F₀F₁-ATPase Family

The two major subdivisions of this family are called F₀F₁-ATPases (or F-type ATPases) and V₀V₁-ATPases (or V-type ATPases) (Figs. 8-4 and 8-5). V₀V₁-ATPases, named for their location in the vacuolar system of eukaryotes, pump protons into organelles and out of Archaea. F₀F₁-ATPases of Bacteria, mitochondria, and chloroplasts generally run in the opposite direction, using proton gradients generated by other membrane proteins to drive ATP synthesis. However, purified F₀F₁-ATPases are freely reversible, using ATP hydrolysis to pump protons or alternatively proton gradients to synthesize ATP. Hence, these enzymes are called both ATP-synthases and F-type ATPases.

Figure 8-4 CRYSTAL STRUCTURE AND MECHANICS OF MITOCHONDRIAL F₁. A, A ribbon diagram viewed from the membrane (bottom) side with α-subunits (red), β-subunits (yellow), and the γ-subunit (blue). All three α-subunits have a bound ATP. The β-subunits are empty or bind ATP or ADP. At the upper right is a space-filling bottom view of the parts of the α- and β-subunits forming the asymmetrical central channel for the γ-subunit (not shown). The electrostatic potential is blue for positive, red for negative, and gray for neutral.B, Oblique view of a ribbon diagram. The γ-subunit forms an antiparallel coiled-coil. At the bottom left is a space-filling model of the γ-subunit. Parts of the surrounding α- and β-subunits are shown as stick diagrams. Note that the surfaces of both the γ-subunit and the channel formed by the α- and β-subunits are hydrophobic and suitable to act as a molecular bearing. C, Drawing of the experimental set-up to show ATP-driven rotation of the γ-subunit relative to the α- and β-subunits. Streptavidin and biotin link the γ-subunit to a bead, which is observed to rotate by light microscopy.

(PDB file: 1BMF. Reference: Abrahams JP, Leslie AGW, Lutter R, Walker JE: Structure at 2.8 Å resolution of F₁-ATPase from bovine heart mitochondria. Nature 370:621–628, 1994.)

Phylogenetic analysis of the subunit polypeptides traces the origin of V-type ATPases to the precursor of all contemporary life forms (see Fig. 1-1). The genes for F-type ATPase subunits arose by divergence in Bacteria after they separated from Archaea. Eukaryotes came to have both F-type ATPases and V-type ATPases when symbiotic Bacteria gave rise to mitochondria and chloroplasts. Two subtle points are of interest here. First, a few Bacteria still have a V-type ATPase. Second, in contrast to the situation in eukaryotes, archaeal V-type ATPases function as ATP synthases similar to mitochondrial and bacterial F-type ATP synthases.

F-type ATPases (ATP Synthases)

F-type ATPases of mitochondria, chloroplasts, and bacterial plasma membranes produce most of the world’s ATP during aerobic metabolism (see Chapter 19). Redox-driven and light-driven pumps create proton gradients to drive ATP synthesis by F-type ATPases. When required by circumstances, many Bacteria use their F-type ATPase to produce a proton gradient at the expense of ATP hydrolysis. Eukaryotes have elaborate mechanisms to inactivate the ATPase and prevent futile cycles of ATP synthesis and hydrolysis. For example, in mitochondria, an inhibitory protein binds the F₁-ATPase if the oxygen supply required to generate the proton gradient is compromised.

F₀F₁-ATPase has two parts (Fig. 8-5). Water-soluble, globular F₁ catalyzes ATP hydrolysis or synthesis. F₀ is embedded in the membrane and passively conducts protons across the lipid bilayer. A stalk connects F₁ to F₀, providing a way to couple proton translocation to ATP synthesis. Given a higher concentration of protons outside a Bacterium or mitochondrion than inside, protons pass through F₀ and drive the synthesis of ATP by F₁. Conversely, in bacteria, ATP hydrolysis by F₁ can drive protons out of the cell.

