Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2 autoreceptor activity

The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show, by application of several super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the DA transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and DA D2 autoreceptor activation in a manner dependent on Ca2+ influx via N-type voltage-gated Ca2+ channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2 autoreceptor, syntaxin-1, and clathrin nanodomains. The data reveal a mechanism for rapid alterations of nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter. [Display omitted] •The dopamine transporter (DAT) is localized to PIP2-enriched nanodomains•Nanoclustered DAT is inward facing, and unclustered DAT is outward facing•Excitatory input disperses DAT from nanodomains in a Ca2+-dependent manner•In contrast, dopamine D2 receptor activity promotes DAT nanodomain localization Lycas et al. use super-resolution microscopy to investigate nanoscale regulation of the dopamine transporter (DAT) in the membrane of presynaptic release sites. The results show conformation-dependent localization of DAT to PIP2-enriched nanodomains and that this localization is inversely regulated by excitatory input and dopamine D2 autoreceptor activity in a Ca2+-dependent manner.