gradient, activity-induced changes in membrane polarity can dramatically influence transport rates. In addition, the transporter may contribute to dopamine release when the neuron depolarizes. DAT–Cav coupling Preliminary evidence suggests that the dopamine transporter couples to L-type voltage-gated calcium channels (particularly Cav1.2 and Cav1.3), which are expressed in virtually all dopamine neurons. As a result of DAT–Cav coupling, DAT substrates that produce depolarizing currents through the transporter are able to open calcium channels that are coupled to the transporter, resulting in a calcium influx in dopamine neurons. This calcium influx is believed to induce CAMKII-mediated phosphorylation of the dopamine transporter as a downstream effect; since DAT phosphorylation by CAMKII results in dopamine efflux in vivo, activation of transporter-coupled calcium channels is a potential mechanism by which certain drugs (e.g., amphetamine) trigger neurotransmitter release. Protein structure The initial determination of the membrane topology of DAT was based upon hydrophobic sequence analysis and sequence similarities with the GABA transporter. These methods predicted twelve transmembrane domains (TMD) with a large extracellular loop between the third and fourth TMDs. Further characterization of this protein used proteases, which digest proteins into smaller fragments, and glycosylation, which occurs only on extracellular loops, and largely verified the initial predictions of membrane topology. The exact structure of the Drosophila melanogaster dopamine transporter (dDAT) was elucidated in 2013 by X-ray crystallography. Location and distribution Regional distribution of DAT has been found in areas of the brain with established dopaminergic circuitry, including the nigrostriatal, mesolimbic, and mesocortical pathways. The nuclei that make up these pathways have distinct patterns of expression. Gene expression patterns in the adult mouse show high expression in the substantia nigra pars compacta. DAT in the mesocortical pathway, labeled with radioactive antibodies, was found to be enriched in dendrites and cell bodies of neurons in the substantia nigra pars compacta and ventral tegmental area. This pattern makes sense for a protein that regulates dopamine levels in the synapse. Staining in the striatum and nucleus accumbens of the mesolimbic pathway was dense and heterogeneous. In the striatum, DAT is localized in the plasma membrane of axon terminals. Double immunocytochemistry demonstrated DAT colocalization with two other markers of nigrostriatal terminals, tyrosine hydroxylase and D2 dopamine receptors. The latter was thus demonstrated to be an autoreceptor on cells that release dopamine. TAAR1 is a presynaptic intracellular receptor that is also colocalized with DAT and which has the opposite effect of the