Determination of the Rate-Limiting Step in Fatty Acid Transport

Abstract

The generally accepted model of free fatty acid (FA) transport through cellular membranes occurs in three steps, adsorption of the FA onto the membrane, translocation across the membrane ("flip-flop"), and subsequent desorption of the FA into the cytosol. There still exists some dispute as to the identity of the rate-limiting step of FA transport. In the present study, sumfrequency vibrational spectroscopy (SFVS) was used to directly measure the rate of stearic acid (SA) flip-flop in planar supported lipid bilayers (PSLBs) comprised of 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC). The impact of SA on the physical properties of binary mixtures of SA and DSPC was investigated via Π−A isotherms from which the excess free energies of mixing and compression moduli were calculated. The manner in which these physical changes influenced the rates of SA and DSPC flip-flop was subsequently examined using SFVS. The rates of SA and DSPC flip-flop revealed that SA flip-flops independently of DSPC and on much faster time scales than its phospholipid counterpart. SFVS was also used to probe the rate of protein-unassisted SA desorption from hybrid supported lipid bilayers (HSLBs), allowing for the first decoupled measurement of the rates of desorption and flip-flop. These results provide strong evidence for desorption being the rate-limiting step in FA transport through the membrane in the absence of proteins.

In Silico-Based Experiments on Mechanistic Interactions between Several Intestinal Permeation Enhancers with a Lipid Bilayer Model

“…The occurrence of flip-flop events in the membrane for fatty acid molecules with a neutral headgroup was also observed in the past in both experimental and computational studies. 63,64 The total number of expulsion events and flip-flop events for each PE at various concentrations are presented in Figure 3B,C, respectively. An increasing number of expulsion or flip-flop events with increasing concentration for caprylate, caprate, and SNAC molecules can be observed, along with occasional expulsion events occurring for laurate and no expulsion or flip-flop events for sucrose monolaurate molecules (Figure 3B).…”

Molecule‐Membrane Interactions in Biological Cells Studied with Second Harmonic Light Scattering

“…Consequently, the time‐dependent rise and fall of the measured SHS signal is quantitatively related to the molecular densities on the outer and inner surfaces and can be analyzed to reveal the rates of molecular adsorption and transport across the membrane. Following this demonstration, a number of studies have since examined the effects of lipid composition and a variety of environmental factors on the propensity and characteristics of molecular transport across membranes in model liposome systems …”

“…It was expected that the rate of transport across the CM should be similar to those obtained from prior liposome‐based studies . In both cases, the molecular probe has to directly diffuse across the lipid bilayer.…”

“…It was expected that the rate of transport across the CM shouldb es imilar to those obtained from prior liposome-based studies. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] In both cases, the molecular probe has to directly diffusea cross the lipid bilayer.S till, the deduced average moleculart ransport rate for the bacterial CM is found to be an order of magnitude slower compared to unilamellar liposomes (ca. 10 À3 s À1 )w hose lipid composition was homogeneous.…”

“…Following this demonstration,anumber of studies have since examined the effects of lipid composition and av ariety of environmental factorso nt he propensity and characteristics of molecular transport across membranes in model liposome systems. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] The studies on liposomes suggested the feasibility that SHS could be used for monitoring molecular transport across membranesi nl iving biological cells. Indeed, in 2010, our laboratory reportedt he first demonstration that SHS could be applied to study molecular transport across membranes in living cells.…”