Bilayer permeation

Despite the abovementioned difficulties, kinetic models reproducing typical micellar kinetics have found widespread use and typically reproduce micellar reactivity well. Whereas these models are described here in terms of micellar kinetics, they can equally be adopted for the analysis of most vesicular rate effects, as long as bilayer permeation is either slow or fast compared to the rate of reaction. The issue of bilayer permeation-dependent rates of reaction has been addressed in detail by Moss et and will not be discussed here. A brief overview of the basic kinetic... 

Most drug substances and substances of interest to health and environmental risk assessors enter cells by passive permeation (diffusion). In this process, a substance dissolves in the membrane lipid bilayer, permeates through the membrane, and enters into the cytoplasm of the cell. The substance thus must be soluble in lipids. The process is passive because the rate and extent to which a substance will enter a cell by this means depends on its concentration outside and inside the cell. The net movement is from the region of higher concentration to that of lower concentration. Unlike the cell membrane, which is chiefly lipid, the extracellular and intracellular spaces separated by the membrane are aqueous. The higher the concentration of substance outside of the cell, and the more soluble the substance in the membrane lipid bilayer, the greater will be the tendency for the substance to diffuse across the membrane and enter the cytoplasm. The rate and extent of diffusion will decrease as the concentration of the substance inside the cell increases until, eventually, equilibrium is reached. 

As noted previously, real membranes are characterized by nonuniform free-energy profiles for bilayer permeation. Consequently, molecular dynamics simulations have been used to treat the motion of a solute through a bilayer membrane. These calculations accurately reproduce experimentally estimated values of diffusion 143]. However, because this approach is mathematically complex, it has been applied only to the permeation of relatively small molecules. Approximate methods have been developed based on a simple kinetic scheme for the diffusion of solute molecules M in a four compartment system ... 

This is further illustrated by the free-energy profile for bilayer permeation by a simple hydrophobe, methane, determined from MD simulation (it is impossible to obtain such profiles experimentally) (Fig. 1). Distinct barriers for the penetration of headgroups, and a clear preference for localization in the hydrophobic hydrocarbon center are observed. Although one can easily determine general regions of the bilayer, distinct, discrete compartments cannot be observed. Rather, the bilayer shows a smoothly changing profile. 

One of the principal constituents of membranes, water, is often ignored. Luckily, because of its fast convergence rates, the structure and dynamics of water at the bilayer interface have been studied extensively. For some time, based on the known slower bilayer permeation rate of hydrophobic cations relative to hydrophobic anions, it has been speculated that there is a membrane dipole potential . The genesis of this positive potential has been the subject of much speculation. Experimental studies have shown it to be largely due to the orientation of ester carbonyl groups, which are common in glycerol based lipids. However, a substantial component could only be explained by a contribution due to oriented water molecules at the bilayer surface. A simulation conducted in such a way as to remove artifacts due to electrostatics showed this clearly to be the case. At the headgroup/water... 

As for the lipid bilayer permeation, several phenomena could be responsible, namely dissolution of the hydrophobic part ofthe polycation... 

This review addresses the issues of the chemical and physical processes whereby inorganic anions and cations are selectively retained by or passed through cell membranes. The channel and carrier mechanisms of membranes permeation are treated by means of model systems. The models are the planar lipid bilayer for the cell membrane, Gramicidin for the channel mechanism, and Valinomycin for the carrier mechanism. 

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

Lipophilicity is intuitively felt to be a key parameter in predicting and interpreting permeability and thus the number of types of lipophilicity systems under study has grown enormously over the years to increase the chances of finding good mimics of biomembrane models. However, the relationship between lipophilicity descriptors and the membrane permeation process is not clear. Membrane permeation is due to two main components the partition rate constant between the lipid leaflet and the aqueous environment and the flip-flop rate constant between the two lipid leaflets in the bilayer [13]. Since the flip-flop is supposed to be rate limiting in the permeation process, permeation is determined by the partition coefficient between the lipid and the aqueous phase (which can easily be determined by log D) and the flip-flop rate constant, which may or may not depend on lipophilicity and if it does so depend, on which lipophilicity scale should it be based ... 

Thomae, A. V., Wunderli-Allenspach, H., Kramer, S. D. Permeation of aromatic carboxylic adds across lipid bilayers the pH-partition hypothesis revisited. Biophys. J. 2005, 89,1802-1811. 

Seventy years later, this theory largely holds true, although periodically challenged [67, 68]. Observation of transmembrane permeability of ionic species was initially explained by the formation of neutral ion-pair [69, 70]. A comprehensive review of the physicochemical properties influencing permeation has been written by Malkia et al. [5]. The reality is that, despite many studies, the effect of ionization on permeation is still a matter of discussion and active research. In contrast, it became clear that bulk-phase partitioning measurements are not adequate to describe bilayer partitioning [71-73]. 

