Herborg, F. H. | bioRxiv Science

bioRxiv2021

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 dopamine 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 by DA D2-autoreceptor activation in 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. Summarized, the data reveal a mechanism for rapid alterations in nanoscopic DAT distribution and show a striking link of this to the conformational state of the transporter.

neuroscience↗

bioRxiv2021

Disrupted PDZ-domain binding motif of the dopamine transporter uniquely alters nanoscale distribution, dopamine homeostasis and reward motivation

The dopamine transporter (DAT) is part of a presynaptic multi-protein network involving interactions with scaffold proteins via its C-terminal PDZ-domain binding sequence. In a mouse model expressing DAT with mutated PDZ binding sequence (DAT-AAA), we previously demonstrated the importance of this binding sequence for striatal expression of DAT. Here we show by application of direct Stochastic Reconstruction Microscopy (dSTORM) not only that the striatal level of transporter is reduced in DAT-AAA mice, but also that the nanoscale distribution of the transporter is altered with a higher propensity of DAT-AAA to localize to irregular nanodomains in dopaminergic terminals. In parallel, we observe mesostriatal dopamine (DA) adaptations and changes in DA-related behaviors different from those seen in other genetic DAT mouse models. DA levels in striatum are reduced to [~]45% of wild type (WT), accompanied by elevated DA turnover. Nonetheless, Fast-Scan Cyclic Voltammetry recordings on striatal slices reveal a larger amplitude and prolonged clearance rate of evoked DA release in DAT-AAA mice compared to WT mice. Autoradiography and radioligand binding show reduced DA D2 receptor levels while immunohistochemistry and autoradiography show unchanged DA D1 receptor levels. In behavioral experiments, we observe enhanced self-administration of liquid food under both a fixed-ratio (FR1) and progressive-ratio (PR) schedule of reinforcement, but a reduction compared to WT when using cocaine as reinforcer. Summarized, our data demonstrate how disruption of PDZ-domain interactions causes changes in DAT expression and its nanoscopic distribution that in turn alter DA clearance dynamics.

neuroscience↗