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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 15.5Active Transport by ATP-Powered Pumps

We turn now to the ATP-powered pumps that transport ions and various small molecules against their concentration gradients. The general structures of the four principal classes of these transport proteins are depicted in Figure 15-10, and their properties are summarized in Table 15-2. Note that the P, F, and V classes transport ions only, whereas the ABC superfamily class transports small molecules as well as ions.

Figure 15-10. The four classes of ATP-powered transport proteins.

Figure 15-10

The four classes of ATP-powered transport proteins. P-class pumps are composed of two different polypeptides, α and β, and become phosphorylated as part of the transport cycle. The (more...)

Table 15-2. Comparison of Major Classes of ATP-Powered Ion and Small-Molecule Pumps.

Table 15-2

Comparison of Major Classes of ATP-Powered Ion and Small-Molecule Pumps.

P-class ion pumps contain a transmembrane catalytic α subunit, which contains an ATP-binding site, and usually a smaller β subunit, which may have regulatory functions. Many of these pumps are tetramers composed of two α and two β subunits. During the transport process, at least one of the α subunits is phosphorylated (hence the label “P”), and the transported ions are thought to move through the phosphorylated subunit. This class includes the Na+/K+ ATPase in the plasma membrane, which maintains the Na+ and K+ gradients typical of animal cells, and several Ca2+ ATPases, which pump Ca2+ ions out of the cytosol into the external medium or into the lumen of the sarcoplasmic reticulum (SR) of muscle cells. Another member of the P class, found in acid-secreting cells of the mammalian stomach, transports protons (H+ ions) out of and K+ ions into the cell. The H+ pump that maintains the membrane electric potential in plant, fungal, and bacterial cells also belongs to this class.

The structures of F-class and V-class ion pumps are similar to each other but unrelated to and more complicated than P-class pumps. F- and V-class pumps contain at least three kinds of transmembrane proteins and five kinds of extrinsic polypeptides that form the cytosolic domain. Several of the transmembrane and extrinsic subunits in F-class and V-class pumps exhibit sequence homology, and each pair of homologous subunits is thought to have evolved from a common polypeptide.

All known V and F pumps transport only protons in a process that does not involve a phosphoprotein intermediate. V-class pumps generally function to maintain the low pH of plant vacuoles and of lysosomes and other acidic vesicles in animal cells by using the energy released by ATP hydrolysis to pump protons from the cytosolic to the exoplasmic face of the membrane against the proton electrochemical gradient. F-class pumps are found in bacterial plasma membranes and in mitochondria and chloroplasts. In contrast to V pumps, they generally function to power the synthesis of ATP from ADP and Pi by movement of protons from the exoplasmic to the cytosolic face of the membrane down the proton electrochemical gradient. Because of their importance in ATP synthesis in chloroplasts and mitochondria, F-class proton pumps are treated separately in the next chapter.

The final class of ATP-powered transport proteins is larger and more diverse than the other classes. Referred to as the ABC (ATP-binding cassette) superfamily, this class includes more than 100 different transport proteins found in organisms ranging from bacteria to humans. Each ABC protein is specific for a single substrate or group of related substrates including ions, sugars, peptides, polysaccharides, and even proteins. All ABC transport proteins share a common organization consisting of four “core” domains: two transmembrane (T) domains, forming the passageway through which transported molecules cross the membrane, and two cytosolic ATP-binding (A) domains. In some ABC proteins, the core domains are present in four separate polypeptides; in others, the core domains are fused into one or two multidomain polypeptides.

All classes of ATP-powered pumps have one or more binding sites for ATP, and these are always on the cytosolic face of the membrane (see Figure 15-10). Although these proteins are often called ATPases, they normally do not hydrolyze ATP into ADP and Pi unless ions or other molecules are simultaneously transported. Because of the tight coupling between ATP hydrolysis and transport, the energy stored in the phosphoanhydride bond is not dissipated. Thus ATP-powered transport proteins are able to collect the free energy released during ATP hydrolysis and use it to move ions or other molecules uphill against a potential or concentration gradient.

