Chapter 10

Membrane Transport

"Drawbridge at Arles with a Group of Washerwomen" (1888) by Vincent van Gogh
(Rikjsmuseum Kroller-Muller; photo by Erich Lessing/Art Resource)

Transport processes are vitally important to all life forms because all cells must exchange materials with their environment. Cells must obviously have ways to bring nutrient molecules into the cell and ways to send waste products and toxic substances out. Also, inorganic electrolytes must be able to pass in and out of cells and across organelle membranes. All cells maintain concentration gradients of various metabolites across their plasma membranes and also across the membranes of intracellular organelles. By their very nature, cells maintain a very large amount of potential energy in the form of such concentration gradients. Sodium and potassium ion gradients across the plasma membrane mediate the transmission of nerve impulses and the normal functions of the brain, heart, kidneys, and liver, among other organs. Storage and release of calcium from cellular compartments controls muscle contraction, and also the response of many cells to hormonal signals. High acid concentrations in the stomach are required for the digestion of food. Extremely high hydrogen ion gradients are maintained across the plasma membranes of the mucosal cells lining the stomach in order to maintain high acid levels in the stomach yet protect the cells that constitute the stomach walls from the deleterious effects of such acid.
            In this chapter, we shall consider the molecules and mechanisms that mediate these transport activities. In nearly every case, the molecule or ion transported is water-soluble, yet moves across the hydrophobic, impermeable lipid membrane at a rate high enough to serve the metabolic and physiologic needs of the cell. This perplexing problem is solved in each case by a specific transport protein. The transported species either diffuses through a channel-forming protein or is carried by a carrier protein. Transport proteins are all classed as integral membrane proteins (Chapter 9), ranging in size from small peptides to large, multisubunit protein complexes.
            Some transport proteins merely provide a path for the transported species, whereas others couple an enzymatic reaction with the transport event. In all cases, transport behavior depends on the interactions of the transport protein not only with solvent water but with the lipid milieu of the membrane as well. The dynamic and asymmetric nature of the membrane and its components (Chapter 9) plays an important part in the function of these transport systems.
            From a thermodynamic and kinetic perspective, there are only three types of membrane transport processes: passive diffusion, facilitated diffusion, and active transport. To be thoroughly appreciated, membrane transport phenomena must be considered in terms of thermodynamics. Some of the important kinetic considerations also will be discussed.

10.1 · Passive Diffusion

Passive diffusion is the simplest transport process. In passive diffusion, the transported species moves across the membrane in the thermodynamically favored direction without the help of any specific transport system/molecule. For an uncharged molecule, passive diffusion is an entropic process, in which movement of molecules across the membrane proceeds until the concentration of the substance on both sides of the membrane is the same. For an uncharged molecule, the free energy difference between side 1 and side 2 of a membrane (Figure 10.1) is given by

Figure 10.1 · Passive diffusion of an uncharged species across a membrane depends only on the concentrations (C₁ and C₂) on the two sides of the membrane.

              (10.1)

The difference in concentrations, [C₂] - [C₁], is termed the concentration gradient, and DG here is the chemical potential difference.

Passive Diffusion of a Charged Species

For a charged species, the situation is slightly more complicated. In this case, the movement of a molecule across a membrane depends on its electrochemical potential. This is given by

            DG=G₂-G₁=RT ln +ZDc           (10.2)

where Z is the charge on the transported species, Á is Faraday’s constant (the charge on 1 mole of electrons = 96,485 coulombs/mol = 96,485 joules/volt · mol, because 1 volt = 1 joule/coulomb), and Dy is the electric potential difference (that is, voltage difference) across the membrane. The second term in the expression thus accounts for the movement of a charge across a potential difference. Note that the effect of this second term on DG depends on the magnitude and the sign of both Z and Dy . For example, as shown in Figure 10.2, if side 2 has a higher potential than side 1 (so that Dy is positive), for a negatively charged ion the term Z Á Dy makes a negative contribution to DG.

Figure 10.2 · The passive diffusion of a charged species across a membrane depends upon the concentration and also on the charge of the particle, Z, and the electrical potential difference across the membrane, Dy.

            In other words, the negative charge is spontaneously attracted to the more positive potential— and DG is negative. In any case, if the sum of the two terms on the right side of Equation 10.2 is a negative number, transport of the ion in question from side 1 to side 2 would occur spontaneously. The driving force for passive transport is the DG term for the transported species itself.

10.2 · Facilitated Diffusion

The transport of many substances across simple lipid bilayer membranes via passive diffusion is far too slow to sustain life processes. On the other hand, the transport rates for many ions and small molecules across actual biological membranes is much higher than anticipated from passive diffusion alone. This difference is due to specific proteins in the membrane that facilitate transport of these species across the membrane. Similar proteins capable of effecting facilitated diffusion of a variety of solutes are present in essentially all natural membranes. Such proteins have two features in common: (a) they facilitate net movement of solutes only in the thermodynamically favored direction (that is, DG < 0), and (b) they display a measurable affinity and specificity for the transported solute. Consequently, facilitated diffusion rates display saturation behavior similar to that observed with substrate binding by enzymes (Chapter 14). Such behavior provides a simple means for distinguishing between passive diffusion and facilitated diffusion experimentally. The dependence of transport rate on solute concentration takes the form of a rectangular hyperbola (Figure 10.3), so that the transport rate approaches a limiting value, V_max , at very high solute concentration. Figure 10.3 also shows the graphical behavior exhibited by simple passive diffusion. Because passive diffusion does not involve formation of a specific solute:protein complex, the plot of rate versus concentration is linear, not hyperbolic.

Figure 10.3 · Passive diffusion and facilitated diffusion may be distinguished graphically. The plots for facilitated diffusion are similar to plots of enzyme-catalyzed processes (Chapter 14) and they display saturation behavior.

Glucose Transport in Erythrocytes Occurs by Facilitated Diffusion

Many transport processes in a variety of cells occur by facilitated diffusion. Table 10.1 lists just a few of these. The glucose transporter of erythrocytes illustrates many of the important features of facilitated transport systems. Although glucose transport operates variously by passive diffusion, facilitated diffusion, or active transport mechanisms, depending on the particular cell, the glucose transport system of erythrocytes (red blood cells) operates exclusively by facilitated diffusion.

