Altered Membrane Mechanics Provides a Receptor-Independent Pathway for Serotonin Action

Abstract

Serotonin, an important signaling molecule in humans, has an unexpectedly high lipid membrane affinity. The significance of this finding has evoked considerable speculation. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, providing an activity pathway completely independent of serotonin receptors. Atomic force microscopy shows that serotonin makes artificial lipid bilayers softer, and induces nucleation of liquid disordered domains inside the raft-like liquid-ordered domains. Solid-state NMR spectroscopy corroborates this data at the atomic level, revealing a homogeneous decrease in the order parameter of the lipid chains in the presence of serotonin. In the RN46A immortalized serotonergic neuronal cell line, extracellular serotonin enhances transferrin receptor endocytosis, even in the presence of broad-spectrum serotonin receptor and transporter inhibitors. Similarly, it increases the membrane binding and internalization of oligomeric peptides. Our results uncover a mode of serotonin-membrane interaction that can potentiate key cellular processes in a receptor-independent fashion.

Keywords: lipid bilayers; membrane modulation; neurotransmission; serotonin-membrane interaction; volume transmission.

Figure 1

Probing the stiffness of membrane lipid bilayers by AFM force indentation measurements. (A) A typical indentation force curve obtained from supported POPC/POPG/cholesterol (1:1:1) lipid bilayer on mica. The force applied by the cantilever is plotted as a function of the z‐piezo displacement. The discontinuity in the force curve marks the point of indentation (red arrow). Inset: magnified image shows an indentation force of 2.2 nn. (B) Representative histogram of indentation forces before (black) and after (red) serotonin incubation on a negatively charged (PPC111) bilayer. (C) Average values of force histograms. (D), (E) representative histograms of indentation forces measured on the ordered and disordered domains, respectively on a neutral biphasic (DEC221) bilayer. (F) Average values of force histograms before (black) and after (red) serotonin incubation.

Figure 2

AFM images displaying time‐dependent nucleation of disordered domains within the ordered domains in phase separated lipid bilayers (DEC221) (A) 0 min (B) 20 min, (C) 120 min, and (D) 300 min after incubation with 4.1 mm of serotonin. Insets: magnified images of the region with white dashed border, showing growth of disordered domain inside ordered domains. Scale bar 0.5 μm. Height is false color coded. Color bar indicates the relative height.

Figure 3

Probing the distribution and the effect of serotonin in lipid bilayer by solid‐state NMR. (A) Average lipid chain length of POPC‐d ₃₁: POPG:cholesterol (1:1:1) and POPC:POPG‐d ₃₁:cholesterol (1:1:1) with 0, 10, 25 mol % of serotonin at (i) 25 °C and (ii) 37 °C. (B) ²H NMR average order parameters of the above mentioned membrane composition. (C) schematic representation of serotonin interaction with POPC lipid chain segment. (D,E,F) are the ¹H NOESY NMR cross‐relaxation rates representing the contact probability between the individual protons of serotonin labelled in (C) with the respective lipid segments.

Figure 4

Effect of serotonin on the interaction of RN46A cells with amylin (IAPP) and transferrin. (A,B) Confocal images of the binding and internalization of Rhodamine (Rh)‐labelled IAPP oligomers to RN46A cells in the absence (A) and in the presence (B) of serotonin. (C,D) Confocal images of transferrin receptor internalization by RN46A cells showing AlexaFluor488‐transferrin distribution in absence (C) and in presence (D) of serotonin. (E) analysis of the extent of binding of different probes in absence (black) and in presence (red) of serotonin. Cells are pre‐treated with 5‐HT1A receptor antagonist, WAY100635 (10 μm), 5‐HT2 receptor antagonists Methysergide (10 μm) and Ketanserin (10 μm), and SERT inhibitor Fluoxetine (10 μm).