The energy expended by cells to maintain the concentration gradients of Na+, K+, H+, and Ca2+ across the plasma and intracellular membranes is considerable. In nerve and kidney cells, for example, up to 25 percent of the ATP produced by the cell is used for ion transport; in human erythrocytes, up to 50 percent of the available ATP is used for this purpose. In cells treated with poisons that inhibit the aerobic production of ATP (e.g., 2,4-dinitrophenol), the ion concentration inside the cell gradually approaches that of the exterior environment as the ions move through plasma membrane channels down their electric and concentration gradients. Eventually treated cells die: partly because protein synthesis requires a high concentration of K+ ions and partly because in the absence of a Na+ gradient across the cell membrane, a cell cannot import certain nutrients such as amino acids. Studies on the effects of such poisons provided early evidence for the existence of ion pumps. In this section, we discuss in some detail examples of the P, V, and ABC classes of ATP-powered pumps.

Plasma-Membrane Ca2+ ATPase Exports Ca2+ Ions from Cells

As discussed in Chapter 20, small increases in the concentration of free Ca2+ ions in the cytosol trigger a variety of cellular responses. In order for Ca2+ to function in intracellular signaling, its cytosolic concentration usually must be kept below 0.1 – 0.2 μM. (Although some cytosolic Ca2+ is bound to negatively charged groups, it is the concentration of free, unbound Ca2+ that is critical to its signaling function.) The plasma membranes of animal, yeast, and probably plant cells contain Ca2+ ATPases that transport Ca2+ out of the cell against its electrochemical gradient. These P-class ion pumps help maintain the concentration of free Ca2+ ions in the cytosol at a low level.

In addition to a catalytic α subunit containing an ATP-binding site, as found in other P-class pumps, plasmamembrane Ca2+ ATPases also contain the Ca2+-binding regulatory protein calmodulin. A rise in cytosolic Ca2+ induces the binding of Ca2+ ions to calmodulin, which triggers an allosteric activation of the Ca2+ ATPase; as a result, the export of Ca2+ ions from the cell accelerates, and the original low cytosolic concentration of free Ca2+ is restored rapidly.

Muscle Ca2+ ATPase Pumps Ca2+ Ions from the Cytosol into the Sarcoplasmic Reticulum

Besides the plasma-membrane Ca2+ ATPase, muscle cells contain a second, different Ca2+ ATPase that transports Ca2+ from the cytosol into the lumen of the sarcoplasmic reticulum (SR), an internal organelle that concentrates and stores Ca2+ ions. As discussed in Chapter 18, the SR and its calcium pump (referred to as the muscle calcium pump) are critical in muscle contraction and relaxation: release of Ca2+ ions from the SR into the muscle cytosol causes contraction, and the rapid removal of Ca2+ ions from the cytosol by the muscle calcium pump induces relaxation.

Because the muscle calcium pump constitutes more than 80 percent of the integral protein in SR membranes, it is easily purified and characterized. Each transmembrane catalytic α subunit has a molecular weight of 100,000 and transports two Ca2+ ions per ATP hydrolyzed. In the cytosol of muscle cells, the free Ca2+ concentration ranges from 10−7 M (resting cells) to more than 10−6 M (contracting cells), whereas the total Ca2+ concentration in the SR lumen can be as high as 10−2 M. Sites on the cytosolic surface of the muscle calcium pump have a very high affinity for Ca2+ (Km = 10−7 M), allowing the pump to transport Ca2+ efficiently from the cytosol into the SR against the steep concentration gradient.

The concentration of free Ca2+ within the sarcoplasmic reticulum is actually much less than the total concentration of 10−2 M. Two soluble proteins in the lumen of SR vesicles bind Ca2+ and serve as a reservoir for intracellular Ca2+, thereby reducing the concentration of free Ca2+ ions in the SR vesicles, and consequently decreasing the energy needed to pump Ca2+ ions into them from the cytosol. The activity of the muscle Ca2+ ATPase is so regulated that if the free Ca2+ concentration in the cytosol becomes too high, the rate of calcium pumping increases until the cytosolic Ca2+ concentration is reduced to less than 1 μM. Thus in muscle cells, the calcium pump in the SR membrane can supplement the activity of the plasma-membrane pump, assuring that the cytosolic concentration of free Ca2+ remains below 1 μM.

