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. 2020 Oct 23:11:578868.
doi: 10.3389/fphys.2020.578868. eCollection 2020.

Serotonin Alters the Phase Equilibrium of a Ternary Mixture of Phospholipids and Cholesterol

Oskar Engberg  1 , Anna Bochicchio  2 , Astrid F Brandner  2 , Ankur Gupta  3 , Simli Dey  3 , Rainer A Böckmann  2 , Sudipta Maiti  3 , Daniel Huster  1   3
Affiliations

Serotonin Alters the Phase Equilibrium of a Ternary Mixture of Phospholipids and Cholesterol

Oskar Engberg et al. Front Physiol. .

Abstract

Unsaturated and saturated phospholipids tend to laterally segregate, especially in the presence of cholesterol. Small molecules such as neurotransmitters, toxins, drugs etc. possibly modulate this lateral segregation. The small aromatic neurotransmitter serotonin (5-HT) has been found to bind to membranes. We studied the lipid structure and packing of a ternary membrane mixture consisting of palmitoyl-oleoyl-phosphatidylcholine, palmitoyl-sphingomyelin, and cholesterol at a molar ratio of 4/4/2 in the absence and in the presence of 5-HT, using a combination of solid-state 2H NMR, atomic force microscopy, and atomistic molecular dynamics (MD) simulations. Both NMR and MD report formation of a liquid ordered (L o ) and a liquid disordered (L d ) phase coexistence with small domains. Lipid exchange between the domains was fast such that single component 2H NMR spectra are detected over a wide temperature range. A drastic restructuring of the domains was induced when 5-HT is added to the membranes at a 9 mol% concentration relative to the lipids. 2H NMR spectra of all components of the mixture showed two prominent contributions indicative of molecules of the same kind residing both in the disordered and the ordered phase. Compared to the data in the absence of 5-HT, the lipid chain order in the disordered phase was further decreased in the presence of 5-HT. Likewise, addition of serotonin increased lipid chain order within the ordered phase. These characteristic lipid chain order changes were confirmed by MD simulations. The 5-HT-induced larger difference in lipid chain order results in more pronounced differences in the hydrophobic thickness of the individual membrane domains. The correspondingly enlarged hydrophobic mismatch between ordered and disordered phases is assumed to increase the line tension at the domain boundary, which drives the system into formation of larger size domains. These results not only demonstrate that small membrane binding molecules such as neurotransmitters have a profound impact on essential membrane properties. It also suggests a mechanism by which the interaction of small molecules with membranes can influence the function of membrane proteins and non-cognate receptors. Altered membrane properties may modify lateral sorting of membrane protein, membrane protein conformation, and thus influence their function as suspected for neurotransmitters, local anesthetics, and other small drug molecules.

Keywords: 2H NMR spectroscopy; domain size; line tension; molecular dynamics simulation; raft mixture.

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Figures

FIGURE 1
FIGURE 1
Temperature dependence of the 2H NMR spectra of a ternary POPC/PSM/Chol mixture (molar ratio 4/4/2) hydrated to 50 wt% aqueous buffer (K2PO4 20 mM, 100 mM NaCl, 0.1 mM EGTA, pH 7.4) in the absence (black spectra in the left columns) and in the presence of 9 mol% 5-HT (colored spectra in the right columns) at various temperatures as indicated. (A–D) 2H NMR spectra were recorded for each component of the mixture: (A) POPC-d31, (B) PSM-d31, (C) Chol-d6, (D) 5-HT-d4.
FIGURE 2
FIGURE 2
2H NMR spectra of deuterated POPC-d31 (A) PSM-d31 (B), Chol-d6 (C), and 5-HT-d4 (D) of the POPC/PSM/Chol mixture (molar ratio 4/4/2) mixture in the absence (left column) and in the presence of 9 mol% 5-HT (middle column) at a temperature of 30°C. Note that the 2H NMR spectrum shown in the left column of panel (D) was acquired for 5-HT in pure POPC membranes. The right column shows numerical simulations of the 2H NMR spectra.
FIGURE 3
FIGURE 3
Experimental order parameter profiles and average chain length of (A) POPC-d31 and (B) PSM-d31 in a POPC/PSM/Chol (4/4/2) mixture in the absence and in the presence of 9 mol% 5-HT determined from 2H NMR experiments carried out at a temperature of 30°C. In the presence of 5-HT, two phases with distinctly different order parameters are observed. Phase I represents a disordered and phase II an ordered phase. The insets show the projected average chain length <L> of the palmitoyl chain of POPC or PSM in the absence (black bars) and in the presence of 9 mol% 5-HT (phase I filled colored bars, phase II empty colored bars). The error bars in the insets represent the error estimate of the chain length determination from 2H NMR spectra (Holte et al., 1996).
FIGURE 4
FIGURE 4
Order parameter profiles of POPC-d31 (A,B) and PSM-d31 (C,D) in a POPC/PSM/Chol (4/4/2) mixture in the absence and in the presence of 9 mol% 5-HT determined from a MD simulation at a temperature of 30°C. Upon the addition of 5-HT, an increased difference in the order parameters between the two phases was observed. I indicates the disordered and II the ordered phase or domain.
FIGURE 5
FIGURE 5
Comparison of the average order parameters of the sn-1 chain of POPC (A) and the acyl chain of PSM (B) in a POPC/PSM/Chol (4/4/2) mixture in the presence and in the absence of 5-HT determined by 2H NMR experiments (filled bars) and the MD simulations (open bars). Note that in the absence of 5-HT only one averaged lipid phase is observed experimentally and the same bars are reported.
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