearance. In humans, DAT is also a major target for prescribed and abused psychostimulants. Multiple structural determinants of DAT function and regulation have been defined, though largely these findings have arisen from heterologous expression or ex vivo cell culture studies. Loss of function mutations in the gene encoding the Caenhorhabditis elegans DAT (dat-1) produces rapid immobility when animals are placed in water, a phenotype termed Swimming-induced paralysis (Swip). The ability of a DA neuron-expressed, GFP-tagged DAT-1 fusion protein (GFP::DAT-1) to localize to synapses and rescue Swip in these animals provides a facile approach to define sequences supporting DAT somatic export and function in vivo. In prior studies, we found that truncation of the last 25 amino acids of the DAT-1 C-terminus (Δ25) precludes Swip rescue, supported by a deficit in GFP::DAT-1 synaptic localization. Here, we further defined the elements within Δ25 required for DAT-1 export and function in vivo. We identified two conserved motifs (584KW585 and 591PYRKR595) where mutation results in a failure of GFP::DAT-1 to be efficiently exported to synapses and restore DAT-1 function. The 584KW585 motif conforms to a sequence proposed to support SEC24 binding, ER export from the endoplasmic reticulum (ER), and surface expression of mammalian DAT proteins, whereas the 591PYRKR595 sequence conforms to a 3R motif identified as a SEC24 binding site in vertebrate G-protein coupled receptors. Consistent with a potential role of SEC24 orthologs in DAT-1 export, we demonstrated DA neuron-specific expression of a sec-24.2 transcriptional reporter. Mutations of the orthologous C-terminal sequences in human DAT (hDAT) significantly reduced transporter surface expression and DA uptake, despite normal hDAT protein expression. Although, hDAT mutants retained SEC24 interactions, as defined in co-immunoprecipitation studies. However, these mutations disrupted the ability of SEC24D to enhance hDAT surface expression. Our studies document an essential role of conserved DAT C-terminal sequences in transporter somatic export and synaptic localization in vivo, that add further support for important roles for SEC24 family members in efficient transporter trafficking.

Pharmacochaperoning in a Drosophila model system rescues human dopamine transporter variants associated with infantile/juvenile parkinsonism

“…In fact, we recently identified a compound with pharmacochaperoning activity by analyzing a series of partial substrates ( 49 ). Second, the current study shows that D. melanogaster can be used as a model system to test candidate pharmacochaperones within a reasonable time frame; Caenorhabditis elegans is also being explored as an alternative model organism ( 50 , 51 ). Third, an additional class of compounds can be envisaged by analogy with the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7), where the corrector lumacaftor remedies the folding deficiency of some mutants ( 52 ) and the potentiator ivacaftor potentiates the action of cAMP on channel gating and thus enhances their Cl − conductance ( 53 ).…”

Dynamic control of the dopamine transporter in neurotransmission and homeostasis

“…Supporting the role of SEC24-mediated ER export in DAT axonal targeting, siRNA knockdown against SEC24D in heterologous cell lines slows down dopamine reuptake 106 . In C. elegans , deletion or mutagenesis of a conserved sequence 591 PYRKR 595 in the DAT-1 C′-terminus leads to somatic retention 107 . The PYRKR sequence contains a 3 R motif that mediates the interaction between SEC24 and α2β-adrenergic receptor 107 , 108 , suggesting the failure of DAT-1 synaptic localization might be due to reduced interaction with SEC24-1.…”

“…In C. elegans , deletion or mutagenesis of a conserved sequence 591 PYRKR 595 in the DAT-1 C′-terminus leads to somatic retention 107 . The PYRKR sequence contains a 3 R motif that mediates the interaction between SEC24 and α2β-adrenergic receptor 107 , 108 , suggesting the failure of DAT-1 synaptic localization might be due to reduced interaction with SEC24-1. In HEK293 cells, mutation of the corresponding sequence in DAT does not affect its interaction with SEC24D, nor significantly reduces DAT surface expression, but does disrupt the ability of SEC24D to enhance DAT surface expression 107 .…”

Dopamine-dependent, swimming-induced paralysis arises as a consequence of loss of function mutations in the RUNX transcription factor RNT-1

“…One possibility for the incomplete ability of GFP::DAT1 to rescue the Swip of vt34 animals is an impact of the vt34 mutation on transporter trafficking, limiting export of the fusion protein to synapses. Indeed, our prior imaging studies [20, 42] demonstrated perturbed synaptic trafficking of C-terminal truncations of GFP:DAT that also fail to rescue the Swip and 6-OHDA insensitivity of dat-1 mutants. As previously described, quantitation of the distribution of GFP:DAT-1 in N2 head DA neurons demonstrates that expression of GFP::DAT-1 is highest in synapses, followed by the cell soma and then in dendrites (Fig 2C and 2D).…”