Membrane Mobility and Microdomain Association of the Dopamine Transporter Studied with Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching

Abstract

To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of approximately 3.6 x 10(-9) cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-beta2AR (beta2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the Vmax and KM values for [3H]dopamine uptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity.

Dopamine Transporter Endocytic Trafficking in Striatal Dopaminergic Neurons: Differential Dependence on Dynamin and the Actin Cytoskeleton

“…Previous studies reported that DAT partitions between lipid raft and nonraft plasma membrane microdomains (Adkins et al, 2007;Foster et al, 2008;Cremona et al, 2011;Navaroli et al, 2011), and that flotillin-1 retains DAT in lipid rafts (Cremona et al, 2011;Sorkina et al, 2013) and is required for PKC-stimulated DAT endocytosis (Cremona et al, 2011) in HEK cells. Thus, we asked whether the endocytic-willing DAT surface pool preferentially arises from either raft or nonraft loci in SK-N-MC cells.…”

“…To date, DAT trafficking studies have been performed primarily in non-neuronal cell lines or cultured midbrain neurons derived from P0-P2 rodents, which may not faithfully recapitulate endocytic mechanisms inherent in adult dopaminergic neurons. Studies comparing DAT surface dynamics in HEK versus Neuro2A cells similarly found striking differences in DAT lateral mobility between the two cell types (Adkins et al, 2007). Indeed, our current results comparing PC12 cells, SK-N-MC cells and striatal slices clearly demonstrate that DAT's dynamin dependence is highly sensitive to cellular context (Figs.1-3).Interestingly,SK-N-MCcellsidentically recapitulated striatal slice results, suggesting that SK-N-MC neuroblastoma is a reliable culture model for constitutive and PKC-stimulated DAT internalization studies.…”

Dopamine Transporter Activity Is Modulated by α-Synuclein

“…DAT activity has been shown to be regulated by the stabilization or distribution of the transporter in the cholesterol-rich membrane microdomains (40,54,55). Because ␣-synuclein has been shown to recruit the GM1 gangliosides to the plasma membrane (57), the measured increase in co-localization of DAT with the CTx-B-labeled GM1 gangliosides in Fig.…”

“…␣-Synuclein Alters Membrane Microdomain Distribution of DAT-DAT is distributed in the cholesterol-rich and cholesterol-poor membrane microdomains termed membrane rafts (40,54,55). Previous studies support the idea that membrane microdomain distribution of DAT influences its conformational state and its function (54,55).…”

“…4B). However, when a large protein interacts with a small protein, be it a stable or transient interaction, the mobility of the smaller protein is expected to decrease in both the lateral mobility and mobile fraction, although the larger protein may experience little to no change in mobility (40,41). We aimed to test this hypothesis with DAT (Ͼ75 kDa) and ␣-synuclein (14 kDa).…”