Pioneering biochemical studies and the crystal structure of F₁ (Fig. 8-4) suggested that rotation of a protein shaft couples proton fluxes in F₀ to ATP synthesis or, alternatively, couples ATP hydrolysis in F₁ to proton pumping by F₀. In the simplest case, bacterial F₁ consists of five different types of polypeptides in the ratio a₃b₃gDe. Mitochondrial F₁ has additional subunits. The α- and β-subunits are folded similarly and arranged alternately like segments of a orange. The γ-subunit is folded into a long, antiparallel, α-helical coiled-coil that forms a shaft. This hydrophobic shaft fits tightly in a hydrophobic sleeve in the middle of the hexamer of α- and β-subunits. To accommodate the asymmetrical shaft, each of the surrounding α- and β-subunits has a slightly different conformation. Each α- and β-subunit has an adenine nucleotide-binding site at the interface with its neighbor. ATP bound stably to α-subunits does not participate in catalysis. Nucleotide-binding sites on β-subunits catalyze ATP synthesis and hydrolysis.

Mechanical rotation of the γ-subunit inside F₁ is tightly coupled to ATP synthesis or hydrolysis. A proton gradient across the membrane can drive a flux of protons through the F₀ complex. This drives clockwise (when viewed from F₀) rotation of the γ-subunit inside the ab hexamer, like the camshaft in a motor. The mechanical force produced by the asymmetric camshaft drives conformational changes in β-subunits that synthesize ATP (Figs. 8-5 and 8-6). When the machine operates in the other direction, ATP hydrolysis drives counterclockwise rotation of the shaft, which can pump protons through F₀.

Figure 8-6 THE BINDING CHANGE MODEL FOR ATP SYNTHESIS BY F₁. Each of the three β-subunits differs in conformation and affinity for nucleotides. The three subunits cycle in succession from the loose (L) state (that binds ADP and P_i), to the tight (T) state (that favors ATP synthesis) to the open (O) state (that releases ATP). Energy provided by the electrochemical gradient of protons (DmH⁺) is required for the γ-subunit to drive each successive transition from loose to tight.

(Reference: Boyer PD: The ATP synthase: A splendid molecular machine. Annu Rev Biochem 66:717–749, 1997.)

Light microscopy is used to observe rotation directly (Fig. 8-4C). F₁ is attached to a glass coverslip, and a tiny bead or actin filament is attached to the free end of the γ-subunit. ATP hydrolysis by the β-subunits drives the rotation of the bead or filament on the γ-subunit. If the bead is magnetic, a rotating magnet field can be used to drive the shaft and synthesize ATP from ADP and phosphate. The γ-subunit rotates at a maximum rate of about 130 times per second (8000 rpm) in mitochondria and twice as fast in chloroplasts.

During ATP hydrolysis or synthesis, the catalytic sites on the β-subunits participate cooperatively, in a sequence of steps coupled to rotation of the shaft. The β-subunits can be in one of three conformations—open, loose, and tight—designating their increasing affinities for adenine nucleotides. At any time in an F₁ molecule, one β-subunit is open, one is loose, and one is tight. All three subunits pass in lock step through the sequence of three states.

In hydrolyzing ATP, the rate-limiting step is ATP bind-ing to the open β-subunit. The energy from ATP binding causes a conformational change that drives an 80° counterclockwise rotation of γ-subunit in less than a millisecond and favors hydrolysis of ATP on the β-subunit that just previously bound ATP. After about 2 milliseconds, another reaction, possibly ADP dissociation from the third β-subunit, rapidly rotates the γ-subunit another 40°, completing a 120° step of the motor.

In synthesizing ATP, the β-subunit in the loose conformation binds ADP and inorganic phosphate (P_i). Energy provided by a 120° rotation of the γ-subunit converts this site to the tight conformation. In the environment created by the active site, ADP and P_i spontaneously form ATP. Energy from a subsequent 120° of the shaft drives the tight state to the open state, which allows ATP to dissociate.

