ABCB1 - P-glycoprotein 170

P-gp was first described by Juliano et al. in 1976, who found that chinese hamster ovary cells selected for resistance to colchicine showed cross resistance to a broad range of drugs (Juliano and Ling 1976). The authors detected a transport protein in the cell membranes of the resistant cells that was not found in wild type cells and named it P-glycoprotein 170. Besides this localization at tumor cell membranes, where p-gp is responsible for the development of multi drug resistance in chemotherapy, p-gp was found in several tissues of the human body. It is expressed at the biliary canalicular membrane of hepatocytes as well as at the brush border membrane of the intestinal epithelium (Thiebaut et al. 1987).

In kidney, abundant p-gp is found at the luminal membrane of the proximal tubules. In liver, kidney and intestine, p-gp has excretory and detoxifying functions. Moreover, p-gp is expressed in the brain. It was detected both in the choroid plexus (Rao et al. 1999) and at the BBB (Thiebaut et al. 1989). The precise localization of p-gp at the BBB is a topic of controversy. On the one hand, there is evidence for the expression of p-gp at the luminal membranes of endothelial cells of the brain capillaries. Beaulieu et al.

(1997) detected strong enrichment of p-gp by Western blotting in brain capillary luminal membranes, compared with brain capillaries (17-fold) and whole membranes (400-500-fold). On the other hand, other work groups used the MRK-16 MAb against human p-gp for the detection of the transport protein. According to their findings p-gp expression at the BBB is similar to the localization of glial fibrillary acidic protein (GFAP) which is found in astrocytes. Hence, they postulated that p-gp is localized at the astrocyte foot processes (Pardridge et al. 1997). In the meantime several studies have demonstrated that p-gp is co-localized at the astrocytes as well as at the luminal membrane of the endothelial cells (Decleves et al. 2000). However, much more p-gp is expressed at the endothelial cell membrane compared to astrocytes foot processes. Furthermore, the co-cultivation of astrocytes and brain capillary endothelial cells (BCEC) in in vitro models of the BBB led to an increased p-gp expression compared to BCEC monolayer models indicating an important role of the astrocytes regarding the expression of p-gp (Gaillard et al. 2000). Due to the localization of p-gp at the endothelial membrane of the capillaries the role of p-gp is the protection of the brain from foreign and toxic compounds.

1.3 ABC transporters 25

Figure 1.5: Predicted membrane topology of human p-gp. The ABC transport protein p-gp is thought to be organized in two hydrophobic halves, each half consisting of six TMDs and one cytoplasmic ATP-binding domain (NBD). The protein is glycosylated at three sites on the first extracellular loop.

A model for the predicted structure of p-gp is presented in Fig. 1.5. P-gp is a 170 kDa transmembrane protein that is encoded by the MDR1 gene. This gene consists of 27 exons distributed over 100 kb and is located on the long arm of chromosome 7 (Fardel et al. 1996). The human p-gp is composed of 1280 amino acids. They are organized in two halves each containing 610 amino acids in 6 hydrophobic transmembrane domains (TMD) and one hydrophilic NBD. The protein is N-glycosylated on the first extracellular loop in three different locations. This glycosylation appears to be necessary for the effectiveness of the protein (Ramakrishnan 2003). The TMDs play an important role for the substrate recognition as single mutations in all transmembrane regions affect the transport function of the protein either directly through alteration of the binding site or by interference in the conformational changes (Litman et al. 2001). Therefore, the binding sites are broad interaction regions. Using photoaffinity labelling studies and epitope mapping with iodomycin (Demmer et al. 1997) two major regions were identified within the TMDs: TMD 5 and 6 in the N-terminal half, and TMD 11 and 12 in the C-terminal half. However, there are much more interaction points between substrates and amino acids. These are particularly localized at TMD 4 and 10 and have been discovered by means of cysteine scanning mutagenesis of all TMDs combined with thiol modification (Loo and Clarke 2000; 2001, Van Veem and Callaghan 2003). Within these TMDs, specific amino acids

26 Introduction are responsible for substrate recognition. For example, charged substrates are able to interact with the π face of aromatic residues in tyrosine, phenylalanine and tryptophan.

This binding is as strong as electrostatic interactions between ion pairs (Dougherty 1996, Kwan et al. 2000). Until now, at least four binding sites are identified by several work groups (Martin et al. 2000, Ekins et al. 2002, Wang et al. 2003). These binding sites are in part transport sites, but regulatory sites also exist. Both site types appear to switch between high and low affinity conformations (Martin et al. 2000).

