Multi-faceted transport (Ferte, 2000) One of the most puzzling things about Pgp is its ability to transport an astonishing range of compounds. Lipophilic weak bases, lipophilic cations, neutral polycyclic compounds, amphiphiles, short chain phospholipids, and hydrophobic peptides are all known to be transported by Pgp. An even more startling discovery is that different members of these classes of are also capable of inhibiting Pgp function. This lack of substrate selectivity is relatively surprising and has made it difficult to apply experimental knowledge to clinical oncology; it is rather difficult to effectively eliminate Pgp function when the signal for functional activation is not know. However, one thing that all Pgp substrates share is their ability to easily traverse the lipid region of the plasma membrane. Fluorescent labeling has provided strong evidence than Pgp binds its substrates in the lipid phase of the bilayer, not in the aqueous exterior of the cell as is the case with most transporters. As a result, analysis of lipid biochemistry may be beneficial in the overall understanding of Pgp.

Membrane movement (Ferte, 2000). The movement of a compound across a membrane is often divided into three steps: partition within the extracellular region of the membrane, diffusion through the hydrophobic interior of the bilayer, and desorption across the intracellular region of the membrane (Figure 18). Mathematical quantitation of this process is found in the equation: P = P x D/e where P is the permeability (cm/s), P is the solute partition coefficient, D is the diffusion coefficient and e is the membrane thickness.

Figure 18

Pgp and membrane interactions (Ferte, 2000). Both Pgp substrates and inhibitors have fairly high P values as determined through evaluation in isotropic phase systems, by convention involving octanol and water. Compounds that interact with Pgp typically have a high affinity for the hydrophobic regions of the plasma membrane as opposed to the extracellular aqueous environment. In contrast, Pgp substrates have a low D value while Pgp inhibitors tend to have a much higher D value. Pgp substrates spend more time in the lipid region of the bilayer, allowing the rate of Pgp activity to exceed the rate of intracellular diffusion. Pgp inhibitors are able to escape the plasma membrane and gain access to the cell before they are pumped out. In conjunction with this idea, an inverse correlation has been found with the degree of lipophilicity; the more lipophilic a compound, the more likely it is to be a Pgp modulator. Highly lipophilic compounds are able to move readily through the plasma membrane and become inhibitors while less lipophilic compounds remain in the bilayer long enough to become Pgp substrates. This information is potentially useful in a clinical setting, since extensive pharmacological studies have shown that the degree to which a chemotherapy drug will be extruded from tumor cells is directly related to its lipophilicity.

Pgp and lipid composition (Ferte, 2000). Shortly after the initial characterization of MDR in cancer cells, the phenomenon was thought to be due to a reduced permeability of the plasma membrane for anti-cancer agents. After Pgp was discovered, characterized and genetically, it was assumed that Pgp pumping was responsible for the MDR phenotype. However, a recent adds an additional degree of complexity to Pgp study - one of the MDR genes is responsible for the production of a phosphatidylcholine translocase (enzyme which translocates proteins between the two leaflets of the plasma membrane) expressed in the bile canicular membrane (Figure 19a). This result suggests that plasma membrane lipid composition is also related to MDR and Pgp expression in the membrane.

Figure 19b

This idea is further supported by the fact that drug-sensitive cell lines have altered levels of neutral lipids, phospholipids, cholesterol and fatty acids as compared to normal cell lines (Figure 19b). In addition, MDR cell membranes appear to be more fragile, most likely due to the increase in complex phospholipids and higher rate of membrane turnover.

Figure 19a

Since the diffusion coefficient of various compounds can distinguish between Pgp substrates and inhibitors, it is likely that altered lipid membrane composition could contribute to the development of MDR. However, the exact nature of this correlation is still subject to several possible explanations. Changes in lipid may represent a genetic link to Pgp expression or they may be a phenotypic response to overexpressed levels of Pgp.

Pgp structure and lipid interactions (Ferte, 2000). Analysis of Pgp structure and function, in light of known lipid membrane properties, has revealed two hydrophobic domains within the protein. Each region appears to contain six alpha helices and two large loops which could extend into the cytoplasm. These cytoplasmic loops could contain the nucleotide binding sites (Figure 20). Results from several studies support this model, and the flexibility of the membrane helix regions is thought to be of functional significance, perhaps allowing movement of the nucleotide binding site upon interaction with ATP.

Figure 20

However, an alternate model has also been proposed with the transmembrane region composed of two beta barrels. As a result of conformational constraints, this model moves the nucleotide binding regions closer to the cytoplasm than the alpha helix model.

Figure 21

A conformational change induced by ATP binding could account for some of the conflicting structures proposed for the exact nature of Pgp transmembrane structure. Until the Pgp membrane-spanning region is crystallized, the specific details of the structure will remain unknown.

Pgp and detergents (Ferte, 2000). An interesting finding was that detergents, including those commonly used in protein reconstitution studies, are capable of interacting with Pgp. Cell lines which were drug-resistant can be chemosensitized as a result of treatment with a detergent, and specific amphiphilic molecules have been found to modulate cytotoxicity and intracellular accumulation of toxic compounds. Experimental analysis shows that the detergents, through their ability to interact with both the hydrophobic and polar regions of the plasma membrane, probably interfere with the catalytic ATPase activity of Pgp. At solubilizing concentrations, drug-induced ATPase activity was almost completely eliminated. These results indicate that detergents most likely block the nucleotide binding sites necessary for Pgp function.

Figure 22

Pgp function and membrane lipids (Ferte, 2000). Further analysis of Pgp-membrane lipid interactions indicates that certain membrane-associated lipids - phosphatidyl-ethanolamine, phosphatidyl-serine and phosphatidyl-choline - are required for proper Pgp function (Figure 23). In contrast, excess exogenous phospholipids, dipalmitoyl-phosphatidylethanolamine, or egg phosphatidylcholine can inhibit Pgp function. Overall lipid composition can also affect Pgp function - the lipid environment of E. coli prevents Pgp-induced drug efflux while the lipid membrane in sheep liver and brain, tissues known to be highly effected by chemotoxic agents, stimulates Pgp-induced drug efflux.

Figure 23

Pgp and cholesterol (Ferte, 2000). Cholesterol, a membrane lipid of particular interest because of its role in heart disease, can also play a role in modulating Pgp function. Cholesterol does alter membrane fluidity and is also capable of binding at the lipid-protein interface of Pgp in the plasma membrane. One study reports a biphasic effect upon Pgp, as increasing cholesterol concentration in the membrane up to a weight ratio of 20% results in increased Pgp activity. Increased concentration above 20% decreases Pgp activity (Figure 24). These results support the fact that cholesterol typically lowers membrane permeability, thus decreasing the diffusion coefficient and increasing Pgp transport for compounds attempting to enter the cell. However, cholesterol can also bind to Pgp and there is preliminary evidence that Pgp is involved with cholesterol metabolism (see next section). As a result, extremely high levels of cholesterol may actually inhibit Pgp function.

Figure 24

Pgp and lipid metabolism (Ferte, 2000). Pgp also appears to play a role in lipid metabolism. Pgp is capable of transporting short-chain as well as some long-chain lipid analogs. As a result, Pgp may be involved with transporting lipid precursors in lipid biosynthesis and catabolism. This is affirmed by the previously-mentioned discovery that one of the genes encoded in the MDR region of chromosome 3 is actually a lipid-specific translocase. A role for Pgp as such a flippase also explains some of the conflicting evidence regarding the exact structure and function of the protein. In addition, several other ABC proteins are known to participate in lipid translocation, making it likely that Pgp has at least some involvement with regulation of lipid membrane metabolism and composition.