Cell membrane cholesterol affects serotonin transporter efflux due to altered transporter oligomerization

The human monoamine transporters (MATs) for serotonin (SERT), dopamine (DAT), and norepinephrine (NET) play a key role in neurotransmission by transporting neurotransmitters from the synaptic cleft back into the neuron. MATs are embedded in the cell membrane's lipid bilayer, encompassing cholesterol, phospholipids, and sphingolipids as main components. Membrane cholesterol association has been shown for all MATs impacting transporter conformation, substrate affinity, transport velocity, and turnover rates. In the present study, we compared the regulatory impact of cholesterol on the uptake and efflux function, binding affinity, and transporter oligomerization across all three MATs. We observed that cholesterol depletion impairs transporter-mediated uptake in human transporter-transfected HEK293 cells and reduces the binding affinity of all MATs. Electrophysiological investigations in SERT-expressing cells revealed that cholesterol alterations affect the transition of the transporter from the outward to the inward-facing conformation in the presence of substrate. Upon cholesterol depletion, FRET imaging and single molecule microscopy studies indicated altered oligomerization behavior exclusively for SERT. Interestingly, reduction of membrane cholesterol selectively increased amphetamine-induced efflux via SERT, while efflux via DAT and NET was reduced. This effect was diminished in a mutant with reduced PIP₂ binding capacity. Hence, the increased efflux at SERT due to cholesterol depletion appears to depend on the ability of PIP₂ to bind to SERT. Thus, we hypothesize that the interaction profile between cholesterol and MATs may fine-tune the transporter functionality and influence MAT-dependent disorders.