The current model of the mechanism of action of the Ca2+ ATPase in the SR membrane is outlined in Figure 15-11. Coupling of ATP hydrolysis with ion pumping involves several steps that must occur in a defined order. When the protein is in one conformation, termed E1, two Ca2+ ions bind in sequence to high-affinity sites on the cytosolic surface (step 1). Then an ATP binds to its site on the cytosolic surface; in a reaction requiring that a Mg2+ ion be tightly complexed to the ATP, the bound ATP is hydrolyzed to ADP and the liberated phosphate is transferred to a specific aspartate residue in the protein, forming a high-energy acyl phosphate bond, denoted by E1~P (step 2). The protein then changes its conformation to E2 – P, generating two lowaffinity Ca2+-binding sites on the exoplasmic surface, which faces the SR lumen; this conformational change simultaneously propels the two Ca2+ ions through the protein to these sites (step 3) and inactivates the high-affinity Ca2+-binding sites on the cytosolic face. The Ca2+ ions then dissociate from the exoplasmic surface of the protein (step 4). Following this, the aspartyl-phosphate bond in E2 – P is hydrolyzed, causing E2 to revert to E1, a change that inactivates the exoplasmic-facing Ca2+-binding sites and regenerates the cytosolicfacing Ca2+-binding sites (step 5).

Figure 15-11. Model of the mechanism of action of muscle Ca2+ ATPase, which is located in the sarcoplasmic reticulum (SR) membrane.

Figure 15-11

Model of the mechanism of action of muscle Ca2+ ATPase, which is located in the sarcoplasmic reticulum (SR) membrane. Only one of the two α subunits of this P-class pump is (more...)

Thus phosphorylation of the muscle calcium pump by ATP favors conversion of E1 to E2, and dephosphorylation favors the conversion of E2 to E1. While only E2 – P, not E1~P, is actually hydrolyzed, the free energy of hydrolysis of the aspartyl-phosphate bond in E1~P is greater than that for E2 – P. The reduction in free energy of the aspartyl-phosphate bond in E2 – P, relative to E1~P, can be said to power the E1 → E2 conformational change. The affinity of Ca2+ for the cytosolic-facing binding sites in E1 is a thousandfold greater than the affinity of Ca2+ for the exoplasmic-facing sites in E2; this difference enables the protein to transport Ca2+ unidirectionally from the cytosol, where it binds tightly to the pump, to the exoplasm, where it is released.

Much evidence supports the model depicted in Figure 15-11. For instance, the muscle calcium pump has been isolated with phosphate linked to an aspartate residue, and spectroscopic studies have detected slight alterations in protein conformation during the E1 → E2 conversion. On the basis of the protein’s amino acid sequence and various biochemical studies, investigators proposed the structural model for the catalytic α subunit shown in Figure 15-12. The membrane-spanning α helices are thought to form the passageway through which Ca2+ ions move. The bulk of the subunit consists of cytosolic globular domains that are involved in ATP binding, phosphorylation of aspartate, and energy transduction. These domains are connected by “stalks” to the membrane-embedded domain.

Figure 15-12. Schematic structural model for the catalytic (α) subunit of muscle Ca2+ ATPase.

Figure 15-12

Schematic structural model for the catalytic (α) subunit of muscle Ca2+ ATPase. The 10 transmembrane α helices are thought to form a channel through which Ca2+ ions move. (more...)

As noted previously, all P-class ion pumps, regardless of which ion they transport, are phosphorylated during the transport process. The amino acid sequences around the phosphorylated aspartate in the catalytic α subunit are highly conserved in all proteins of this type. Thus the mechanistic model in Figure 15-11 probably is generally applicable to all these ATP-powered ion pumps. In addition, the α subunits of all the P pumps examined to date have a similar molecular weight and, as deduced from their amino acid sequences derived from cDNA clones, have a similar arrangement of transmembrane α helices (see Figure 15-12). These findings strongly suggest that all these proteins evolved from a common precursor, although they now transport different ions.

