A Quantitative Model for the Dependence of Solute Permeability on Peptide and Cholesterol Content in Biomembranes

Abstract

The influence of varying concentrations of a transmembrane peptide, gramicidin A (gA), and cholesterol (Chol) on the passive permeation of p-methylhippuric acid (MHA) and alpha-carbamoyl-p-methylhippuric acid (CMHA) across egg-lecithin membranes (EPC) has been investigated in vesicle efflux experiments. Incorporation of 0.25 volume fraction of gA in its nonchannel conformation increased the permeability coefficient (Pm) for CMHA by a factor of 6.0 +/- 1.8 but did not alter Pm for MHA, a more lipophilic permeant. In contrast, incorporation of 0.26 volume fraction Chol with no added protein decreased the Pm values for both CMHA and MHA by similar factors of 4.2 +/- 1.1 and 3.5 +/- 1.2, respectively. A quantitative structure-transport model has been developed to account for the dependence of Pm on the membrane concentrations of gA and Chol in terms of induced changes in both membrane chain ordering and hydrophobicity. Chain ordering is assumed to affect Pm for both permeants similarly since they are comparable in molecular size, while changes in Pm ratios in the presence of gA or Chol are attributed to alterations in membrane hydrophobicity. Changes in lipid chain ordering were detected by monitoring membrane fluidity using fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene incorporated into the membranes. The influence of additives on membrane hydrophobicity, which governs Pm ratios through effects on solute partitioning into the barrier domain, were rationalized within the framework of regular solution theory using solubility parameters as a measure of membrane hydrophobicity. Fits of the Pm ratios using the theoretical model yielded solubility parameters for gA and Chol in EPC membranes of 13.2 and 7.7 (cal/ml)(1/2), respectively, suggesting that gA decreases the barrier domain hydrophobicity while Chol has a minimal effect on barrier hydrophobicity. After correcting for barrier domain hydrophobicity, permeability decrements due to membrane ordering induced by gA or Chol were found to exhibit a strong correlation with membrane order as predicted by free-surface-area theory, regardless of whether gA or Chol is used as the ordering agent.

Permeability of Fluid-Phase Phospholipid Bilayers: Assessment and Useful Correlations for Permeability Screening and Other Applications

“…Observed permeabilities generally decrease with increasing permeant MW more rapidly than in a bulk liquid. Thus, the coefficients K lip/w and D lip ⊥ are properties that reflect not only the chemical (solvent) nature of the lipid, but are also influenced by ordering of the hydrocarbon chains, 17,26,28,31,[39][40][41] as is discussed at length below. The subscript "⊥" for "perpendicular" in the symbol D lip ⊥ is significant, because-given the anisotropy of the microstructure-the lateral diffusion coefficient D lip describing motion parallel to the plane of the bilayer is generally different.…”

“…The most comprehensive data set for egg lecithin emerging from a single laboratory is that developed collectively in a series of papers by Xiang, Anderson, and coworkers, 16,17,26,28,29,[32][33][34][35]50 summarized in Table 1. It comprises four sets of compounds, namely: (1) p-toluic acid and a series of six related compounds with different chemical substitutions of a hydrogen atom on the "-methyl group, 29 (2) a corresponding series based on p-methylhippuric acid, 33 (3) a set of 15 other selected compounds, 26 and (4) a set of "peptidelike small organic molecules" studied most recently (including new peptide derivatives of p-toluic and pmethylhippuric acids).…”

“…28,35 The temperature applicable to all measurements is 25 • C. Of the 39 data points, 37 apply to pure egg lecithin, and two additional ones quantify the effects of 28.6 mol % cholesterol on permeability for p-methylhippuric acid and "-carbamoylp-methylhippuric acid. 17 In their data reduction, Xiang, Anderson, and coworkers treat carefully the existence of unionized and ionized forms of their weak acid solutes, which leads to pH dependence of apparent permeability coefficients, as well as mass transfer resistances associated with unstirred layers in diffusion cells. All reported permeability coefficients cited here are values referring specifically to the unionized form.…”

“…The chain ordering effect is usually quantified in terms of an exponential decrease in permeability with increasing projected area (as opposed to volume) of the permeant molecule, that is, area taken up in the plane of the bilayer by a molecule aligned more or less perpendicularly to it. 17 Experimental results are listed as mean value ± standard deviation. Data points are listed only for conditions under which the phase is liquid crystalline ("L," fluid).…”

“…The focus on phospholipids is logical because viable cell membranes are bilayers of such polar lipids (with intercalated cholesterol, membrane proteins, and other constituents). [15][16][17][18] Although active transport should not be discounted, a focus on passive diffusion is also appropriate "[i]n light of recent conclusions that 80%-95% of commercial drugs are absorbed primarily by passive diffusion" 5 (see cited assessments 4,19 ).…”