The membrane-embedded F_o complex is a second rotary motor consisting of 12 to 15 protein subunits in the ratio ab₂c_9–12. The c-subunits are simple hairpins of two α-helices. A ring of c-subunits is attached to γ-subunit. The single α-subunit provides a channel for protons to move across the lipid bilayer. This proton channel is thought to be divided into two physically separated parts. To cross the membrane, a proton enters one side of the channel, transfers to aspartate 61 (Asp61) of a c-subunit. Rotation of the ring of c-subunits relative to the α-subunit aligns a protonated Asp61 with the second half of the channel in the α-subunit, allowing the proton to escape on the opposite side from the membrane from which it came. Each ATP synthesized or hydrolyzed is coupled to the transport of three or four protons. Given three ATPs synthesized/hydrolyzed per complete F₁ cycle, 9 to 12 copies of c-subunits in various types of F₀ provide the correct stoichiometry if each transports a single proton.

All of these transitions are reversible in bacterial F₀F₁. With input of energy from proton translocations, F₁ moves through this cycle of states and produces ATP. Alternatively, these reactions can pump protons at the expense of ATP hydrolysis. Reaction of the chemical DCCD with Asp61 on a single c-subunit blocks H⁺ conduction and ATP hydrolysis or synthesis. This inhibi-tion emphasizes the tight coupling of all the subunits of F₀F₁.

V-Type ATPases

Vacuolar ATPases (Fig. 8-5) are found in the membranes bounding acidic compartments in eukaryotic cells, including clathrin-coated vesicles, endosomes, lysosomes (see Chapter 22), Golgi apparatus (see Chapter 21), secretory vesicles (including synaptic vesicles), and plant vacuoles. V-type pumps are also present in the plasma membranes of cells specialized to secrete protons, such as osteoclasts (see Fig. 32-6), macrophages, and kidney tubule intercalated cells.

V-type pumps have two functions. First, they acidify all the compartments listed here using rotation of the c-subunits to drive proton translocation. The acidic pH promotes ligand dissociation from receptors in endosomes and activates lysosomal hydrolases, as well as many other reactions (see Chapter 22). Second, proton gradients across these membranes provide the energy source to drive H⁺−coupled transport of other solutes by carriers, such as the uptake of neurotransmitters by synaptic vesicles (see Figs. 11-8 and 11-9).

Like F-type pumps, V-type ATPases are rotary motors. Most protein subunits of V-type pumps are homologs of their counterparts from F-type pumps. An ancient gene duplication made the c-subunits of eukaryotic V-type pumps twice as large as those in F-type pumps. Six copies of V-type c-subunits provide the same total number of membrane-spanning helices as the 12 F-type c-subunits, but each has only one glutamate equivalent to Asp61. Accordingly, V-type pumps transport only 1 to 2 H⁺ per ATP hydrolyzed.

P-Type Cation Pumps: e₁E₂-ATPases

All living organisms depend on P-type ATPases (Table 8-2) to pump cations across membranes. Their name comes from the fact that they utilize a high-energy covalent β-aspartyl phosphate intermediate. They are also called e₁E₂-ATPases from a description of the conformational changes that they undergo during the course of their mechanism.

Eukaryotic P-type ATPases generate primary ion gradients across the plasma membrane that are required for the function of ion channels (see Chapter 10) and most cation-coupled transport mediated by carrier proteins (see Chapter 9). In animal cells, Na⁺K⁺-ATPase produces the primary gradients of Na⁺ and k⁺. In plants and fungi, the functional homolog H⁺−ATPase generates a proton gradient. Production of these primary ion gradients is expensive, consuming up to 25% of total cellular ATP. Other eukaryotic P-type ATPases acidify the stomach and clear cytoplasm of the second messenger, Ca²⁺ (see Fig. 26-12). Bacterial P-type ATPases scavenge k⁺ and Mg²⁺ from the medium and export Ca²⁺, Cu²⁺, and toxic heavy metals.

