Characterizing the Role of Cysteine-5 in the Human Dopamine Transporter (DAT) Through a Naïve Drosophila DAT Model
Conference Year
2024
Abstract
Dysregulation of dopamine homeostasis has been implicated in neurological and mental disorders such as addiction, ADHD, narcolepsy, Parkinsons, motor dysregulation, and schizophrenia. The dopamine (DA) transporter (DAT) plays a critical role in dopaminergic signaling via reuptake of synaptic DA. DAT function has been found to be regulated by post-translational modifications (PTMs) including palmitoylation, which can occur on cysteines in both the N and C termini and modulate DAT kinetics and plasmalemmal expression. To investigate the palmitoylation status and functional contribution of Cys 5 in hDAT in an isolated system, we introduced a Cys at the homologous position in Drosophila DAT (dDAT which cannot be palmitoylated as it lacks cysteines in its termini. dDAT and G5C mutant constructs were expressed in HEK 293-MSR and LLCPK cell lines and assessed for surface expression, DA transport, and palmitoylation using surface biotinylation, Western blots, [ 3H]DA uptake, and acyl-biotin exchange (ABE) assays. Our results show that G5C is expressed on the surface of the cell and exhibits enhanced DA transport versus dDAT. Efforts to establish differences in palmitoylation via an acyl-biotin exchange assay are underway. G5C is a promising construct to bolster current findings that palmitoylation correlates with increased kinetics of hDAT.
Primary Faculty Mentor Name
James Stafford
Graduate Student Mentors
Dr. Keith Loren Henry from UND
Status
Undergraduate
Student College
College of Agriculture and Life Sciences
Program/Major
Molecular Genetics
Primary Research Category
Life Sciences
Characterizing the Role of Cysteine-5 in the Human Dopamine Transporter (DAT) Through a Naïve Drosophila DAT Model
Dysregulation of dopamine homeostasis has been implicated in neurological and mental disorders such as addiction, ADHD, narcolepsy, Parkinsons, motor dysregulation, and schizophrenia. The dopamine (DA) transporter (DAT) plays a critical role in dopaminergic signaling via reuptake of synaptic DA. DAT function has been found to be regulated by post-translational modifications (PTMs) including palmitoylation, which can occur on cysteines in both the N and C termini and modulate DAT kinetics and plasmalemmal expression. To investigate the palmitoylation status and functional contribution of Cys 5 in hDAT in an isolated system, we introduced a Cys at the homologous position in Drosophila DAT (dDAT which cannot be palmitoylated as it lacks cysteines in its termini. dDAT and G5C mutant constructs were expressed in HEK 293-MSR and LLCPK cell lines and assessed for surface expression, DA transport, and palmitoylation using surface biotinylation, Western blots, [ 3H]DA uptake, and acyl-biotin exchange (ABE) assays. Our results show that G5C is expressed on the surface of the cell and exhibits enhanced DA transport versus dDAT. Efforts to establish differences in palmitoylation via an acyl-biotin exchange assay are underway. G5C is a promising construct to bolster current findings that palmitoylation correlates with increased kinetics of hDAT.