Figure 10.4 · SDS-gel electrophoresis of erythrocyte membrane proteins (top) and a densitometer tracing of the same gel (bottom). The region of the gel between band 4.2 and band 5 is referred to as zone 4.5 or “band 4.5.” The bands are numbered from the top of the gel (high molecular weights) to the bottom (low molecular weights). Band 3 is the anion-transporting protein and band 4.5 is the glucose transporter. The dashed line shows the staining of the gel by periodic acid – Schiff’s reagent (PAS), which stains carbohydrates. Three “PAS bands” (PAS-1, PAS-2, PAS-3) indicate the positions of glycoproteins in the gel.
(Photo courtesy of Theodore Steck , University of Chicago )

The erythrocyte glucose transporter has a molecular mass of approximately 55 kD and is found on SDS polyacrylamide electrophoresis gels (Figure 10.4) as band 4.5. Typical erythrocytes contain around 500,000 copies of this protein. The active form of this transport protein in the erythrocyte membrane is a trimer. Hydropathy analysis of the amino acid sequence of the erythrocyte glucose transporter has provided a model for the structure of the protein (Figure 10.5).

Figure 10.5 · A model for the arrangement of the glucose transport protein in the erythrocyte membrane. Hydropathy analysis is consistent with 12 transmembrane helical segments.

In this model, the protein spans the membrane 12 times, with both the N- and C-termini located on the cytoplasmic side. Trans-membrane segments M7, M8, and M11 comprise a hydrophilic transmembrane channel, with segments M9 and M10 forming a relatively hydrophobic pocket adjacent to the glucose-binding site. Cytochalasin B, a fungal metabolite (Figure 10.6), is a competitive inhibitor of glucose transport. The mechanism of glucose transport is not well understood. An alternating conformation model, in which the glucose-binding site is alternately exposed to the cytoplasmic and extracellular surfaces of the membrane, has been proposed but remains controversial. Many other glucose transport proteins with sequences that are homologous to the erythrocyte glucose transporter have been identified in muscle, liver, and most other animal tissues. The reduced ability of insulin to stimulate glucose transport in diabetic patients is due to reduced expression of some, but not all, of these glucose transport proteins.

Figure 10.6 · The structure of cytochalasin B.

The Anion Transporter of Erythrocytes Also Operates by Facilitated Diffusion

The anion transport system is another facilitated diffusion system of the eryth-rocyte membrane. Chloride and bicarbonate (HCO₃²) ions are exchanged across the red cell membrane by a 95-kD transmembrane protein. This protein is abundant in the red cell membrane and is represented by band 3 on SDS electrophoresis gels (Figure 10.4). The gene for the human erythrocyte anion transporter has been sequenced and hydropathy analysis has yielded a model for the arrangement of the protein in the red cell membrane (Figure 10.7). The model has 14 transmembrane segments, and the sequence includes 3 regions: a hydrophilic, cytoplasmic domain (residues 1 through 403) that interacts with numerous cytoplasmic and membrane proteins; a hydrophobic domain (residues 404 through 882) that comprises the anion transporting channel; and an acidic, C-terminal domain (residues 883 through 911). This transport system facilitates a one-for-one exchange of chloride and bicarbonate, so that the net transport process is electrically neutral. The net direction of anion flow through this protein depends on the sum of the chloride and bicarbonate concentration gradients. Typically, carbon dioxide is collected by red cells in respiring tissues and is then carried in the blood to the lungs, where bicarbonate diffuses out of the erythrocytes in exchange for Cl^- ions.

Figure 10.7 · A model for the arrangement of the anion transport protein in the membrane, based on hydropathy analysis.

10.3 · Active Transport Systems

Passive and facilitated diffusion systems are relatively simple, in the sense that the transported species flow downhill energetically, that is, from high concentration to low concentration. However, other transport processes in biological systems must be driven in an energetic sense. In these cases, the transported species moves from low concentration to high concentration, and thus the transport requires energy input. As such, it is considered an active transport system. The most common energy input is ATP hydrolysis (see Chapter 3), with hydrolysis being tightly coupled to the transport event. Other energy sources also drive active transport processes, including light energy and the energy stored in ion gradients (concentration differences of an ion (or solute) across a membrane represent an energized state (see Chapter 21)). The original ion gradient is said to arise from a primary active transport process, and the transport that depends on the ion gradient for its energy input is referred to as a secondary active transport process (see discussion of amino acid and sugar transport, Section 10.6). When transport results in a net movement of electric charge across the membrane, it is referred to as an electrogenic transport process. If no net movement of charge occurs during transport, the process is electrically neutral.

All Active Transport Systems Are Energy-Coupling Devices

Hydrolysis of ATP is essentially a chemical process, whereas movement of species across a membrane is a mechanical process (that is, movement). An active transport process that depends on ATP hydrolysis thus couples chemical free energy to mechanical (translational) free energy. The bacterio-rhodopsin protein in Halobacterium halobium couples light energy and mechanical energy. Oxidative phosphorylation (Chapter 21) involves coupling between electron transport, proton translocation, and the capture of chemical energy in the form of ATP synthesis. Similarly, the overall process of photosynthesis (Chapter 22) amounts to a coupling between captured light energy, proton translocation, and chemical energy stored in ATP.

10.4 · Transport Processes Driven by ATP

Monovalent Cation Transport: Na⁺,K⁺-ATPase

All animal cells actively extrude Na⁺ ions and accumulate K⁺ ions. These two transport processes are driven by Na⁺,K ⁺-ATPase , also known as the sodium pump, an integral protein of the plasma membrane. Most animal cells maintain cytosolic concentrations of Na⁺ and K⁺ of 10 mM and 100 mM , respectively. The extracellular milieu typically contains about 100 to 140 mM Na⁺ and 5 to 10 mM K⁺. Potassium is required within the cell to activate a variety of processes, whereas high intracellular sodium concentrations are inhibitory. The transmembrane gradients of Na⁺ and K⁺ and the attendant gradients of Cl^- and other ions provide the means by which neurons communicate (see Chapter 34). They also serve to regulate cellular volume and shape. Animal cells also depend upon these Na⁺ and K⁺ gradients to drive transport processes involving amino acids, sugars, nucleotides, and other substances. In fact, maintenance of these Na⁺ and K⁺ gradients consumes large amounts of energy in animal cells—20 to 40% of total metabolic energy in many cases and up to 70% in neural tissue.

            The Na⁺- and K⁺-dependent ATPase comprises two subunits, an a-subunit of 1016 residues (120 kD ) and a 35-kD b-subunit. The sodium pump actively pumps three Na⁺ ions out of the cell and two K¹ ions into the cell per ATP hydrolyzed:

ATP^4- + H₂O + 3Na⁺( inside) + 2K⁺(outside) ® ADP^3- +

H₂PO₄^- + 3Na⁺( outside) + 2K⁺(inside)   (10.3)

ATP hydrolysis occurs on the cytoplasmic side of the membrane (Figure 10.8), and the net movement of one positive charge outward per cycle makes the sodium pump electrogenic in nature.

Figure 10.8 · A schematic diagram of the Na⁺,K ⁺-ATPase in mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na⁺ ions are transported out of the cell, and K⁺ ions are transported in. The transport stoichiometry is 3 Na⁺ out and 2 K⁺ in per ATP hydrolyzed. The specific inhibitor ouabain (Figure 7.12) and other cardiac glycosides inhibit Na⁺,K ⁺-ATPase by binding on the extracellular surface of the pump protein.

            Hydropathy analysis of the amino acid sequences of the a - and b-subunits and chemical modification studies have led to a model for the arrangement of the ATPase in the plasma membrane (Figure 10.9).

Figure 10.9 · A model for the arrangement of Na⁺,K ⁺-ATPase in the plasma membrane. The large cytoplasmic domain between transmembrane segments 4 and 5 contains the ATP-binding site and the aspartate residue that is phosphorylated during the catalytic cycle. The b-subunit contains one transmembrane segment and a large extracellular carboxy -terminal segment.

The model describes 10 transmembrane a-helices in the a-subunit, with two large cytoplasmic domains. The larger of these, between transmembrane segments 4 and 5, has been implicated as the ATP-binding domain. The enzyme is covalently phosphorylated at an aspartate residue on the a-subunit in the course of ATP hydrolysis. The covalent E-P intermediate was trapped and identified using tritiated sodium borohydride (Figure 10.10).

Figure 10.10 · The reaction of tritiated sodium borohydride with the aspartyl phosphate at the active site of Na⁺,K⁺-ATPase . Acid hydrolysis of the enzyme following phosphorylation and sodium borohydride treatment yields a tripeptide containing serine, homoserine (derived from the aspartyl-phosphate), and lysine as shown. The site of phosphorylation is Asp³⁶⁹ in the large cytoplasmic domain of the ATPase .

            A minimal mechanism for Na⁺,K⁺-ATPase postulates that the enzyme cycles between two principal conformations, denoted E₁ and E₂ (Figure 10.11). E₁ has a high affinity for Na⁺ and ATP and is rapidly phosphorylated in the presence of Mg²⁺ to form E₁-P, a state which contains three occluded Na⁺ ions (occluded in the sense that they are tightly bound and not easily dissociated from the enzyme in this conformation). A conformation change yields E₂-P, a form of the enzyme with relatively low affinity for Na⁺, but a high affinity for K⁺. This state presumably releases 3 Na⁺ ions and binds 2 K⁺ ions on the outside of the cell. Dephosphorylation leaves E₂K₂, a form of the enzyme with two occluded K⁺ ions. A conformation change, which appears to be accelerated by the binding of ATP (with a relatively low affinity), releases the bound K⁺ inside the cell and returns the enzyme to the E1:ATP state. Enzyme forms with occluded cations represent states of the enzyme with cations bound in the transport channel. The alternation between high and low affinities for Na⁺, K⁺, and ATP serves to tightly couple the hydrolysis of ATP and ion binding and transport.

Figure 10.11 · A mechanism for Na⁺,K⁺-ATPase . The model assumes two principal conformations, E₁ and E₂. Binding of Na⁺ ions to E₁ is followed by phosphorylation and release of ADP. Na⁺ ions are transported and released and K⁺ ions are bound before dephosphorylation of the enzyme. Transport and release of K⁺ ions complete the cycle.

Na⁺,K⁺-ATPase Is Inhibited by Cardiac Glycosides

Plant and animal steroids such as ouabain (Figure 10.12) specifically inhibit Na⁺,K⁺-ATPase and ion transport. These substances are traditionally referred to as cardiac glycosides or cardiotonic steroids, both names derived from the potent effects of these molecules on the heart. These molecules all possess a cis-configuration of the C-D ring junction, an unsaturated lactone ring (5- or 6-membered) in the b-configuration at C-17, and a b-OH at C-14. There may be one or more sugar residues at C-3. The sugar(s) are not required for inhibition, but do contribute to water solubility of the molecule. Cardiac glycosides bind exclusively to the extracellular surface of Na⁺,K⁺-ATPase when it is in the E₂-P state, forming a very stable E₂-P (cardiac glycoside) complex.

Figure 10.12 · The structures of several cardiac glycosides. The lactose rings are yellow.

            Medical researchers studying high blood pressure have consistently found that people with hypertension have high blood levels of some sort of Na⁺,K⁺-ATPase inhibitor. In such patients, inhibition of the sodium pump in the cells lining the blood vessel wall results in accumulation of sodium and calcium in these cells and the narrowing of the vessels to create hypertension. An 8-year study aimed at the isolation and identification of the agent responsible for these effects by researchers at the University of Maryland Medical School and the Upjohn Laboratories in Michigan recently yielded a surprising result. Mass spectrometric analysis of compounds isolated from many hundreds of gallons of blood plasma has revealed that the hypertensive agent is ouabain itself or a closely related molecule!

Calcium Transport: Ca²⁺-ATPase

Calcium, an ion acting as a cellular signal in virtually all cells (see Chapter 34), plays a special role in muscles. It is the signal that stimulates muscles to contract (Chapter 17). In the resting state, the levels of Ca²⁺ near the muscle fibers are very low (approximately 0.1 mM ), and nearly all of the calcium ion in muscles is sequestered inside a complex network of vesicles called the sarcoplasmic reticulum, or SR (see Figure 17.2). Nerve impulses induce the sarcoplasmic reticulum membrane to quickly release large amounts of Ca²⁺, with cytosolic levels rising to approximately 10 mM . At these levels, Ca²⁺ stimulates contraction. Relaxation of the muscle requires that cytosolic Ca²⁺ levels be reduced to their resting levels. This is accomplished by an ATP-driven Ca²⁺ transport protein known as the Ca²⁺-ATPase. This enzyme is the most abundant protein in the SR membrane, accounting for 70 to 80% of the SR protein. Ca²⁺-ATPase bears many similarities to the Na⁺,K⁺-ATPase. It has an a-subunit of the same approximate size, it forms a covalent E-P intermediate during ATP hydrolysis, and its mechanism of ATP hydrolysis and ion transport is similar in many ways to that of the sodium pump.

Figure 10.13 · Some of the sequence homologies in the nucleotide binding and phosphorylation domains of Na⁺,K⁺-ATPase , Ca²⁺-ATPase, and gastric H⁺, K⁺-ATPase.
(Adapted from Jørgensen , P. L., and Andersen, J. P., 1988. Structural basis for E₁ - E₂ conformational transitions in Na⁺,K ⁺-pump and Ca²⁺-pump proteins. Journal of Membrane Biology 103:95–120)

            The amino acid sequence of the a -subunit is homologous with the sodium pump a-subunit, particularly around the phosphorylation site and the ATP-binding site (Figure 10.13). Ten transmembrane helical segments are predicted from hydropathy analysis, as well as a “stalk” consisting of five helical segments (Figure 10.14). This stalk lies between the membrane surface and the globular cytoplasmic domain containing the nucleotide-binding domain and the site of phosphorylation. The E-P formed by SR Ca²⁺-ATPase is an aspartyl phosphate like that of Na⁺,K⁺-ATPase , in this case Asp residue 351.

Figure 10.14 · The arrangement of Ca²⁺-ATPase in the sarcoplasmic reticulum membrane. Ten transmembrane segments are postulated on the basis of hydropathy analysis.

            Two Ca²⁺ ions are transported into the SR per ATP hydrolyzed by this enzyme, and the mechanism (Figure 10.15) appears to involve two major conformations, E₁ and E₂, just as the Na⁺,K⁺-ATPase mechanism does. Calcium ions are strongly occluded in the E₁-Ca₂-P state, and these occluded ions do not dissociate from the enzyme until the enzyme converts to the E₂-Ca₂-P state, which has a very low affinity for Ca²⁺. In the E₁-Ca₂-P state, the transported Ca²⁺ ions are bound in the transport channel.

Figure 10.15 · A mechanism for Ca²⁺-ATPase from sarcoplasmic reticulum. Note the similarity to the mechanism of Na⁺,K ⁺-ATPase (see also Figure 10.11). (“Out” here represents the cytosol ; “in” represents the lumen of the SR.)

The Gastric H⁺,K⁺-ATPase

Production of protons is a fundamental activity of cellular metabolism, and proton production plays a special role in the stomach. The highly acidic environment of the stomach is essential for the digestion of food in all animals. The pH of the stomach fluid is normally 0.8 to 1. The pH of the parietal cells of the gastric mucosa in mammals is approximately 7.4. This represents a pH gradient across the mucosal cell membrane of 6.6, the largest known transmembrane gradient in eukaryotic cells. This enormous gradient must be maintained constantly so that food can be digested in the stomach without damage to the cells and organs adjacent to the stomach. The gradient of H⁺ is maintained by an H⁺,K ⁺-ATPase , which uses the energy of hydrolysis of ATP to pump H⁺ out of the mucosal cells and into the stomach interior in exchange for K⁺ ions. This transport is electrically neutral, and the K⁺ that is transported into the mucosal cell is subsequently pumped back out of the cell together with Cl^- in a second electroneutral process (Figure 10.16). Thus, the net transport effected by these two systems is the movement of HCl into the interior of the stomach. (Only a small amount of K⁺ is needed because it is recycled.) The H⁺,K⁺-ATPase bears many similarities to the plasma membrane Na⁺,K⁺-ATPase and the SR Ca²⁺-ATPase described above. It has a similar molecular weight, forms an E-P intermediate, and many parts of its peptide sequence are homologous with the Na⁺,K⁺-ATPase and Ca²⁺-ATPase (Figure 10.13).

Figure 10.16 · The H⁺,K ⁺-ATPase of gastric mucosal cells mediates proton transport into the stomach. Potassium ions are recycled by means of an associated K⁺/Cl ^- cotransport system. The action of these two pumps results in net transport of H⁺ and Cl ^- into the stomach.

Bone Remodeling by Osteoclast Proton Pumps

Other proton-translocating ATPases exist in eukaryotic and prokaryotic systems. Vacuolar ATPases are found in vacuoles, lysosomes, endosomes, Golgi, chromaffin granules, and coated vesicles. Various H⁺-transporting ATPases occur in yeast and bacteria as well. H⁺-transporting ATPases found in osteoclasts (multinucleate cells that break down bone during normal bone remodeling) provide a source of circulating calcium for soft tissues such as nerves and muscles. About 5% of bone mass in the human body undergoes remodeling at any given time. Once growth is complete, the body balances formation of new bone tissue by cells called osteoblasts with resorption of existing bone matrix by osteoclasts. Osteoclasts possess proton pumps—similar to vacuolar ATPases—on the portion of the plasma membrane that attaches to the bone. This region of the osteoclast membrane is called the ruffled border. The osteoclast attaches to the bone in the manner of a cup turned upside down on a saucer (Figure 10.17), leaving an extracellular space between the bone surface and the cell. The H⁺-ATPases in the ruffled border pump protons into this space, creating an acidic solution that dissolves the bone mineral matrix. Bone mineral is primarily an inorganic mixture of calcium carbonate and hydroxy-apatite (calcium phosphate). In this case, transport of protons out of the osteoclasts lowers the pH of the extracellular space near the bone to about 4, solubilizing the hydroxyapatite.

Figure 10.17 · Proton pumps cluster on the ruffled border of osteoclast cells and function to pump protons into the space between the cell membrane and the bone surface. High proton concentration in this space dissolves the mineral matrix of the bone.

ATPases That Transport Peptides and Drugs

Species other than protons and inorganic ions are also transported across certain membranes by specialized ATPases. Yeast (Saccharomyces cerevisiae ) has one such system. Yeasts exist in two haploid mating types, designated a and a. Each mating type produces a mating factor (a-factor or a-factor, respectively) and responds to the mating factor of the opposite type. The a-factor is a peptide that is inserted into the ER during translation on the ribosome. a-Factor is glycosylated in the ER and then secreted from the cell. On the other hand, the a-factor is a 12-amino acid peptide made from a short precursor. Export of this peptide from the cell is carried out by a 1290-residue protein, which consists of two identical halves joined together—a tandem duplication. Each half contains six putative transmembrane segments arranged in pairs, and a conserved hydrophilic cytoplasmic domain containing a consensus sequence for an ATP-binding site (Figure 10.18). This protein uses the energy of ATP hydrolysis to export the 12-residue a-factor from the cell. In yeast cells that produce mutant forms of the a-factor ATPase , a-factor is not excreted and accumulates to high levels inside the cell.

Figure 10.18 · A model for the structure of the a-factor transport protein in the yeast plasma membrane. Gene duplication has yielded a protein with two identical halves, each half containing six transmembrane helical segments and an ATP-binding site. Like the yeast a-factor transporter, the multidrug transporter is postulated to have 12 transmembrane helices and 2 ATP-binding sites.

            Proteins very similar to the yeast a-factor transporter have been identified in a variety of prokaryotic and eukaryotic cells, and one of these appears to be responsible for the acquisition of drug resistance in many human malignancies. Clinical treatment of human cancer often involves chemotherapy, the treatment with one or more drugs that selectively inhibit the growth and proliferation of tumorous tissue. However, the efficacy of a given chemotherapeutic drug often decreases with time, owing to an acquired resistance. Even worse, the acquired resistance to a single drug usually results in a simultaneous resistance to a wide spectrum of drugs with little structural or even functional similarity to the original drug, a phenomenon referred to as multidrug resistance, or MDR. This perplexing problem has been traced to the induced expression of a 170-kD plasma membrane glycoprotein known as the P-glycoprotein or the MDR ATPase. Like the yeast a-factor transporter, MDR ATPase is a tandem repeat, each half consisting of a hydrophobic sequence with six transmembrane segments followed by a hydrophilic, cytoplasmic sequence containing a consensus ATP-binding site (Figure 10.18). The protein uses the energy of ATP hydrolysis to actively transport a wide variety of drugs (Figure 10.19) out of the cell. Ironically, it is probably part of a sophisticated protection system for the cell and the organism. Organic molecules of various types and structures that might diffuse across the plasma membrane are apparently recognized by this protein and actively extruded from the cell. Despite the cancer-fighting nature of chemotherapeutic agents, the MDR ATPase recognizes these agents as cellular intruders and rapidly removes them. It is not yet understood how this large protein can recognize, bind, and transport such a broad group of diverse molecules, but it is known that the yeast a-factor ATPase and the MDR ATPase are just two members of a superfamily of transport proteins, many of whose functions are not yet understood.

Figure 10.19 · Some of the cytotoxic drugs that are transported by the MDR ATPase .

10.5 · Transport Processes Driven by Light

As noted previously, certain biological transport processes are driven by light energy rather than by ATP. Two well-characterized systems are bacte-riorhodopsin, the light-driven H⁺-pump, and halorhodopsin, the light-driven Cl^--pump, of Halobacterium halobium, an archaebacterium that thrives in high-salt media. H. halobium grows optimally at an NaCl concentration of 4.3 M. It was extensively characterized by Walther Stoeckenius , who found it growing prolifically in the salt pools near San Francisco Bay, where salt is commercially extracted from seawater. H. halobium carries out normal respiration if oxygen and metabolic energy sources are plentiful. However, when these substrates are lacking, H. halobium survives by using bacteriorhodopsin and halorhodopsin to capture light energy. In oxygen- and nutrient-deficient conditions, purple patches appear on the surface of H. halobium (Figure 10.20).

Figure 10.20 · A schematic drawing of Halobacterium halobium . The purple patches contain bacteriorhodopsin (bR ).

These purple patches of membrane are 75% protein, the only protein being bacteriorhodopsin (bR). The purple color arises from a retinal molecule that is covalently bound in a Schiff base linkage with an Î-NH₂ group of Lys²¹⁶ on each bacteriorhodopsin protein (Figure 10.21). Bacteriorhodopsin is a 26-kD transmembrane protein that packs so densely in the membrane that it naturally forms a two-dimensional crystal in the plane of the membrane. The structure of bR has been elucidated by image enhancement analysis of electron microscopic data, which reveals seven transmembrane helical protein segments. The retinal moiety lies parallel to the membrane plane, about 1 nm below the membrane’s outer surface (Figure 9.15).

Figure 10.21 · The Schiff base linkage between the retinal chromophore and Lys²¹⁶.

A Model for Light-Driven Proton Transport

Figure 10.22 · The reaction cycle of bacteriorhodopsin. The intermediate states are indicated by letters, with subscripts to indicate the absorption maxima of the states. Also indicated for each state is the configuration of the retinal chromophore (all-trans or 13-cis) and the protonation state of the Schiff base (C=N: or C=N⁺H).

The mechanism of the light-driven transport of protons by bacteriorhodopsin is complex, but a partial model has emerged (Figure 10.22). A series of intermediate states, named for the wavelengths (in nm) of their absorption spectra, has been identified. Absorption of a photon of light by the bR₅₆₈ form (in which the Schiff base at Lys²¹⁶ is protonated ) converts the retinal from the all-trans configuration to the 13-cis isomer. Passage through several different intermediate states results in outward transport of 2 H⁺ ions per photon absorbed, and the return of the bound retinal to the all-trans configuration. It appears that the transported protons are in fact protons from the protonated Schiff base. The proton gradient thus established represents chemical energy that can be used by H. halobium to drive ATP synthesis and the movement of molecules across the cell membrane (see Chapter 21).

Light-Driven Chloride Transport in H. halobium

Figure 10.23 · The folding of halorhodopsin with the transmembrane segments indicated. The only lysine residue in the protein is Lys²⁴², to which the retinal chromophore is covalently linked.

Anion transport, on the other hand, is handled by a second light-driven ion pump in the H. halobium membrane. The inward transport of Cl^- ion is mediated by halorhodopsin, a 27-kD protein whose primary structure and arrangement in the membrane (Figure 10.23) is very similar to that of bacte-riorhodopsin . Although halorhodopsin does not exist naturally as a tightly packed two-dimensional crystal in the membrane, it does have a retinal chromophore , bound covalently at Lys²⁴², the only lysine in the protein. The transmembrane portion of halorhodopsin is 36% homologous with bacteriorhodopsin. The conserved residues are concentrated in the central core formed in both proteins by the seven transmembrane helices (Figure 10.24).

Figure 10.24 · A helical wheel model of halorhodopsin. The amino acids facing the polar, hydrophilic core of the protein are shown. Of these 60 residues, 36 are conserved between halorhodopsin and bacteriorhodopsin .
(Adapted from Oesterhelt , D., and Tittor , J., 1989. Trends in Biochemical Sciences 14:57–61.)

Like bacteriorhodopsin, halorhodopsin undergoes a cycle of light-driven conformational changes (Figure 10.25), but no deprotonation of the Schiff base occurs during the halorhodopsin photocycle. Given the striking similarity of structures for these two proteins, it is intriguing to ask why bacteriorhodopsin pumps H⁺ but not Cl^- and why halorhodopsin pumps Cl^- but not H⁺. The first question may be answered by the work of H.G. Khorana and his coworkers, who replaced Asp⁸⁵ and Asp⁹⁶ in bacteriorhodopsin with asparagine and found that either substitution caused a drastic reduction in H⁺ transport. Dieter Oesterhelt and coworkers have shown that Asp⁸⁵ and Asp⁹⁶ are important in the deprotonation and reprotonation, respectively, of the Schiff base in bacteriorhodopsin. The absence of these two crucial residues in halorhodopsin may explain why the latter protein can’t reversibly deprotonate the Schiff base and why halorhodopsin doesn’t pump protons.

Figure 10.25 · The photocycle of light-adapted halorhodopsin (hR ), shown in the presence and absence of chloride. The superscripts indicate the maxima of the difference spectra between hR and the intermediates.

10.6 · Transport Processes Driven by Ion Gradients

Amino Acid and Sugar Transport

The gradients of H⁺, Na⁺, and other cations and anions established by ATPases and other energy sources can be used for secondary active transport of various substrates. The best-understood systems use Na⁺ or H⁺ gradients to transport amino acids and sugars in certain cells. Many of these systems operate as symports, with the ion and the transported amino acid or sugar moving in the same direction (that is, into the cell). In antiport processes, the ion and the other transported species move in opposite directions. (For example, the anion transporter of erythrocytes is an antiport.) Proton symport proteins are used by E. coli and other bacteria to accumulate lactose, arabinose, ribose, and a variety of amino acids. E. coli also possesses Na⁺-symport systems for melibiose as well as for glutamate and other amino acids.

            Table 10.2 lists several systems that transport amino acids into mammalian cells. The accumulation of neutral amino acids in the liver by System A represents an important metabolic process. Thus, plasma membrane transport of alanine is the rate-limiting step in hepatic alanine metabolism. This system is normally expressed at low levels in the liver, but substrate deprivation and hormonal activation both stimulate System A expression.

10.7 · Group Translocation

Certain bacteria possess a novel and versatile system for the inward transport of certain sugars. In this process, the sugar becomes phosphorylated during its transport across the membrane; that is, transport and phosphorylation are tightly coupled. This type of process, in which a chemical modification accompanies transport, has been denoted group translocation. Several such systems are known, but the best understood is the phosphoenolpyruvate:glucose phosphotransferase system, or simply the phosphotransferase system (or PTS), discovered by Saul Roseman of Johns Hopkins University in 1964. The advantage of this system lies in the fact that the sugars, once phosphorylated , are trapped in the cell. Membranes are permeable to simple sugars but impermeable to sugar phosphates, which are negatively charged. The overall reaction for the phosphotransferase is:

The subscripts illustrate an important point: the phosphoryl transfer occurs entirely on the inside surface of the bacterial membrane.

            Several unique features distinguish the phosphotransferase. First, phosphoenolpyruvate is both the phosphoryl donor and the energy source for sugar transport. Second, four different proteins are required for this transport. Two of these proteins (Enzyme I and HPr ) are general and are required for the phosphorylation of all PTS-transported sugars. The other two proteins (Enzyme II and Enzyme III) are specific for the particular sugar to be transported.

Figure 10.26 · Glucose transport in E. coli is mediated by the PEP-dependent phosphotransferase system. Enzyme I is phosphorylated in the first step by PEP. Successive phosphoryl transfers to HPr and Enzyme III in Steps 2 and 3 are followed by transport and phosphorylation of glucose. Enzyme II is the sugar transport channel.

            The first step in the phosphotransferase reaction (Figure 10.26) is the phosphorylation of Enzyme I by PEP to form a reactive phosphohistidine intermediate (Figure 10.27). This is followed by phosphoryl transfer to a histidyl residue of HPr, followed by phosphorylation of Enzyme III. At the same time, the sugar to be transported is bound on the outside surface of the cell by Enzyme II, which constitutes the sugar transport channel. As the sugar is moved to the inside surface of the membrane, the phosphoryl group is transferred from Enzyme III to the sugar, forming the desired sugar phosphate, which is released into the cytoplasm. (In some cases, for example the E. coli mannitol system, no Enzyme III has been identified. In these cases, the C-terminal end of the relevant Enzyme II, which resembles an Enzyme III-type sequence, substitutes for Enzyme III. )

Figure 10.27 · The path of the phosphoryl group through the PTS mechanism. Reactive phosphohistidine intermediates of Enzyme I, HPr , and Enzyme III transfer phosphoryl groups from PEP to the transported sugar.

10.8 · Specialized Membrane Pores

Porins in Gram-Negative Bacterial Membranes

The membrane transport systems described previously (and many others like them) are relatively specific and function to transport either a single substrate or a very limited range of substrates under normal conditions. At the same time, several rather nonspecific systems also carry out transport processes. One such class of nonspecific transport proteins is found in the outer membranes of Gram-negative bacteria and mitochondria. Low-molecular-weight nutrients and certain other molecules, such as some antibiotics, cross this outer membrane, but larger molecules such as proteins cannot. The ability of the outer membrane to act as a molecular sieve is due to proteins called porins (Chapter 9). Alternatively, these molecules have been referred to as peptidoglycan -associated proteins or simply matrix proteins. General porins form nonspecific pores across the outer membrane and sort molecules according to molecular size, whereas specific porins contain binding sites for particular substrates. Porins from several organisms have been isolated and characterized (Table 10.3). Molecular masses of the porins generally range from 30 kD to 50 kD . Most (but not all) porins are arranged in the outer membrane as trimers of identical subunits. The molecular exclusion limits clearly depend on the size of the pore formed by the porin molecule. The pores formed by E. coli and S. typhimurium porins are relatively small, but porin F from Pseudomonas aeruginosa creates a much larger pore, with an exclusion limit of approximately  6 kD . Specific porins LamB and Tsx of E. coli and porins P and DI of P. aeruginosa possess specific binding sites for maltose and related oligosaccharides (Table 10.4), nucleosides, anions, and glucose, respectively.

            Porins show high degrees of sequence homology and similarity. The most intriguing feature of porin secondary and tertiary structure is this: In contrast to nearly all other membrane proteins that adopt a-helical structures in the transmembrane segments, porins show little or no evidence of a-helical domains and segments. Instead, the porins and other outer membrane proteins adopt b-sheet structures for their membrane-spanning segments. Models of membrane insertion, which involve b-strands arranged perpendicular to the membrane plane, have been proposed for several porins (Figure 10.28).

Figure 10.28 · A model for the arrangement of the porin PhoE in the outer membrane of E. coli. The transmembrane segments are strands of b-sheet.

The crystal structure of the porin from Rhodobacter capsulatus shows a trimer in which each monomer forms a pore (Figure 10.29). The monomer pore consists of a 16-stranded b-barrel that traverses the membrane as a tube. The tube is narrowed near the center by peptide chain segments protruding from the inner wall of the barrel. These chain segments form an “eyelet” about 1 nm long and 0.6 to 1 nm across. The eyelet is postulated to determine the exclusion limit for particles diffusing through the pore.

Figure 10.29 · Three-dimensional reconstruction of porin from Rhodobacter capsulatus . Drawings of (a) side view of porin monomer showing b-sheet structure. (b) Top view and (c) nearly top view of porin trimer

            Porins and the other outer membrane proteins of Gram-negative bacteria appear to be the only known membrane proteins that have chosen the b-strand over the a-helix. Why might this be? Among other reasons, there is an advantage of genetic economy in the use of b-strands to traverse the membrane instead of a-helices. An a-helix requires 21 to 25 amino acid residues to span a typical biological membrane; a b-strand can cross the same membrane with 9 to 11 residues. Therefore, a given amount of genetic information could encode a larger number of membrane-spanning segments using a b-strand motif instead of a-helical arrays. Further, b-strands can present alternating hydrophobic and hydrophilic R groups along their length, with hydrophobic R groups facing the lipid bilayer and hydrophilic R groups facing the water-filled channel (Chapter 9).

The Pore-Forming Toxins

Many organisms produce lethal molecules known as pore-forming toxins, which insert themselves in a host cell’s plasma membrane to form a channel or pore. Pores formed by such toxins can kill the host cell by collapsing ion gradients or by facilitating the entry of toxic agents into the cell. Produced by a variety of organisms and directed toward a similarly diverse range of target cells, these toxins nonetheless share certain features in common. The structures of these remarkable toxins have provided valuable insights into the mechanisms of their membrane insertion and also into the architecture of membrane proteins.
            Colicins are pore-forming proteins, produced by certain strains of E. coli, that kill or inhibit the growth of other, competing bacteria and even other strains of E. coli (a process known as allelopathy ). Channel-forming colicins are released as soluble monomers. Upon encountering a host cell, the colicin molecule traverses the bacterial outer membrane and periplasm , then inserts itself into the inner (plasma) membrane. The channel thus formed is monomeric and a single colicin molecule can kill a host cell. The structure of colicin Ia, a 626-residue protein, is shown in Figure 10.30.

Figure 10.30 · The structure of colicin Ia . Colicin Ia , with a total length of 210 Å, spans the periplasmic space of a Gram-negative bacterium host, with the R (receptor-binding) domain (blue) anchored to proteins in the outer membrane and the C domain (violet) forming a channel in the inner membrane. The T (translocation) domain is shown in red. The image on the right shows details of the C domain, including helices 8 and 9 (green), which are highly hydrophobic.

It consists of three domains, termed the T (translocation) domain, the R (receptor-binding) domain, and the C (channel-forming) domain. The T domain mediates translocation across the outer membrane, the R domain binds to an outer-membrane receptor, and the C-domain creates a voltage-gated channel across the inner membrane. The T, R, and C domains are separated by long (160 Å) a-helical segments. The peptide is folded at the R domain, so that the C and T domains are juxtaposed and the two long helices form an underwound antiparallel coiled coil. The protein is unusually elongated—210 Å from end to end—with the T and C domains at one end and R at the other. This unusual design permits colicin Ia to span the periplasmic space (which has an average width of 150 Å) and insert in the inner membrane.

Figure 10.31 · The umbrella model of membrane channel protein insertion. Hydrophobic helices insert directly into the core of the membrane, with amphipathic helices arrayed on the surface like an open umbrella. A trigger signal (low pH or a voltage gradient) draws some of the amphipathic helices into and across the membrane, causing the pore to open.

The nature of the channel-forming domain provides clues to the process of channel formation in the inner membrane. The C domain consists of a 10-helix bundle, with helices 8 and 9 forming an unusually hydrophobic hairpin structure. The other eight helices are amphipathic and serve to stabilize hydrophobic helices 8 and 9 in solution. When this domain inserts in the inner membrane, helices 8 and 9 inject themselves into the hydrophobic membrane core, leaving the other helices behind on the membrane surface (Figure 10.31). Application of a transmembrane potential (voltage) then triggers the amphipathic helices to insert into the membrane, with their hydrophobic faces facing the hydrophobic bilayer and their polar faces forming the channel surface. This model is hypothetical, but it is supported by studies showing that channel opening involves dramatic structural changes and that helices 2 to 5 move across the membrane during channel opening.
            Interestingly, certain other pore-forming toxins possess helix-bundle motifs that may participate in channel formation, in a manner similar to that proposed for colicin Ia . For example, the d-endotoxin produced by Bacillus thuringiensis is toxic to Coleoptera insects (beetles) and is composed of three domains, including a seven-helix bundle, a three-sheet domain, and a b-sandwich. In the seven-helix bundle, helix 5 is highly hydrophobic, and the other six helices are amphipathic . In solution (Figure 10.32), the six amphipathic helices surround helix 5, with their nonpolar faces apposed to helix 5 and their polar faces directed to the solvent. Membrane insertion and channel formation may involve initial insertion of helix 5, as in Figure 10.31, followed by insertion of the amphipathic helices, so that their nonpolar faces contact the bilayer lipids and their polar faces line the channel.

Figure 10.32 · The structures of (a) d-endotoxin (two views) from Bacillus thuringiensis and (b) diphtheria toxin from Corynebacterium diphtheriae . Each of these toxins possesses a bundle of a-helices which is presumed to form the transmembrane channel when the toxin is inserted across the host membrane. In d-endotoxin , helix 5 (white) is surrounded by 6 helices (red) in a 7-helix bundle. In diphtheria toxin, three hydrophobic helices (white) lie at the center of the transmembrane domain (red).

            There are a number of other toxins for which the helical channel model is inappropriate. These include a-hemolysin from Staphylococcus aureus, aerolysin from Aeromonas hydrophila, and the anthrax toxin protective antigen from Bacillus anthracis. The membrane-spanning domains of these proteins do not possess long stretches of hydrophobic residues that could form a-helical transmembrane segments. They do, however, contain substantial peptide segments of alternating hydrophobic and polar residues. Like the porins, such segments can adopt b-strand structures, such that one side of the b-strand is hydrophobic and the other side is polar. Oligomeric association of several such segments can produce a b-barrel motif, with the inside of the barrel lined with polar residues and the outside of the barrel coated with hydrophobic residues — a motif that can be accommodated readily in a bilayer membrane, creating a polar transmembrane channel.
            a-Hemolysin, a 33.2-kD monomer protein, forms a mushroom-shaped heptameric pore, 100 Å in length, with a diameter that ranges from 14 Å to 46 Å (Figure 10.33). In this structure, each monomer contributes two b-strands 65 Å long, which are connected by a hairpin turn. The interior of the 14-stranded b-barrel structure is hydrophilic and the hydrophobic outer surface of the barrel is 28 Å wide. Pores formed by a-hemolysin in human erythrocytes, platelets, and lymphocytes allow rapid Ca²⁺ influx into these cells with toxic consequences.

Figure 10.33 · The structure of the heptameric channel formed by a-hemolysin . Each of the seven subunits contributes a b-sheet hairpin to the transmembrane channel.