Na+/K+ ATPase Maintains the Intracellular Na+ and K+ Concentrations in Animal Cells

A second P-class ion pump that has been studied in considerable detail is the Na+/K+ ATPase present in the plasma membrane of all animal cells. This ion pump is a tetramer of subunit composition α2β2. (Classic Experiment 15.1 describes the discovery of this enzyme.) The β polypeptide is required for newly synthesized α subunits to fold properly in the endoplasmic reticulum but apparently is not involved directly in ion pumping. The α subunit is a 120,000-MW nonglycosylated polypeptide whose amino acid sequence and predicted membrane structure are very similar to those of the muscle Ca2+ ATPase. In particular, the Na+/K+ ATPase has a stalk on the cytosolic face that links domains containing the ATP-binding site and the phosphorylated aspartate to the membrane-embedded domain. The overall process of transport moves three Na+ ions out of and two K+ ions into the cell per ATP molecule split (Figure 15-13a).

Figure 15-13. Models for the structure and function of the Na+/K+ ATPase in the plasma membrane.

Figure 15-13

Models for the structure and function of the Na+/K+ ATPase in the plasma membrane. (a) This P-class pump comprises two copies each of a small (more...)

Several lines of evidence indicate that the Na+/K+ ATPase is responsible for the coupled movement of K+ and Na+ into and out of the cell, respectively. For example, the drug ouabain, which binds to a specific region on the exoplasmic surface of the protein and specifically inhibits its ATPase activity, also prevents cells from maintaining their Na+/K+ balance. Any doubt that the Na+/K+ ATPase is responsible for ion movement was dispelled by the demonstration that the enzyme, when purified from the membrane and inserted into liposomes, propels K+ and Na+ transport in the presence of ATP.

The mechanism of action of the Na+/K+ ATPase, outlined in Figure 15-13b, is similar to that of the muscle calcium pump, except that ions are pumped in both directions across the membrane. In its E1 conformation, the Na+/K+ ATPase has three high-affinity Na+-binding sites and two low-affinity K+-binding sites on the cytosolic-facing surface of the protein. The Km for binding of Na+ to these cytosolic sites is 0.6 mM, a value considerably lower than the intracellular Na+ concentration of ≈12 mM; as a result, Na+ ions normally will fill these sites. Conversely, the affinity of the cytosolic K+-binding sites is low enough that K+ ions, transported inward through the protein, dissociate from E1 into the cytosol despite the high intracellular K+ concentration. During the E1 → E2 transition, the three bound Na+ ions move outward through the protein. Transition to the E2 conformation also generates two high-affinity K+ sites and three low-affinity Na+ sites on the exoplasmic face. Because the Km for K+ binding to these sites (0.2 mM) is considerably lower than the extracellular K+ concentration (4 mM), these sites will fill quickly with K+ ions. In contrast, the three Na+ ions, transported outward through the protein, will dissociate into the extracellular medium from the low-affinity Na+ sites on the exoplasmic surface despite the high extracellular Na+ concentration. Similarly, during the E2 → E1 transition, the two bound K+ ions are transported inward.

V-Class H+ ATPases Pump Protons across Lysosomal and Vacuolar Membranes

All V-class ATPases transport H+ ions only. These proton pumps, present in the membranes of lysosomes, endosomes, and plant vacuoles, function to acidify the lumen of these organelles. The acidity of the lysosomal lumen, usually ≈4.5 – 5.0, can be measured precisely in living cells by use of particles labeled with a pH-sensitive fluorescent dye. Cells phagocytose these particles (see Figure 5-44a) and transfer them to the lysosomes. The ability of different wavelengths of visible light to excite fluorescence is highly dependent on pH, and the lysosomal pH can be calculated from the spectrum of the fluorescence emitted. Maintenance of the 100-fold or more proton gradient between the lysosomal lumen (pH ≈4.5 – 5.0) and the cytosol (pH ≈7.0) depends on ATP production by the cell.

The ATP-powered proton pumps in lysosomal and vacuolar membranes have been isolated, purified, and incorporated into liposomes. As illustrated in Figure 15-10, these V-class proton pumps contain two discrete domains: a cytosolic-facing hydrophilic domain (V1) composed of five different polypeptides and a transmembrane domain (V0) containing 9 – 12 copies of proteolipid c, one copy of protein b, and one copy of protein a. The subunit composition of the cytosolic domain is α3β3γδϵ; the α and β subunits contain the sites where ATP binding and hydrolysis occur. Each transmembrane c subunit is thought to span the membrane two times; the c and a subunits together form the proton-conducting channel. Unlike P-class ion pumps, the V-class H+ ATPases are not phosphorylated and dephosphorylated during proton transport.

