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.
Highlights
Dopamine transporter (DAT) and ␣-synuclein interact, but the mode and mechanism of ␣-synuclein regulation of dopamine transporter (DAT) activity are less known
We found that introducing ␣-synuclein intracellularly induces a Naϩ-independent but ClϪ-sensitive inward current in immortalized DA neurons and DAT-expressing cells, which is eliminated by the DAT antagonist GBR12935 and is absent when the cell is dialyzed with heat-inactivated ␣-synuclein
Regulation of DAT activity plays a key role in extracellular dopamine availability
Summary
Dopamine transporter (DAT) and ␣-synuclein interact, but the mode and mechanism of ␣-synuclein regulation of DAT activity are less known. Taken together our findings are consistent with the interpretation that DAT/␣-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/ ␣-synuclein interaction can modulate dopamine transmission and neuronal function. Lee et al have shown that ␣-synuclein overexpression results in DAT clustering at the plasma membrane and increased DAT function [19], Wersinger and Sidhu [27, 28] have reported a decreased cell surface expression of DAT and inhibition of DAT activity when ␣-synuclein is overexpressed To address these conflicting findings and further explore the functional consequences of DAT/␣-synuclein interaction at the cell surface, we utilized single cell dual pipette whole cell patch clamp recordings to measure DAT-mediated current before and after dialysis of recombinant ␣-synuclein into immortalized DA neurons expressing dopamine transporter. Our finding suggests that overexpression of ␣-synuclein decreases DAT-mediated substrate uptake without decreasing the overall number of transporters at the membrane
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