1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers.

Drew Marquardt,Frederick A Heberle,Erwin London,Tatiana Miti,Georg Pabst,John Katsaras,Barbara Eicher

doi:10.1021/acs.langmuir.6b04485

Abstract

We measured the transbilayer diffusion of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (Lβ′) and fluid (Lα) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-d13 vesicles was monitored using 1H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol–1. In gel-phase DPPC vesicles, flip-flop was not observed over the course of 250 h. Our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. We reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement.

Highlights

The eukaryotic plasma membrane (PM) is characterized by an asymmetric distribution of lipids between the exoplasmic and cytoplasmic bilayer leaflets.[1−3] The physiological fate of cells depends on the strict maintenance of this asymmetry through the interplay of active and passive lipid translocation events.[4]Active mechanisms are thought to rely on the so-called floppases that move newly synthesized lipids from the inner to the outer leaflet and on flippases that restore the asymmetry of passively translocated lipids.[5,6] The selectivity of these enzymes for different classes of lipids regulates compositional asymmetry within the PM.[4]
We find that DPPC flip-flop in the gel phase is too slow to accurately measure, whereas fluid-phase DPPC undergoes flip-flop with a temperature-dependent half time ranging from days to weeks
At all temperatures we examined, the translocation rate in vesicles is slower by orders of magnitude than that previously measured with sum-frequency generation (SFG) on supported DPPC bilayers.[14,16]

Summary

■ INTRODUCTION

The eukaryotic plasma membrane (PM) is characterized by an asymmetric distribution of lipids between the exoplasmic and cytoplasmic bilayer leaflets.[1−3] The physiological fate of cells depends on the strict maintenance of this asymmetry through the interplay of active and passive lipid translocation events.[4]. One contributing factor might be submicron topological defects that are known to exist in SSBs from atomic force microscopy (AFM) imaging studies.[33,37−46] To investigate the influence of defects on flip-flop, we performed random-walk (Monte Carlo) simulations of lateral lipid diffusion in the presence of 100 nm-diameter holes, a typical size observed in AFM images.[46] Figure 4a shows a schematic illustration of such a defect (not to scale) in a supported bilayer. (equivalent to compressing the time axis of Figure 4d by a factor of 103) resulted in complete equilibration of the two leaflets within seconds, even for 99.9% surface coverage This observation is consistent with reports that SSBs prepared by Langmuir−Blodgett/Langmuir−Schaefer (LB/LS) deposition do not support asymmetry at fluid-phase temperatures.[14] We conclude that defect-mediated translocation is a plausible mechanism to reconcile the observed differences between vesicles and SSBs. Transbilayer movement is assumed to occur via unhindered lateral diffusion through the pore formed by lipid headgroups. The inset shows an expanded view in the vicinity of a circular defect, revealing multiple translocation events. (c) Top−down view of simulation snapshots (defects shown as black circles; scale bar, 1 μm) for different bilayer surface coverages: 99.0% (gray), 99.5% (blue) and 99.9% (yellow). (d) Simulated asymmetry decay curves (open symbols) and fits (solid lines) corresponding to the surface coverages in (c), for a lateral lipid diffusion coefficient of 10−3 μm[2] s−1. (e) Lipid translocation rate kf vs lateral diffusion coefficient DT calculated from decay curves corresponding to the surface coverages in (c)

■ DISCUSSION

■ SUMMARY AND CONCLUSIONS

■ EXPERIMENTAL PROCEDURES

■ ACKNOWLEDGMENTS

■ REFERENCES