Similar V-class ATPases are found in the plasma membrane of certain acid-secreting cells. These include osteoclasts, bone-resorbing macrophagelike cells, which bind to a bone and seal off a small segment of extracellular space between the plasma membrane and the surface of the bone. HCl secreted into this space by osteoclasts dissolves the calcium phosphate crystals that give bone its rigidity and strength.

Another example is the mitochondria-rich epithelial cells lining the toad bladder; the apical plasma membrane of these cells contain many V-class H+ ATPases, which function to acidify the urine (Figure 15-14). As we discuss later, the membrane of plant vacuoles contains two proton pumps: a typical V-class H+ ATPase and another one that utilizes the energy released by hydrolysis of inorganic pyrophosphate (PPi) to pump protons into the vacuole. This PPi-hydrolyzing proton pump, believed to be unique to plants, has an amino acid sequence different from any other ion-transporting proteins.

Figure 15-14. The plasma membrane of certain acid-secreting cells contains an almost crystalline array of V-class H+ ATPases.

Figure 15-14

The plasma membrane of certain acid-secreting cells contains an almost crystalline array of V-class H+ ATPases. This electron micrograph is of a platinum replica of the cytosolic (more...)

ATP-powered proton pumps cannot acidify the lumen of an organelle (or the extracellular space) by themselves. The reason for this is that pumping of protons would rapidly cause a buildup of positive charge on the exoplasmic face of the membrane on the inside of the vesicle membrane and a corresponding buildup of negative charges on the cytosolic face. In other words, the pump would generate a voltage across the membrane, exoplasmic face positive, which would prevent movement of protons into the vesicle before a significant H+ concentration gradient had been established. In fact, this is the way that H+ pumps generate an insidenegative potential across plant and yeast plasma membranes. In order for an organelle lumen or an extracellular space (e.g., the outside of an osteoclast) to become acidic, movement of H+ up its concentration gradient must be accompanied by (1) movement of an equal number of anions in the same direction or (2) movement of equal numbers of a different cation in the opposite direction. The first process occurs in lysosomes and plant vacuoles whose membranes contain V-class H+ ATPases and ion channels through which accompanying anions (e.g., Cl) move. The second occurs in the lining of the stomach, which contains a P-class H+/K+ ATPase that pumps one H+ outward and one K+ inward.

The ABC Superfamily Transports a Wide Variety of Substrates

As noted earlier, all members of the very large and diverse ABC superfamily of transport proteins contain two transmembrane (T) domains and two cytosolic ATP-binding (A) domains (see Figure 15-10). The T domains, each built of six membrane-spanning α helices, form the pathway through which the transported substance (substrate) crosses the membrane and determine the substrate specificity of each ABC protein. The sequence of the A domains is ≈30 to 40 percent homologous in all members of this superfamily, indicating a common evolutionary origin. Some ABC proteins also contain a substrate-binding subunit or regulatory subunit.

Bacterial Plasma-Membrane Permeases

The plasma membrane of many bacteria contain numerous permeases that belong to the ABC superfamily. These proteins use the energy released by hydrolysis of ATP to transport specific amino acids, sugars, vitamins, or even peptides into the cell. Since bacteria frequently grow in soil or pond water where the concentration of nutrients is low, these ABC transport proteins allow the cells to concentrate amino acids and other nutrients in the cell against a substantial concentration gradient. Bacterial permeases generally are inducible; that is, the quantity of a transport protein in the cell membrane is regulated by both the concentration of the nutrient in the medium and the metabolic needs of the cell.

In E. coli histidine permease, a typical bacterial ABC protein, the two transmembrane domains and two cytosolic ATP-binding domains are formed by four separate subunits. In gram-negative bacteria such as E. coli, which have an outer membrane, a soluble histidine-binding protein in the periplasmic space assists in transport (Figure 15-15). This soluble protein binds histidine tightly and directs it to the T subunits, through which histidine crosses the membrane powered by ATP hydrolysis. Mutant E. coli cells that are defective in any of the histidine-permease subunits or the soluble binding protein are unable to transport histidine into the cell, but are able to transport other amino acids whose uptake is facilitated by other transport proteins. Such genetic analyses provide strong evidence that histidine permease and similar ABC proteins function to transport solutes into the cell.