The P-type ATPase that is best understood is the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1), which pumps Ca²⁺ out of the cytoplasm into the endoplasmic reticulum (Fig. 8-7). ATP hydrolysis provides energy to move Ca²⁺ across the membrane up a steep concentration gradient. The mechanism is understood in detail, thanks to extensive biochemical analysis and crystal structures of five of the chemical intermediates along the pathway (Fig. 8-8). This analysis was possible because the enzyme is abundant in the sarcoplasmic reticulum of skeletal muscle (see Fig. 39-10), allowing it to be purified in large quantities.

Figure 8-7 Structure of a P-type atpase, the sarcoendoplasmic reticulum calcium–atpase from skeletal muscle. A, The structure of the 2Ca-E1 conformation was determined by X-ray diffraction of crystals formed in the presence of Ca²⁺. Two Ca²⁺ ions bind among four of the ten transmembrane helices near the middle of the membrane bilayer. In the cytoplasm, the N-domain binds nucleotide (ATP) and transfers the γ-phosphate to Asp351 (D351) in the P-domain. (PDB file: 1EUL.)B, Reconstruction of the E2 conformation of the pump from electron micrographs.

(A, Reference: Toyoshima C, Nakasako μ, Nomura H, Ogawa H: Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 Å resolution. Nature 405:647–655, 2000.B, From Toyoshima CH, Sasabe H, Stokes DL: Three-dimensional cryoelectron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane. Nature 362:467–471, 1993.)

Figure 8-8 Reaction mechanism of the sarcoendoplasmic reticulum calcium–atpase. A, Biochemical pathway. The E stands for enzyme, having two conformations: e₁ and e₂. e₁ has the Ca²⁺−binding site oriented toward the cytoplasm. e₂ has the Ca²⁺−binding site oriented toward the lumen of the endoplasmic reticulum. Ca²⁺ binds e₁ on the cytoplasmic side. Subsequent binding of ATP and phosphorylation of the enzyme drive the enzyme toward the e₂ state and transport Ca²⁺ up a steep concentration gradient into the lumen of the endoplasmic reticulum. Dephosphorylation of the enzyme favors a return to the e₁ state.B, Ribbon diagrams of structures along the biochemical pathway. Conformational changes are coupled to ATP hydrolysis and transport of Ca²⁺. Starting on the left side of part A, the pump without bound Ca²⁺ or ATP vacillates between the e₂ and e₁ conformations, alternatively exposing the Ca²⁺−binding sites on the two sides of the membrane. If Ca²⁺ ions are available in the cytoplasm, such as after activation of muscle (see Fig. 39-16), they bind cooperatively to the e₁ conformation with micromolar affinity. Ca²⁺ binding strongly stimulates enzyme activity by favoring Mg-ATP binding to the N-domain. Mg-ATP can bind simultaneously to both the N- and the P-domains, so its presence favors a striking change in conformation that requires the N-domain to rotate and the A-domain to pull on a transmembrane helix that closes a gate between the bound Ca²⁺ and the cytoplasm. This conformation brings the γ-phosphate of ATP into proximity with the side chain of Asp351 and allows formation of the phosphoenzyme intermediate. The equilibrium constant for phosphorylation is near unity, so most of the energy from ATP hydrolysis is stored in a high-energy conformation of the protein. After transfer of γ-phosphate to the enzyme, ADP dissociates. This results in a rotation of the A-domain that moves several transmembrane helices to expose the Ca²⁺−binding sites to the lumen of the endoplasmic reticulum and reduces the affinity for Ca²⁺ by several orders of magnitude. Thus Ca²⁺ dissociates into the lumen, completing its uphill transfer from the cytoplasm. Hydrolysis of the phosphorylated intermediate and dissociation of phosphate reverse the conformational changes in both the cytoplasmic and transmembrane domains, completing the cycle.

(References: Toyoshima C, Nomura H, Tsuda T: Lumenal gating mechanism revealed in calcium pump crystal structures with phosphate analogues. Nature 432:361–368, 2004; and Soerensen T, Moeller JV, Nissen P: Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science 304:1672–1675, 2004. PDB files: 1EUL, 1T5S, 1T5T, IWPG, IIWO.)

