A direct interaction of cholesterol with the dopamine transporter prevents its out-to-inward transition

Monoamine transporters (MATs) carry out neurotransmitter reuptake from the synaptic cleft, a key step in neurotransmission, which is targeted in the treatment of neurological disorders. Cholesterol (CHOL), a major component of the synaptic plasma membrane, has been shown to exhibit a modulatory effect on MATs. Recent crystal structures of the dopamine transporter (DAT) revealed the presence of two conserved CHOL-like molecules, suggesting a functional protein-CHOL direct interaction. Here, we present extensive atomistic molecular dynamics (MD) simulations of DAT in an outward-facing conformation. In the absence of bound CHOL, DAT undergoes structural changes reflecting early events of dopamine transport: transition to an inward-facing conformation. In contrast, in the presence of bound CHOL, these conformational changes are inhibited, seemingly by an immobilization of the intracellular interface of transmembrane helix 1a and 5 by CHOL. We also provide evidence, from coarse grain MD simulations that the CHOL sites observed in the DAT crystal structures are preserved in all human monoamine transporters (dopamine, serotonin and norepinephrine), suggesting that our findings might extend to the entire family.Author summary: It has been revealed that the cellular membrane is an active contributor to biological processes occurring in and across it and can regulate the function of proteins embedded within it. Cholesterol (CHOL) plays a key role in these effects. Notably, CHOL affects neurotransmission. Neurotransmission occurs at the synapse and is central to a proper functioning of the human nervous system. In particular, monoamine transporters (MATs), key players in the process of stopping signaling, are primary targets for treatment of mental disorders. Herein, we address the intriguing mechanism by which the presence of CHOL affects the function of MATs. CHOL is known to modulate the re-uptake of neurotransmitters from the synapse but the mechanism is not known. We looked at the possibility of CHOL effecting MATs by a direct interaction rather than through its effect on the biophysical properties of the embedding membrane. We present clear data indicative of conserved CHOL binding sites on all human MATs and further show how CHOL prevents conformational changes at the intracellular end of transmembrane helix 5 in the human dopamine transporter (hDAT), which we are able to confirm is associated with the outward-to-inward transition of MATs.