III. PERMEATION OF SMALL MOLECULES ACROSS LIPID BILAYERS ROLE OF BILAYER STRUCTURE... 

FIG. 13 A schematic illustration of the effects of the free surface area of lipid bilayer membranes on the permeation of two permeants with the same molecular volume, but different cross-sectional areas, (a) A lower free surface area, (b) A higher free surface area. 

Using liposomes made from phospholipids as models of membrane barriers, Chakrabarti and Deamer [417] characterized the permeabilities of several amino acids and simple ions. Phosphate, sodium and potassium ions displayed effective permeabilities 0.1-1.0 x 10 12 cm/s. Hydrophilic amino acids permeated membranes with coefficients 5.1-5.7 x 10 12 cm/s. More lipophilic amino acids indicated values of 250 -10 x 10-12 cm/s. The investigators proposed that the extremely low permeability rates observed for the polar molecules must be controlled by bilayer fluctuations and transient defects, rather than normal partitioning behavior and Born energy barriers. More recently, similar magnitude values of permeabilities were measured for a series of enkephalin peptides [418]. 

Figure 7.22b shows that hydrophilic molecules, those with log Kj < 1, are much more permeable in octanol than in olive oil. The same may be said in comparison to 2% DOPC and dodecane. Octanol appears to enhance the permeability of hydrophilic molecules, compared to that of DOPC, dodecane, and olive oil. This is dramatically evident in Fig. 7.7, and is confirmed in Figs. 7.8c and 7.22b. The mechanism is not precisely known, but it is reasonable to suspect a shuttle service may be provided by the water clusters in octanol-based PAMPA (perhaps like an inverted micelle equivalent of endocytosis). Thus, it appears that charged molecules can be substantially permeable in the octanol PAMPA. However, do charged molecules permeate phospholipid bilayers to any appreciable extent We will return to this question later, and will cite evidence at least for a partial answer. 

Trimethylaminodiphenylhexatriene chloride (TMADPH Fig. 7.45) is a fluorescent quaternary ammonium molecule that appears to permeate cell membranes [595]. TMADPH fluoresces only when it is in the bilayer, and not when it is dissolved in water. Therefore, its location in cells can be readily followed with an imaging fluorescence microscope. One second after TMADPH is added to the extracellular solution bathing HeLa cell types, the charged molecule fully equilibrates between the external buffer and the extracellular (outer) leaflet bilayer. Washing the cells for one minute removes >95% of the TMADPH from the outer leaflet. If the cells are equilibrated with TMADPH for 10 min at 37°C, followed by a one-minute wash that removed the TMADPH from the outer leaflet, the fluorescent molecule is... 

Walter, A. Gutknecht, J., Monocarboxylic acid permeation through lipid bilayer membranes, J. Membr. Biol. 77, 255-264 (1984). 

The lipid bilayer arrangement of the plasma membrane renders it selectively permeable. Uncharged or nonpolar molecules, such as oxygen, carbon dioxide, and fatty acids, are lipid soluble and may permeate through the membrane quite readily. Charged or polar molecules, such as glucose, proteins, and ions, are water soluble and impermeable, unable to cross the membrane unassisted. These substances require protein channels or carrier molecules to enter or leave the cell. 

Models of lipid bilayers have been employed widely to investigate diffusion properties across membranes through assisted and non-assisted mechanisms. Simple monovalent ions, e.g., Na+, K+, and Cl, have been shown to play a crucial role in intercellular communication. In order to enter the cell, the ion must preliminarily permeate the membrane that acts as an impervious wall towards the cytoplasm. Passive transport of Na+ and Cl ions across membranes has been investigated using a model lipid bilayer that undergoes severe deformations upon translocation of the ions across the aqueous interface [126]. This process is accompanied by thinning defects in the membrane and the formation of water fingers that ensure appropriate hydration of the ion as it permeates the hydrophobic environment. 

To reach such a site, a molecule must permeate through many road blocks formed by cell membranes. These are composed of phospholipid bilayers - oily barriers that greatly attenuate the passage of charged or highly polar molecules. Often, cultured cells, such as Caco-2 or Madin-Darby canine kidney (MDCK) cells [1-4], are used for this purpose, but the tests are costly. Other types of permeability measurements based on artificial membranes have been considered, the aim being to improve efficiency and lowering costs. One such approach, PAMPA, has been described by Kansy et al. [5],... 

Passive diffusion through the lipid bilayer of the epithelium can be described using the partition coefficient between octanol/water (log P) and A log P (the difference between the partition into octanol/water and heptane/ethylene glycol or heptane/ octanol) [157, 158], The lipophilicity of the drug (log P) (or rather log D at a certain pH) can easily be either measured or calculated, and is therefore generally used as a predictor of drug permeability. Recently, a method using artificial membrane permeation (PAMPA) has also been found to describe the passive diffusion in a similar manner to the Caco-2 cell monolayers [159]. 

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