Figure 15-15. Gram-negative bacteria import many solutes by means of ABC proteins (permeases) that utilize a soluble substrate-binding protein present in the periplasmic space.

Figure 15-15

Gram-negative bacteria import many solutes by means of ABC proteins (permeases) that utilize a soluble substrate-binding protein present in the periplasmic space. Depicted here is (more...)

Mammalian MDR Transport Proteins

Image med.jpg A series of rather unexpected observations led to discovery of the first eukaryotic ABC protein. Oncologists noted that tumor cells often became simultaneously resistant to several chemotherapeutic drugs with unrelated chemical structures; similarly, cell biologists observed that cultured cells selected for resistance to one toxic substance (e.g., colchicine, a microtubule inhibitor) frequently became resistant to several other drugs, including the anticancer drug adriamycin. Subsequent studies showed that this resistance is due to enhanced expression of amultidrug-resistance (MDR) transport protein known as MDR1. In this member of the ABC superfamily, all four domains are “fused” into a single 170,000-MW protein (Figure 15-16). This protein uses the energy derived from ATP hydrolysis toexport a large variety of drugs from the cytosol to the extracellular medium. The Mdr1 gene is frequently amplified in multidrug-resistant cells, resulting in a large overproduction of the MDR1 protein.

Figure 15-16. Schematic structural model for mammalian MDR1 protein. In this member of the ABC superfamily, the two transmembrane domains and two cytosolic ATP-binding domains are part of a single polypeptide.

Figure 15-16

Schematic structural model for mammalian MDR1 protein. In this member of the ABC superfamily, the two transmembrane domains and two cytosolic ATP-binding domains are part of a (more...)

Most drugs transported by MDR1 are small hydrophobic molecules, which diffuse from the culture medium across the plasma membrane into the cell. The ATP-powered export of such drugs from the cytosol by MDR1 means a much higher extracellular drug concentration is required to kill cells. That MDR1 is an ATP-powered small-molecule pump has been demonstrated with liposomes containing the purified protein (see Figure 15-4). The ATPase activity of these liposomes is enhanced by different drugs in a dose-dependent manner corresponding to their ability to be transported by MDR1.

Not only does MDR1 transport a varied group of molecules, but all these substrates compete with one another for transport by MDR1. Although the mechanism of action of MDR1-assisted transport has not been definitively demonstrated, the flippase model, depicted in Figure 15-17a, is a likely candidate. Substrates of MDR1 are primarily planar, lipid-soluble molecules with one or more positive charges, and they move spontaneously from the cytosol into the cytosolic-facing leaflet of the plasma membrane. The hydrophobic portion of a substrate molecule is oriented toward the hydrophobic core of the membrane, and the charged portion toward the polar cytosolic face of the membrane and is still in the cytosol. The substrate diffuses laterally until encountering and binding to a site on the MDR1 protein that is within the bilayer. The protein then “flips” the charged substrate molecule into the exoplasmic leaflet, an energetically unfavorable reaction powered by the coupled ATPase activity of MDR1. Once in the exoplasmic face, the substrate diffuses into the aqueous phase on the outside of the cell. Support for the flippase model of transport by MDR1 comes from MDR2, a homologous protein present in the region of the liver cell plasma membrane that faces the bile duct. MDR2 has been shown to flip phospholipids from the cytosolic-facing leaflet of the plasma membrane to the exoplasmic leaflet, thereby generating an excess of phospholipids in the exoplasmic leaflet; these phospholipids peel off into the bile duct and form an essential part of the bile. An alternative pump model also has been proposed for MDR1 (Figure 15-17b). According to this model, drug molecules in the cytosol bind directly to a single small-molecule binding site on the cytosolic face of the MDR1 protein; subsequent ATP hydrolysis powers movement of the bound drug through the protein to the aqueous phase on the outside of the cell by a mechanism similar to that of other ATP-powered pumps.

Figure 15-17. Possible mechanisms of action of the MDR1 protein.

