Cholesterol binding to a conserved site modulates the conformation, pharmacology, and transport kinetics of the human serotonin transporter

Abstract

The serotonin transporter (SERT) is important for reuptake of the neurotransmitter serotonin from the synaptic cleft and is also the target of most antidepressants. It has previously been shown that cholesterol in the membrane bilayer affects the conformation of the serotonin transporter. Although recent crystal structures have identified several potential cholesterol binding sites it is unclear whether any of these potential cholesterol sites are occupieed by cholesterol and functionally relevant. In the present study we focus on the conserved Cholesterol Site 1 (CHOL1) located in a hydrophobic groove between TM1a, TM5 and TM7. By molecular dynamics simulations we demonstrate a strong binding of cholesterol to CHOL1 in a membrane bilayer environment. In biochemical experiments we find that cholesterol depletion induces a more inward-facing conformation favoring substrate analog binding. Consistent with this, we find that mutations in CHOL1 with a negative impact on cholesterol binding induce a more inward-facing conformation and vice versa mutations with a positive impact on cholesterol binding induce a more outward-facing conformation. This shift in transporter conformation dictated by the ability to bind cholesterol in CHOL1 affects the apparent substrate affinity, maximum transport velocity and turn-over rates. Taken together we show that occupation of CHOL1 by cholesterol is of major importance in the transporter conformational equilibrium which in turn dictates ligand potency and serotonin transport activity. Based on our findings, we propose a mechanistic model that incorporates the role of cholesterol binding to CHOL1 in the function of SERT.

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

“…Furthermore, the increased stability of TM5 in the presence of CHOL, the unbinding of CHOL from site 2 and the location of site 1 at the TM1a/TM5 interface, suggests that CHOL bound to site 1 stabilizes an outward-facing conformation of hDAT and thereby prevents the transition of the transporter to an inward-facing conformation. We confirmed this hypothesis in a recent study, where we provide biochemical data to show that CHOL binding to site 1 in the serotonin transporter is key to the conformational changes necessary for transport and ligand binding [ 64 ].…”

Characterizing the lipid-protein interface of the human serotonin transporter by crosslinking mass spectrometry

“…A similar overlap between our experimental results and computational simulations of monoamine transporters (SERT, as well as dopamine and norepinephrine transporters) was observed by Zeppelin et al (50), where it was speculated that cholesterol binding to the N-terminal domain/TM1a and TM5 (adjacent to the TM4TM5 loop) inhibits their necessary movement required for out-to-inward reverse transport. The interaction of cholesterol with TM1a and TM5 was subsequently confirmed by mutagenesis studies (24). The identification of crosslinking sites consistent with predicted and determined cholesterol-specific binding sites suggest that these studies are not strictly stochastic, and that even at low cholesterol availability used in our studies, CX-MS studies are sensitive enough to identify low-abundant binding events.…”

“…Protein structure and/or oligomeric state can change as a function of lipid composition (23). For example, cardiolipin induces protein dimerization of LeuT (46), a paradigmatic monoamine transporter, and SERT structure and function is modulated by cholesterol (24). Thus, it is essential that complementary methodologies be developed to examine membrane protein structure in a lipid environment.…”

Toward a Systematic Structural and Functional Annotation of Solute Carriers Transporters—Example of the SLC6 and SLC7 Families

“…cholesterol-modulated conformational change of the transporter relates to the kinetics variations of the transported substrates (Laursen et al, 2018). Specifically, the authors show that cholesterol binding in site 1 stabilizes the outward open conformation and increases serotonin uptake, while cholesterol depletion shifts the conformational equilibrium toward a more inward open state, and thus prevents serotonin uptake.…”