Transport Proteins

Membrane transporters in general are a large group of membrane proteins that have one (bi-

topic) or more (polytopic) hydrophobic transmembrane segments. These transporters are involved in almost all facets of biological processes in the cell. Their involvement in cellular function can be classified as follows [after Saier (107)]:

1. Mediate entry of all essential nutrients into the cytoplasmic compartment and subsequently into organelles, thus facilitating the metabolism of exogenous sources of carbon, nitrogen, sulfur, and phosphorus.

2. Provide a means for regulation of metabolite concentrations by catalyzing the efflux of end products of metabolic pathways from organelles and cells.

3. Mediate the active extrusion of drugs and other toxic substances from either the cytoplasm or the plasma membrane.

4. Mediate uptake and efflux of ion species that must be maintained at concentrations dramatically different from those in the external milieu.

5. Participate in the secretion of proteins, complex carbohydrates, and lipids into and beyond the cytoplasmic membrane.

6. Transfer of nucleic acids across cell membranes, allowing genetic exchange between organisms and thereby promoting species diversification.

7. Facilitate the uptake and release of phero-mones, hormones, neurotransmitters, and a variety of other signaling molecules that allow a cell to participate in the biological experience of multicellularity.

8. Transporters allow living organisms to conduct biological warfare, secreting, for example, antibiotics, antiviral agents, anti-fungal agents, and toxins of humans and other animals that may confer upon the organisms producing such an agent a selective advantage for survival purposes. Many of these toxins are themselves channel-forming proteins or peptides that serve a cell-disruptive transport function.

Polytopic membrane proteins are indispensable to the cellular uptake and homeosta-sis of many essential nutrients. During the past decade it has become clear that a vast number of drugs share transport pathways with nutrients. Moreover, a critical role has been recognized for transport proteins in the absorption, excretion, and toxicity of drug molecules, as well as in their pharmacokinetic and pharmacodynamic (PK/PD) profiles. Because cellular transporter expression is often regulated by nuclear orphan receptors that simultaneously regulate the translation and expression of metabolic enzymes in the cell (e.g., P-glycoprotein and cytochrome P450 regulation by the pregnane X receptor), they indirectly control drug metabolism. Thus, transport proteins are involved in all facets of drug ADME and ADMET (absorption, distribution, metabolism, excretion, and toxicology), conferring an important field of study for pharmaceutical scientists involved in these areas. As a result, in-depth knowledge of membrane transport systems may be extremely useful in the design of new chemical entities (NCE). After all, it is now well appreciated that the most critical parameter for a new drug to survive the drug development pipeline on its way to the market is its ADMET profile.

Despite the involvement of solute transporters in fundamental cellular processes, most are poorly characterized at the molecular level. As a result, we are unable to predictthe interaction of drugs with this important class of membrane proteins a priori, and detection of drug-transporter interactions remains un-acceptably serendipitous.

This section aims to give an overview of current strategies for modeling transporter systems illustrated by three well-characterized transport systems: (1)the P-glycoprotein efflux pump, a prototypical ABC-transporter and a product of the multidrug resistance (MDR-1,ABC-B1) gene, which exports metabolites as well as drugs from various cell types; (2) the small peptide transporter (PepTl, SLC15A1), which transports di- and tri-pep-tide as well as numerous therapeutic compounds; and (3) the apical sodium-dependent bile acid transporter (ASBT, SLC10A2), which plays a key role in intestinal reabsorption and enterohepatic recycling of bile salts, cholesterol homeostasis, and as a therapeutic target for hypocholesterolemic agents (108-110).