The 100-kD Ca²⁺−ATPase consists of two regions. Ten α-helices cross the membrane bilayer and bind two Ca²⁺ ions side by side in the middle of the membrane. The globular region in the cytoplasm consists of three domains. The N-domain binds ATP and transfers its γ-phosphate to aspartic acid 351 (Asp351) in the P-domain. The A-domain transmits large conformational changes in the cytoplasmic domains to the transmembrane helices, which (6 of 10 helices) alternate between two conformations. The e₁ conformation allows access to the Ca²⁺ binding sites from the cytoplasm (Fig. 8-8A). The e₂ conformation allows access from the lumen of the endoplasmic reticulum (Fig. 8-8B). Each step along the pathway—Ca²⁺ binding, ATP binding, phosphorylation of the enzyme, dissociation of ADP, and hydrolysis of the β-aspartyl phosphate intermediate—causes linked conformational changes in both the cytoplasmic domains and six of the transmembrane helices. The changes in the transmembrane domain alters the affinity of the protein for Ca²⁺ and the exposure of Ca-binding sites on the two sides of the membrane. Figure 8-8 describes the cycle of ATP hydrolysis and Ca²⁺ transport in detail. Note that the transition between the e₁ and e₂ conformations involves an “occluded” state in which the bound Ca²⁺ is not accessible on either side of the membrane. This occluded state allows the pump to transport against a large concentration gradient without a leak.

Because the cycle transfers two Ca²⁺ into the lumen and two H⁺ out, it generates an electrical potential (see Chapter 10). In the steady state, the Ca²⁺ gradient is maintained, but the H⁺ gradient dissipates, owing to H⁺ permeability across the membrane and to the buffering capacity of the lumen. All reactions in the pathway are reversible, so a large gradient of Ca²⁺ across the membrane can drive the synthesis of ATP.

All P-type ATPases consist of homologous α-subunits with large cytoplasmic domains and a minimum of six transmembrane helices. Eukaryotic P-type ATPases, such as the Na⁺K⁺− and Ca²⁺−ATPase s have 10 transmembrane helices. Many P-type ATPase are about 110 kD, like the Ca-ATPase, but some are larger or smaller, owing to variable features. Na⁺K⁺−ATPase and H⁺K⁺−ATPase require a 50-kD glycosylated β-subunit with a single transmembrane segment for both transport and intracellular trafficking.

Other P-type ATPases work the same way as the Ca²⁺−ATPase, but with adaptations to pump other ions. For example, the e₁ conformation of the Na⁺K⁺−ATPase of eukaryotic plasma membranes picks up three Na⁺ from the cytoplasm. The P-E₂ conformation releases these Na⁺ one after another outside the cell and then binds two k⁺. Binding of extracellular k⁺ leads to dephosphorylation of the enzyme and results in the occlusion of k⁺ in the KE₂ conformation. ATP binding leads to the release of this k⁺ on the cytoplasmic side and regenerates the e₁ conformation so that the cycle can start over again.

Some P-type ATPases are involved in human diseases. Mutations in Ca²⁺−ATPase cause muscle stiffness and cramps. Mutations in Cu²⁺−ATPase s cause two inherited diseases: Menkes’ syndrome, in which patients are copper-deficient owing to impaired intestinal absorption, and Wilson’s disease, in which the inability to remove copper from the liver is toxic. Omeprazole and related drugs are used to treat ulcers by inhibiting gastric H⁺K⁺−ATPase. Drugs called cardiac glycosides strengthen the heartbeat by inhibiting the cardiac isoform of Na⁺K⁺−ATPase (see Fig. 11-13 for details). These drugs, which derive originally from the foxglove plant, were among the first used to treat congestive heart failure.

