TY - JOUR
T1 - Cholesterol binding to a conserved site modulates conformation, pharmacology and transport kinetics of the human serotonin transporter
AU - Laursen, Louise
AU - Kristensen, Kristina Birch
AU - Periole, Xavier
AU - Overby, Malene
AU - Müller, Heidi Kaastrup
AU - Schiøtt, Birgit
AU - Sinning, Steffen
N1 - Copyright © 2018, The American Society for Biochemistry and Molecular Biology.
PY - 2018/3/9
Y1 - 2018/3/9
N2 - 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 SERT. Although recent crystal structures have identified several potential cholesterol-binding sites, it is unclear whether any of these potential cholesterol sites are occupied 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 turnover 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.
AB - 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 SERT. Although recent crystal structures have identified several potential cholesterol-binding sites, it is unclear whether any of these potential cholesterol sites are occupied 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 turnover 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.
KW - Journal Article
UR - http://www.scopus.com/inward/record.url?scp=85043700708&partnerID=8YFLogxK
U2 - 10.1074/jbc.M117.809046
DO - 10.1074/jbc.M117.809046
M3 - Journal article
C2 - 29352106
SN - 0021-9258
VL - 293
SP - 3510
EP - 3523
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -