Impact of Cell Membrane Cholesterol Alterations on Function, Oligomerization, and Binding Affinity of Monoamine Transporters

Abstract

Objective and hypothesis Cholesterol is the major lipid constituent of mammalian cell membranes and contributes to membrane fluidity and permeability. Additionally, cholesterol is important for membrane trafficking, lipid and protein sorting, and the formation of lipid nanodomains and lipid rafts. Changes in membrane cholesterol concentration may affect the function and regulation of the neurotransmitter transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET). Therefore, alterations of cholesterol levels may potentially contribute to the development and progression of various psychiatric diseases. Methods Cell membranes of SERT, DAT, and NET-expressing human embryonic kidney (HEK) 293 cells were depleted of or replenished with cholesterol using methyl-β-cyclodextrin (MBCD) or cholesterol-saturated MBCD, respectively. In these modified cells, uptake and efflux function was first quantified by liquid scintillation counting of tritiated neurotransmitters. In a next step, the oligomerization of SERT, DAT, and NET at the plasma membrane was assessed with Förster resonance energy transfer (FRET). Additionally, the binding affinity of specific radioactively labeled inhibitors at the neurotransmitter transporters was determined by liquid scintillation counting. Results Depletion of membrane cholesterol reduced monoamine uptake for all tested transporters in a concentration-dependent manner. Cholesterol replenishment slightly increased the uptake kinetics of SERT but had no effect on DAT and NET function. However, cholesterol depletion significantly reduced the stimulant-induced dopamine and norepinephrine efflux, whereas cholesterol replenishment had no effect. In contrast, cholesterol depletion and replenishment significantly increased and decreased the stimulant-induced serotonin efflux, respectively. Furthermore, the membrane surface oligomerization of SERT, but not of DAT and NET, was significantly increased after cholesterol depletion. Cholesterol replenishment had no effect on either transporter. After cholesterol depletion, the binding affinity of specific inhibitors at SERT, DAT, and NET was significantly diminished after cholesterol depletion. Cholesterol replenishment resulted in increased binding affinity at DAT and NET, but not at SERT. Conclusion Membrane cholesterol alterations similarly affected uptake function and binding affinities at SERT, DAT, and NET. However, drug-induced serotonin efflux and SERT oligomerization was increased due to cholesterol depletion, which was not observed for DAT and NET. This indicates the possibility that SERT but not DAT and NET-mediated efflux is dependent on the oligomeric state of the transporters. Furthermore, these findings suggest different effects of cholesterol alterations in psychiatric diseases, depending on the monoaminergic systems involved.