ABC Transporters

ABC transporters form the largest and most diverse family of ATP-powered pumps (Table 8-2). They are found in all known organisms, so the founding gene must have originated in the common ancestor of all living things. The genome of baker’s yeast encodes at least 30 ABC transporters, compared with 16 P-type ATPases, one F-type ATPase, and one V-type ATPase. ABC transporters are the largest gene family in the colon bacterium Escherichia coli. In eukaryotes particular family members are located in the plasma membrane, endoplasmic reticulum, and, most likely, other membranes.

Each ABC transporter is specific for one or a few related substrates, but the family as a whole has an enormous range of substrates, including inorganic ions, sugars, amino acids, complex polysaccharides, peptides, and even proteins. Given diverse substrates, the sequences of the transmembrane domains of ABC transporters have diverged much more than their cytoplasmic domains. Specialized members of the family act as ion channels (e.g., the cystic fibrosis transmembrane regulator, CFTR) or regulate other membrane proteins, such as the sulfonylurea receptor.

ABC transporters have a modular design that includes two transmembrane domains and two cytoplasmic domains that hydrolyze ATP (Fig. 8-9A). Each transmembrane domain consists of a bundle of α-helices that spans the bilayer: typically six times but up to ten times in some examples. Two sequences in the nucleotide-binding domain give the family its name (ATP-binding cassette). The Walker A motif (GXXGXGKS/T, where X is any residue) is also called a P loop, since it binds the γ-phosphate of ATP in ABC transporters and other ATP-binding proteins. The Walker B motif (RX_6–8F₄D, where F is any hydrophobic residue) interacts with the Mg²⁺ bound to ATP. Motif B is typically separated from motif A by 100 to 150 residues along the sequence.

Figure 8-9 domain architecture of abc transporters. A, Bacterial transporters.B, Eukaryotic transporters. Each transporter has two ATP-binding domains in the cytoplasm (purple circles) and two transmembrane domains, each consisting of 6 to 10 α-helices (blue or pink squares). CFTR has an additional regulatory (R) domain in the cytoplasm. The four domains required for activity may be four separate polypeptides or may be incorporated in several ways into polypeptides with two or four domains.

Various “experiments” of nature during evolution show that the four independently folded domains of an ABC transporter can be assembled by association of up to four subunits or by folding of a single polypeptide (Fig. 8-9). Gram-negative bacterial transporters often utilize periplasmic subunits that bind and concentrate transported substrates in the vicinity of the pump. Some vertebrate ABC transporters include an additional cytoplasmic R-domain in the single polypeptide for regulation by phosphorylation.

The crystal structure of the E. coli BtuCD vitamin B₁₂ transporter (Fig. 8-10) suggests how ABC transporters might work. The molecule consists of four subunits: two copies of the transmembrane BtuC subunit and two copies of the nucleotide-binding BtuD subunit. Each transmembrane subunit consists of ten transmembrane helices. The interface between the BtuC subunits forms a large chamber open outside the cell (the periplasmic space in this case). The chamber is lined by hydrophobic side chains that are expected to interact with vitamin B₁₂. The chamber penetrates more than halfway across the lipid bilayer but is blocked on the cytoplasmic side by residues that form a gate. The nucleotide-binding cytoplasmic subunits form large interfaces with their partner transmembrane subunits and have a small but highly conserved interface with the other nucleotide-binding subunit. This small interface positions ATP-binding sites of the two subunits next to each other. A periplasmic protein binds vitamin B₁₂ and presents it to the transporter.

Figure 8-10 Structure and proposed mechanism of the E. coli BtuCD vitamin B₁₂ transporter. A, Ribbon diagram of the crystal structure of BtuCD. Two identical BtuC subunits (blue) traverse the membrane. Two identical BtuD subunits (red) bind and hydrolyze ATP. A periplasmic protein BtuF binds vitamin B₁₂ and delivers it to BtuCD. (PDB file: 1L7V.)B, A hypothesis for the mechanism of vitamin B₁₂ transport: BtuCD begins free of both vitamin B₁₂ and ATP; vitamin B₁₂ binding to BtuC promotes ATP binding to BtuD; during a transition state, ATP hydrolysis is coupled to conformational changes that open the gate of BtuC, admitting vitamin B₁₂ into the cytoplasm. Release of ADP and phosphate returns the transporter to its initial state.