In contrast to the TMDs, the NBDs are not integrated in the process of substrate recognition. NBDs are involved in the transport mechanism of ABC transporters. Liu and Sharom (1996) demonstrated by 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) labelling of cysteine residues located within the Walker A motif of the NBD that the NBDs change their positions relative to the cell membrane in the presence of p-gp substrates. MIANS is fluorescent in an aqueous environment, but in lipophilic solutions the fluorescence is quenched. Therefore, the quenching of the MIANS fluorescence during the efflux process indicates conformational changes of the transporter including the NBDs.

These changes can be seen directly by cryo-electron microscopy of p-gp trapped in different stages of the transport cycle (Rosenberg et al. 2001b). The energy for these changes which are essential for the releasing of a compound, is provided by hydrolysis of ATP at the NBDs. The binding and hydrolysis of ATP are crucial steps in the transport process, which can be divided in four steps (see Fig. 1.6). First, the substrate has to be recognized and bound by p-gp. ATP binds to one NBD in this first step. In the second step, ATP hydrolysis at the NBD leads to the aforementioned conformational changes. The affinity of the binding site is switched to the low affinity status and the drug binding site is oriented to the extracellular site to release the substrate. Uptake of a second ATP molecule in the third step results in a further conformational change at the NBD, whereas the drug binding site remains in the low affinity status. In the last step after hydrolysis of the second ATP molecule the protein returns to the original conformation with high affinity status of the binding site to recognize a new substrate. ATP hydrolysis takes place during the second and the fourth step. To supply the energy for the conformational changes of the TMDs, the NBDs have to be in contact with the TMDs. This exchange takes places via the intracellular loops (Van Veem and Callaghan 2003).

1.3 ABC transporters 27

Figure 1.6: Proposed mechanism of p-gp function. The ellipses represent the substrate-binding face of the protein, the extra- and the intracellular location. The square describes the intracellular site with reduced affinity. The NBDs are represented by two over-lapping circles indicating that both sites are required for ATP hydrolysis (according to Druley et al. (2001), Sauna and Ambudkar (2001)).

It is important to note that both NBDs are required for the efflux process. For a complete transport cycle, both NBDs have to be activated by the binding of ATP, but it is indifferent which NBD binds to ATP first. Sauna and Ambudkar (2000) proved this by investigation of the affinity of [I¹²⁵]iodoarylazidoprazosin for p-gp in the presence of the nonhydrolyzable nucleotide 5’-adenylylimidodiphosphate and vanadate, as well as for p-gp that is trapped in transition-state conformation by ADP and vanadate. The affinity is clearly reduced (> 30-fold) to low affinity state if p-gp is trapped, whereas it is not influenced by the nonhydrolyzable compounds. Furthermore, they found an inverse correlation between ADP release and the recovery of substrate binding. Therefore, Sauna et al. summarized that one ATP molecule is necessary for the efflux of a bound substrate resulting in the aforementioned change to the low affinity status. In addition, a second ATP molecule is required to reset the status of the binding sites to the high affinity conformation in order to prepare the next catalytic cycle.

Three different models are described in the literature concerning the mechanism of transport at the multiple drug binding sites of ABC transporters. The "altering

ac-28 Introduction cess/single site model" (Bruggemann et al. 1992, Martin et al. 2000) suggests that the sites are localized within a single binding region which is alternately oriented to the ex-tracellular or to the inex-tracellular site. Dey et al. (1997) postulated in the "fixed two side model" the existence of two static binding regions. The high affinity "on"-site is located in the C-terminal half of the protein, whereas the low affinity "off"-site is found in the N-terminal half. The bound substrate moves from the "on" to the "off" site during the first ATP hydrolysis. This model agrees well with the process of ATP hydrolysis described by Sauna and Ambudkar (2000) mentioned above. Furthermore, the "altering two-site (two-cylinder engine) model" is a combination of the aforementioned models; two distinct binding regions are alternately exposed to the inner and outer membrane. Here, ATP hydrolysis leads to simultaneous or sequential movement of the two regions (van Veen et al. 2000). The differences between the three models may be related to the different localization of the substrate binding sites.

The establishment of a complete transport model is difficult since many factors that influence the transport function both directly and indirectly have to be taken into ac-count. These factors include conformational changes, the existence of multiple binding sites, the role of NBDs, as well as the variable effects of p-gp inhibitors (Litman et al.

2001). Nowadays, there is more knowledge about the number of binding sites, their in-teractions among each other, and various pharmacophores of substrates. However, only little research is focused on the reactions of the protein caused by substrate binding which is important for the design of new modulators and for the development of agents with low p-gp affinity.