1 5.1 The ATP-Binding Cassette (ABC) and I Solute Carrier (SLC) Genetic Superfamilies

1 Organic solutes such as nutrients (amino acids, sugars, vitamins, and bile acids), neurotransmitters, and drugs are transferred across cellular membranes by specialized transport systems. These systems encompass integral membrane proteins that shuttle substrates across the membrane by either a passive process (channels, facilitated transporters) or an active process (carriers), the latter energized directly by the hydrolysis of ATP or indirectly by coupling to the cotransport of a counterion down its electrochemical gradient (e.g., Na+,

Our understanding of the biochemistry and molecular biology of mammalian transport proteins has significantly advanced since the development of expression cloning techniques. Initial studies in Xenopus Jeavis oocytes by Hediger and colleagues resulted in the isolation of the intestinal sodium-dependent glucose transporter SGLT1 (111,112). To date, sequence information and functional data derived from numerous transporters have revealed unifying designs, similar energy-coupling mechanisms, and common evolutionary origins (107). The plethora of isolated transporters motivated the Human Gene Nomenclature Committee to classify these proteins into a distinct genetic super-family named SLC (for SoLute Carrier). Currently, the SLC class contains 37 families with 205 members and is rapidly expanding (http:// The ABC superfamily contains 7 families with 48 members (113); class B contains the well-known multidrug-resistance gene (MDR1) derived P-glycoprotein (ABCB1), whereas class C is composed of members of the multidrug-resistance protein (MRP) subfamily. With the completion of the Human Genome Project, it can be anticipated that a vast number of membrane transport proteins will be identified without known physiological function.

Paulsen and colleagues (114, 115) determined the distribution of membrane transport proteins for all organisms with completely sequenced genomes and identified 81 distinct families. Two superfamilies, the ATP-binding cassette (ABC) and major facilitator

(MFS) superfamilies account for nearly 50%of all transporters in each organism. The other half of these genes will be members of the SLC superfamily. Furthermore, Paulsen predicts that 15% of all genes in the human genome will code for transport proteins. With a current number of estimated sequence-tagged sites (STS) of 30,000 (116), we can expect an additional 4500 membrane transporters to emerge. Thus, the SLC superfamily is anticipated to consist of at least 2300 members; at this moment, only a fraction (10%) has been characterized in certain detail (i.e., membrane topology, substrate specificity, organ expression pattern). Table 8.1 present a concise overview of ABC and SLC members that have been classified and characterized.

The current status of transporter nomenclature and classification is in a similar state of disarray to that which the cytochrome P450 enzyme field found itself in during the early 1980s. However, the Human Gene Nomenclature Committee (HUGO) has taken on the task of directing gene classification and defining distinct subclasses in the ABC and SLC families. Eventually, this may eradicate the rampant use of trivial names for these classes of proteins.

5.2 Therapeutic Implications of Membrane Transporters

It has been generally acknowledged that transporters play an important role in clinical pharmacology (Table 8.2). Several classes of pharmacologically active compounds share transport pathways with nutrients (117). A substantial role has been recognized for transport proteins in oral absorption and drug bioavailability (118) ;drug resistance [e.g., efflux of antineoplastic compounds from tumor cells mediated by multidrug resistance (MDR) gene products (119, 120)]; excretion of drugs and their metabolites, mediated by transporters in the kidney and liver; drug toxicity (121); and drug pharmacokinetics and pharmacodynamics (122-124). Furthermore, the pathophysiology of several hereditary diseases (i.e., clearly defined phenotypes shown to be inherited as monogenic Mendelian traits) has been attributed to mutations in transport proteins. Most of these mutations in human genes and genetic disorders have been recorded and can

Table 8.1 Overview of the ABC and SLC Genetic Superfamilies: Nomenclature and Expression

Gene Symbol


Trivial Names

Organ Expression

ATP-BindingCassette Family

Cholesterol efflux regulatory proteins, photoreceptor proteins Efflux transporters, multiple drug resistance fibrosis transconductance regulators, multiple drug resistance associated proteins Cholesterol, fatty acid transporters, adrenoleukodystrophy-associated L inhibitor TNF-a-stimulated ABC-member, non-

membrane-bound Eye pigment transmembrane permeases



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