Determination of the Rate-Limiting Step in Fatty Acid Transport.

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, sum-frequency 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-3-phosphocholine (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.

Structural Origins of Cholesterol Accelerated Lipid Flip-Flop Studied by Sum-Frequency Vibrational Spectroscopy.

The unique structure of cholesterol and its role in modulating lipid translocation (flip-flop) were examined using sum-frequency vibrational spectroscopy (SFVS). Two structural analogues of cholesterol--cholestanol and cholestene--were examined to explore the influence of ring rigidity and amphiphilicity on controlling distearoylphosphocholine (DSPC) flip-flop. Kinetic rates for DSPC flip-flop were determined as a function of sterol concentration and temperature. All three sterols increased the rate of DSPC flip-flop in a concentration-dependent manner following the order cholestene > cholestanol > cholesterol. Rates of DSPC flip-flop were used to calculate the thermodynamic activation free energy barrier (ΔG(‡)) in the presence of cholesterol, cholestanol, and cholestene. The acyl chain gauche content of DSPC, mean lipid area, and membrane compressibility were correlated to observed trends in ΔG(‡). ΔG(‡) for DSPC flip-flop showed a strong positive correlation with the molar compression modulus (K*) of the membrane, influenced by the type and concentration of the sterol added. Interestingly, cholesterol is distinctive in maintaining invariant membrane compressibility over the range of 2-10 mol %. The results in this study demonstrate that the compression modulus of a membrane plays a significant role in moderating ΔG(‡) and the kinetics of native, protein-free, lipid translocation in membranes.