Methamphetamine (METH) is one of the most addictive and neurotoxic drugs in existence whose societal impact is on the rise. The molecular mechanisms underlying the effects of METH on the dopamine transporter (DAT), the major molecular target of several psychoactive drugs, are poorly understood. Importantly, due to structural similarities between METH and amphetamine (AMPH), METH regulation of DAT is generally inferred from studies characterizing AMPH. Therefore, the biophysical properties and underlying molecular mechanisms of METH-exposed DAT are virtually unknown. METH primarily exerts its addictive properties by producing large elevations in extracellular striatal dopamine (DA). The DAT is a neurotransmitter transporter that regulates the magnitude and duration of synaptic signaling by clearing released DA from the synapse. However, DAT also mediates DA release via reverse transport (efflux) and can operate in a channel mode, which dramatically increases DA flux. METH mediates DA efflux via DAT, and revealing the mechanisms for this efflux is critical in understanding METH addiction and neurotoxicity. The proposed studies will test the hypotheses that METH regulates extracellular DA by: stabilizing DAT channel mode activity to increase DA efflux, decreasing DA uptake, and/or modifying DAT cell surface distribution in a voltage- and phosphorylation-dependent manner, and that these coordinated events account for the highly addictive nature and neurotoxicity of METH when compared with structural congeners, like AMPH. We will test these hypotheses, all of which are supported by promising preliminary data, with the following specific aims: 1) Determine the biophysical and molecular mechanisms underlying METH-induced DA efflux relative to AMPH, 2) Test the hypothesis that METH targets a phosphorylated state of DAT to regulate DA efflux, substrate uptake, and DAT surface distribution, 3) Compare METH-induced with AMPH-induced current-to-substrate ratios 4) Measure METH-provoked DAT surface mobility as a function of DAT N-terminal phosphorylation. We will achieve these aims in midbrain dopaminergic neurons and DAT expressing oocytes using whole-cell, cell-attached, and cell-detached patch clamp with simultaneous amperometry to measure DA efflux; and use the fluorescent substrate ASP+ to monitor DAT-dependent uptake. We anticipate that our findings will identify mechanisms for novel therapeutic strategies that may prevent or reverse METH toxicity/addiction, as well as suggest unique targets for other neurological diseases whose etiology includes dysfunction of the dopaminergic system.