Dopamine Transporter Activation Reduces Kv2.1 Activation Potential and Cluster Size

Abstract

Functional interactions between transmembrane proteins fine‐tune neuronal excitability in response to changing homeostatic demands. Dysregulation of neuronal excitability is thought to impose a significant energetic challenge on neurons, and it is implicated as a contributing factor in the demise of substantia nigra (SNc) dopamine neurons in Parkinson's Disease (PD). The objective of this study is to examine whether or not a novel interaction discovered by our lab between the dopamine transporter (DAT) and Kv2.1 has the potential to modulate the excitability (and thusly vulnerability) of SNc dopamine neurons. This is of particular interest because DAT can be targeted by numerous FDA‐approved compounds (bupropion, amphetamine, methylphenidate), eliminating a significant hurdle should the interaction reveal neuroprotective potential. DAT efficiently reuptakes dopamine along with co‐substrates Na+ and Cl− to recycle the released monoamine. This process triggers multiple concurrent events such as the induction of an inward depolarizing current as well as an increase in intracellular Ca2+, both of which impact neuronal activity. In this study, we have identified a novel functional interaction between DAT and the delayed‐rectifier K+ channel Kv2.1. Kv2.1 repolarizes dopamine neurons and excitotoxic stimuli elicit a negative shift in its activation potential such that neuronal excitability is decreased. This shift in activation voltage is coupled to a loss of channel phosphorylation as well as a dispersal of membrane‐bound Kv2.1 clusters. Examining DAT and Kv2.1 distribution in SNc and ventral tegmental area (VTA) dopamine neurons revealed increased colocalization of the two proteins in the axon hillock compared to the soma of SNc DA neurons, but there was no difference in sub‐cellular colocalization in the less vulnerable VTA dopamine neurons, suggesting a heightened modulatory potential of this interaction in the SNc. Functionally, we found that DAT activation induces a negative shift in Kv2.1's activation potential (VEH = 5.61 ± 2.8 mV; METH = 2.675 ± 2.6 mV; n=8, p=0.0292) as well as the canonical decrease in Kv2.1 cluster size (VEH = 0.2871 ± 0.05 μm3, n = 2541 clusters; AMPH = 0.1418 ± 0.03 μm3, n = 1486 clusters; Cyto‐D = 0.1123 ± 0.04 μm3, n = 419 clusters), suggesting that DAT can be targeted to modulate excitability through its downstream effects on Kv2.1. Additionally, we found DAT activation weakens the interaction between the two proteins measured via FRET microscopy (VEH = 12.48 ± 2.48 % n=10; AMPH = 6.55 ± 1.76% n=14; p=.0484), further suggesting that the axon hillock of SNc dopamine neurons represents a uniquely promising site to target this interaction. Ongoing experiments utilizing high‐speed in vitro imaging coupled with single neuron recordings and ex vivo two‐photon microscopy aim to identify the functional significance of the DAT/Kv2.1 interaction in the axon hillock of SNc dopamine neurons.Support or Funding InformationNINDS Interdisciplinary T32 in Movement Disorders & Neurorestoration (T32‐NS082168), R01DA026947‐08S1/NIDA (HK), 1S10OD020026‐01/NIH (HK)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.