Figure 15-17

Possible mechanisms of action of the MDR1 protein. (a) The flippase model proposes that a lipid-soluble molecule first dissolves in the cytosolic-facing leaflet of the plasma membrane (more...)

MDR1 protein is expressed in abundance in the liver, intestines, and kidney — sites from which natural toxic products are removed from the body. Thus the natural function of MDR1 may be to transport a variety of natural and metabolic toxins into the bile, intestinal lumen, or forming urine. During the course of its evolution, MDR1 appears to have coincidentally acquired the ability to transport drugs whose structures are similar to those of these toxins. Tumors derived from these cell types, such as hepatomas (liver cancers), frequently are resistant to virtually all chemotherapeutic agents and thus difficult to treat, presumably because the tumors exhibit increased expression of the MDR1 or MDR2 proteins.

Cystic Fibrosis Transmembrane Regulator (CFTR) Protein

Image med.jpg Discovery of another ABC transport protein came from studies of cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease of Caucasians. This disease is caused by a mutation in the CFTR gene, which encodes a chloride-channel protein that is regulated by cyclic AMP (cAMP), an intracellular second messenger. These Clchannels are present in the apical plasma membranes of epithelial cells in the lung, sweat glands, pancreas, and other tissues. An increase in cAMP stimulates Cltransport by such cells from normal individuals, but not from CF individuals who have a defective CFTR protein.

The sequence and predicted structure of the encoded CFTR protein, based on analysis of the cloned gene, are very similar to those of MDR1 protein except for the presence of an additional domain, the regulatory (R) domain, on the cytosolic face. The Cl-channel activity of CFTR protein clearly is enhanced by binding of ATP. Moreover, as detailed in Chapter 20, cAMP activates a protein kinase that phosphorylates, and thereby activates, CFTR. When purified CFTR protein is incorporated into liposomes, it forms Cl channels with properties similar to those in normal epithelial cells. And when the wild-type CFTR protein is expressed by recombinant techniques in cultured epithelial cells from CF patients, the cells recover normal Cl-channel activity. This latter result raises the possibility that gene therapy might reverse the course of cystic fibrosis.

Since CFTR protein is similar to MDR1 in structure, it may also function as an ATP-powered pump of some as-yet unidentified molecule. In any case, much remains to be learned about this fascinating class of ABC transport proteins.


  •  Four types of membrane transport proteins couple the energy-releasing hydrolysis of ATP with the energy-requiring transport of substances against their concentration gradient (see Figure 15-10 and Table 15-2).
  •  In P-class pumps, phosphorylation of the α subunit and a change in conformational states are essential for coupled transport of H+, Na+, K+, or Ca2+ ions (see Figures 15-11 and 15-13).
  •  The P-class Na+/K+ ATPase pumps three Na+ ions out of and two K+ ions into the cell per ATP hydrolyzed. A homolog, the Ca2+ ATPase, pumps two Ca2+ ions out of the cell or, in muscle, into the sarcoplasmic reticulum per ATP hydrolyzed. The combined action of these pumps in animal cells creates an intracellular ion milieu of high K+, low Ca2+, and low Na+ very different from the extracellular fluid milieu of high Na+, high Ca2+, and low K+.
  •  In the multisubunit V-and F-class ATPases, which pump protons exclusively, a phosphorylated protein is not an intermediate in transport.
  •  A V-class H+ pump in animal lysosomal and endosomal membranes and plant vacuole membranes is responsible for maintaining a lower pH inside the organelles than in the surrounding cytosol.
  •  All members of the large and diverse ABC superfamily of transport proteins contain four core domains: two transmembrane domains, which form a pathway for solute movement and determine substrate specificity, and two cytosolic ATP-binding domains.
  •  The ABC superfamily includes bacterial amino acid and sugar permeases (see Figure 15-15); the mammalian MDR1 protein, which exports a wide array of drugs from cells; and CFTR protein, a Cl channel that is defective in cystic fibrosis.
  •  According to the flippase model of MDR1 activity, a substrate molecule diffuses into the cytosolic leaflet of the plasma membrane, then is flipped to the exoplasmic leaflet in an ATP-powered process, and finally diffuses from the membrane into the extracellular space (see Figure 15-17a).

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