Importance of cholesterol in dopamine transporter function

Abstract

The conformation and function of the dopamine transporter (DAT) can be affected by manipulating membrane cholesterol, yet there is no agreement as to the impact of cholesterol on the activity of lipid-raft localized DATs compared to non-raft DATs. Given the paucity of information regarding the impact of cholesterol on substrate efflux by the DAT, this study explores its influence on the kinetics of DAT-mediated DA efflux induced by dextroamphetamine, as measured by rotating disk electrode voltammetry (RDEV). Treatment with methyl-β-cyclodextrin (mβCD), which effectively depletes total membrane cholesterol- uniformly affecting cholesterol-DAT interactions in both raft and non-raft membrane domains- reduced both DA uptake and efflux rate. In contrast, disruption of raft localized DAT by cholesterol chelation with nystatin had no effect, arguing against a vital role for raft-localized DAT in substrate uptake or efflux. Supra-normal repletion of cholesterol depleted cells with the analogue desmosterol, a non-raft promoting sterol, was as effective as cholesterol itself in restoring transport rates. Further studies with Zn2+ and the conformationally-biased W84L DAT mutant supported the idea that cholesterol is important for maintaining the outward-facing DAT with normal rates of conformational interconversions. Collectively, these results point to a role for direct cholesterol-DAT interactions in regulating DAT function.

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…The role PKC plays in the interactions between DAT and flotillin-1/membrane rafts still remains unclear; however. Cremona et al (2011) also reported that flotillin-1 is required for AMPH-triggered, DAT-dependent DA efflux, a process others have reported to depend on DAT localization to cholesterol-rich raft domains (Jones et al, 2012). Together, these studies point to the existence of PKCdependent regulation of DAT protein associations as critical to the response of the transporter to display altered function and/or trafficking.…”

Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant

“…However, it is unclear whether antibody-bound DAT traffics similar to native DAT, as we investigate here. Multiple studies also demonstrate that DAT partitions into cholesterol-rich membrane microdomains (36,(39)(40)(41)(42)(43)(44) and that the membrane raft protein flotillin-1 is required for PKC-and AMPH-mediated DAT internalization (42), consistent with a clathrin-independent endocytic mechanism. However, a separate study reported that flotillin-1 contributes to DAT membrane mobility rather than PKC-stimulated DAT internalization (45).…”

Psychostimulants, Brain Membrane Lipids and Dopamine Transmission

“…Moreover, cholesterol depletion with MβCD significantly reduced the ability of DAT to take up dopamine without altering DAT surface expression in LLC-PK1, HEK293 and N2A cells [40,41,44]. Furthermore, cholesterol depletion with MβCD attenuates the ability of DAT to reverse transport dopamine in HEK293 and N2A cells [41,44]. Accordingly, knockdown of DAT-associated flottilin-1 proteins results in attenuation of amphetamine efflux in primary mesencephalic dopaminergic neurons, further demonstrating the importance of lipid raft localization in DAT function.…”

“…For instance, exogenously increasing membrane cholesterol level in striatal synaptosomes and neuronal-like MN9D cells increases the binding B max values for cocaine analogs but had no effect of DAT surface level [51]. Moreover, cholesterol depletion with MβCD significantly reduced the ability of DAT to take up dopamine without altering DAT surface expression in LLC-PK1, HEK293 and N2A cells [40,41,44]. Furthermore, cholesterol depletion with MβCD attenuates the ability of DAT to reverse transport dopamine in HEK293 and N2A cells [41,44].…”

“…Both raft-and non-raft-associated DAT can undergo constitutive internalization in neuroblastoma SK-N-MC cells [43]. Disruption of lipid rafts with MβCD or filipin in LLC-PK 1 cells does not alter the lateral movement of DAT between rafts and non-rafts [40]; however, treatment with MβCD (5 mM) or nystain (25 μg/ml, a cholesterol chelator in rafts) in HEK293 cells reduces DAT association with lipid raft microdomain [44]. There are conflicting data on the involvement of cholesterol in DAT internalization mediated by protein kinase C (PKC), a key mechanism of DAT internalization.…”