title: α-Synuclein stimulates a dopamine transporter-dependent chloride current and modulates the activity of the transporter.
abstract: Dysregulation of dopamine (DA) homeostasis is implicated in neurodegenerative diseases, drug addiction, and neuropsychiatric disorders. The neuronal plasma membrane dopamine transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. α-Synuclein is a 140-amino acid protein that forms a stable complex with DAT and is linked to the pathogenesis of neurodegenerative disease. To elucidate the potential functional consequences of DAT/α-synuclein interaction, we explored α-synuclein modulation of DAT activity in midbrain dopaminergic neurons obtained from TH::RFP mice, immortalized DA neurons, and a heterologous system expressing DAT. We used dual pipette whole cell patch clamp recording to measure the DAT-mediated current before and after dialysis of recombinant α-synuclein into immortalized DA neurons. Our data suggest that intracellular α-synuclein induces a Na+ independent but Cl--sensitive inward current in DAT-expressing cells. This current is blocked by DAT blocker GBR12935 and is absent when heat-inactivated α-synuclein is dialyzed into these cells. The functional consequence of this interaction on DAT activity was further examined with real-time monitoring of transport function using a fluorescent substrate of DAT, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+). Overexpression of α-synuclein in DAT-positive immortalized DA neurons and CHO cells expressing DAT decreased the magnitude and rate of DAT-mediated substrate uptake without a decrease in the initial binding of the substrate at the plasma membrane. Taken together our findings are consistent with the interpretation that DAT/α-synuclein interaction at the cell surface results in a DAT-dependent, Na+-insensitive, Cl-sensitive inward current with a decrease in substrate uptake, suggesting that DAT/α-synuclein interaction can modulate dopamine transmission and thus neuronal function.
authors: Department of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208.
authors: Jarod
authors: J
authors: Swant
authors: Department of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208.
authors: J Shawn
authors: JS
authors: Goodwin
authors: Department of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208.
authors: Ashley
authors: A
authors: North
authors: James Madison University, Harrisonburg, Virginia 22807.
authors: Ahmad Abdul
authors: AA
authors: Ali
authors: Department of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208.
authors: Joyonna
authors: J
authors: Gamble-George
authors: Department of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208.
authors: Sanika
authors: S
authors: Chirwa
authors: Department of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee 37208. Electronic address: hkhoshbouei@mmc.edu.
authors: Habibeh
authors: H
authors: Khoshbouei
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D051379:Mice; D019636:Neurodegenerative Diseases / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051844:alpha-Synuclein / Q000378:metabolism*
chemical_list: D002712:Chlorides; D050483:Dopamine Plasma Membrane Transport Proteins; D005456:Fluorescent Dyes; D051844:alpha-Synuclein; D004298:Dopamine