A rare, autism‐associated in‐frame deletion in the dopamine transporter exhibits profound functional deficits

Abstract

The neurotransmitter dopamine (DA) mediates fundamental behaviors such as reward, motivation, attention, and cognition. Important to dopamine neurotransmission is the dopamine transporter (DAT), a member of the solute carrier family 6 encoded by the SLC6A3 gene. DAT is a presynaptic membrane protein responsible for the reuptake and recycling of DA following calcium‐mediated vesicular release. DAT dysfunction has been associated with neuropsychiatric disorders including ADHD, schizophrenia, bipolar disorder, and autism spectrum disorders (ASD). DAT is also the target of commonly abused psychostimulants and controlled substances, namely cocaine and amphetamine (AMPH).Recently, our laboratory identified a novel, SLC6A3 variant identified in an ASD proband from whole exome sequencing. The genetic variant is an in‐frame deletion of three nucleotides resulting in a deletion of amino acid Asn336 (ΔN336). Located in the third intracellular loop, N336 is conserved from human to Drosophila, and in silico algorithms predict that ΔN336 confers a functionally damaging effect to DAT. Expression of human DAT (hDAT) ΔN336 in Chinese Hamster Ovary (CHO) cells revealed a near absence of DAT‐dependent DA uptake relative to wildtype DAT, yet surface expression was unchanged. This SLC6A3 variant is permissible to AMPH‐induced efflux, but exhibits diminished DAT‐mediated inward currents promoted by rapid application of AMPH. To understand the basis of the ΔN336 variant's effects on transporter conformational dynamics, we conducted electron paramagnetic resonance (EPR) spectroscopy analyses by introducing the correspondent deletion in the leucine transporter, LeuT. LeuT ΔV269 promotes a closed conformation of the extracellular gate and facilitates the intracellular gate to reside in a “half‐open and inward‐facing” conformation.The results of this study provide new structural and functional insights into the role of DAT structural domains controlling DA transport. In addition, they define how disruption of specific DAT structural interactions and gate conformations translate to deficits in molecular function. Most importantly, this work adds to the growing body of literature implicating altered regulation of DA homeostasis/transport as a potential biological mechanism underlying liability to ASD.Support or Funding InformationAmerican Heart Association Predoctoral Award 17PRE33410145 (A.S), NIH T32 Training Programs T32NS007491 (N.G.C.) and 4TR32GM007628‐29 (J.I.A.) and NIH grant DA038058 (A.G.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.