(Reference: Locher K, Lee A, Rees D: The E. coli BtuCD structure: A framework for ABC transporter architecture and mechanism. Science 296:1091–1098, 2002.)

It is postulated that ATP binding and hydrolysis drive a cycle of conformational changes that opens the gate, allowing vitamin B₁₂ to escape into the cytoplasm. Binding of substrate to the chamber is expected to increase the affinity of the ABC cassettes for ATP. What happens next is less clear, but an attractive hypothesis is that ATP binding and hydrolysis at the two opposed BtuD active sites transiently open the gate and release B₁₂ in the cytoplasm. Release of ADP and phosphate are presumed to be coupled to closure of the gate. Structures of additional intermediates and more biochemical parameters are required to clarify the mechanism.

ABC transporters with similar mechanisms include bacterial permeases that pump nutrients into the cell, and TAP1 and TAP2 that pump peptide fragments of antigenic proteins into the lumen of the endoplasmic reticulum. Some substrates are membrane bound. ABC transporters such as the E. coli “flippase” MsbA move phospholipids from one leaflet of the bilayer to the other, while yeast STE6 transports a small, prenylated pheromone peptide out of the cell.

Other family members are more problematic. The vertebrate cystic fibrosis transmembrane conductance regulator (CFTR [Fig. 8-9B]) looks like a pump but acts like a channel. It allows Cl⁻ to move down its concentration gradient out of the cell. ATP binding and hydrolysis by the nucleotide-binding domains may open and shut this channel. Mutations in CFTR are responsible for cystic fibrosis (see Fig. 11-4). Of more than 1000 CFTR mutations known to cause cystic fibrosis, by far the most common mutation is deletion of phenylalanine 508. This position corresponds to a highly conserved hydrophobic residue in the vitamin B₁₂ transporter that is important for the interaction of BtuD with BtuC. Mutant DF508 CFTR misfolds and is retained in the ER and destroyed, depriving the plasma membrane of chloride channel activity. Depleting Ca²⁺ from the ER by inhibiting the SERCA Ca²⁺ ATPase can apparently allow DF508 CFTR to escape from calcium-dependent chaperones (see Fig. 20-10) and function on the cell surface.

The multiple drug resistance proteins (MDR1 and MDR2) are ABC transporters that provide a challenge for cancer chemotherapy (Fig. 8-11). In about half of the cases in which chemotherapy fails to cure cancer in humans, the cause is the emergence of clones of tumor cells that overexpress an MDR. Normal cells use a low level of MDR1 to export unknown substrates, perhaps a steroid, a phospholipid, or another hydrophobic molecule. MDR can also transport many hydrophobic compounds, including some chemotherapeutic drugs. These drugs enter cells by dissolving in the membrane, and they subsequently poison vital cellular processes. Cells that overexpress MDR survive by pumping the drug out of the cell.

Figure 8-11 multiple drug resistance in cancer chemotherapy. In a population of tumor cells, most are sensitive to kill-ing by a chemotherapeutic drug. However, variants that express high levels of the ABC transporter, MDR, can clear the cytoplasm of the drug. A clone of these variant cells may expand, allowing the tumor to grow in the presence of the drug.

The multiple drug resistance protein 2 (MDR2) is another unconventional pump located in the apical plasma membrane of liver cells. It is a flippase that moves phosphatidylcholine from the inner to the outer half of the lipid bilayer, perhaps in preparation for secretion in bile.

Some even less conventional ABC transporters appear to regulate ion channels. The sulfonylurea receptor (SUR) is an ABC transporter required for the function of an ATP-sensitive potassium channel (K_ATP) that regulates insulin secretion. SUR binds drugs called sulfonylureas that are used to treat forms of diabetes involving inadequate insulin secretion. These drugs activate secretion by inhibiting the k_ATP channel (see Chapter 10).