            Aeromonas hydrophila is a bacterium that causes diarrheal diseases and deep wound infections. These complications arise due to pore formation in sensitive cells by the protein toxin aerolysin. Proteolytic processing of the 52-kD precursor proaerolysin (Figure 10.34) produces the toxic form of the protein, aerolysin. Like a-hemolysin, aerolysin monomers associate to form a heptameric transmembrane pore. Michael Parker and coworkers have proposed that each monomer in this aggregate contributes three b-strands to the b-barrel pore. Each of these b-strands (residues 277 to 287, 290 to 302, and 410 to 422) consists of alternating hydrophobic and polar residues, so that the pore once again places polar residues toward the water-filled channel and nonpolar residues facing the lipid bilayer .

Figure 10.34 · The structure of proaerolysin, produced by Aeromonas hydrophila. Proteolysis of this precursor yields the active form, aerolysin , which is responsible for the pathogenic effects of the bacterium in deep wound infections and diarrheal diseases. Like hemolysin, aerolysin monomers associate to form heptameric membrane pores. The three b-strands that contribute to the formation of the heptameric pore are shown in red. The N-terminal domain (residues 1–80, yellow) is a small lobe that protrudes from the rest of the protein.

            Whether crossing the membrane with aggregates of amphipathic a-helices or b-barrels, these pore-forming toxins represent Nature’s accommodation to a structural challenge facing all protein-based transmembrane channels: the need to provide hydrogen-bonding partners for the polypeptide backbone N-H and C = O groups in an environment (the bilayer interior) that lacks hydrogen-bond donors or acceptors. The solution to this problem is found, of course, in the extensive hydrogen-bonding possibilities of a-helices and b-sheets.

Amphipathic Helices Form Transmembrane Ion Channels

Figure 10.35 · The amino acid sequences of several amphipathic peptide antibiotics. a-Helices formed from these peptides cluster polar residues on one face of the helix, with nonpolar residues at other positions.

Recently, a variety of natural peptides that form transmembrane channels have been identified and characterized. Melittin (Figure 10.35) is a bee venom toxin peptide of 26 residues. The cecropins are peptides induced in Hyalophora cecropia (Figure 10.36) and other related silkworms when challenged by bacterial infections. These peptides are thought to form a-helical aggregates in membranes, creating an ion channel in the center of the aggregate. The unifying feature of these helices is their amphipathic character, with polar residues clustered on one face of the helix and nonpolar residues elsewhere. In the membrane, the polar residues face the ion channel, leaving the nonpolar residues elsewhere on the helix to interact with the hydrophobic interior of the lipid bilayer.

Figure 10.36 · Adult (left) and caterpillar (right) stages of the cecropia moth, Hyalophora cecropia . (left , Greg Neise /Visuals Unlimited; right, Patti Murray/Animals, Animals)

Gap Junctions in Mammalian Cell Membranes

When cells lie adjacent to each other in animal tissues, they are often connected by gap junction structures, which permit the passive flow of small molecules from one cell to the other. Such junctions essentially connect the cells metabolically, providing a means of chemical transfer and communication. In certain tissues, such as heart muscle that is not innervated, gap junctions permit very large numbers of cells to act synchronously. Gap junctions also provide a means for transport of nutrients to cells disconnected from the circulatory system, such as the lens cells of the eye.
            Gap junctions are formed from hexameric arrays of a single 32-kD protein. Each subunit of the array is cylindrical, with a length of 7.5 nm and a diameter of 2.5 nm. The subunits of the hexameric array are normally tilted with respect to the sixfold axis running down the center of the hexamer (Figure 10.37). In this conformation, a central pore having a diameter of about 1.8 to 2.0 nm is created, and small molecules (up to masses of 1 kD to 1.2 kD ) can pass through unimpeded. Proteins, nucleic acids, and other large structures cannot. A complete gap junction is formed from two such hexameric arrays, one from each cell. A twisting, sliding movement of the subunits narrows the channel and closes the gap junction. This closure is a cooperative process, and a localized conformation change at the cytoplasmic end assists in the closing of the channels. Because the closing of the gap junction does not appear to involve massive conformational changes in the individual subunits, the free energy change for closure is small.

Figure 10.37 · Gap junctions consist of hexameric arrays of cylindrical protein subunits in the plasma membrane. The subunit cylinders are tilted with respect to the axis running through the center of the gap junction. A gap junction between cells is formed when two hexameric arrays of subunits in separate cells contact each other and form a pore through which cellular contents may pass. Gap junctions close by means of a twisting, sliding motion in which the subunits decrease their tilt with respect to the central axis. Closure of the gap junction is Ca²⁺-dependent.

            Although gap junctions allow cells to communicate metabolically under normal conditions, the ability to close gap junctions provides the tissue with an important intercellular regulation mechanism. In addition, gap junctions provide a means to protect adjacent cells if one or more cells are damaged or stressed. To these ends, gap junctions are sensitive to membrane potentials, hormonal signals, pH changes, and intracellular calcium levels. Dramatic changes in pH or Ca²⁺ concentration in a cell may be a sign of cellular damage or death. In order to protect neighboring cells from the propagation of such effects, gap junctions close in response to decreased pH or prolonged increases in intracellular Ca²⁺. Under normal conditions of intracellular Ca²⁺ levels (<10^-7 M), gap junctions are open and intercellular communication is maintained. When calcium levels rise to 10^-5 M or higher, the junctions, sensing danger, rapidly close.

10.9 · Ionophore Antibiotics

All of the transport systems examined thus far are relatively large proteins. Several small molecule toxins produced by microorganisms facilitate ion transport across membranes. Due to their relative simplicity, these molecules, the ionophore antibiotics, represent paradigms of the mobile carrier and pore or channel models for membrane transport. Mobile carriers are molecules that form complexes with particular ions and diffuse freely across a lipid membrane (Figure 10.38). Pores or channels, on the other hand, adopt a fixed orientation in a membrane, creating a hole that permits the transmembrane movement of ions. These pores or channels may be formed from monomeric or (more often) multimeric structures in the membrane.

Figure 10.38 · Schematic drawings of mobile carrier and channel ionophores. Carrier ionophores must move from one side of the membrane to the other, acquiring the transported species on one side and releasing it on the other side. Channel ionophores span the entire membrane.

            Carriers and channels may be distinguished on the basis of their temperature dependence. Channels are comparatively insensitive to membrane phase transitions and show only a slight dependence of transport rate on temperature. Mobile carriers, on the other hand, function efficiently above a membrane phase transition, but only poorly below it. Consequently, mobile carrier systems often show dramatic increases in transport rate as the system is heated through its phase transition. Figure 10.39 displays the structures of several of these interesting molecules. As might be anticipated from the variety of structures represented here, these molecules associate with membranes and facilitate transport by different means.

Figure 10.39 · Structures of several ionophore antibiotics. Valinomycin consists of three repeats of a four-unit sequence. Because it contains both peptide and ester bonds, it is referred to as a depsipeptide.

Valinomycin Is a Mobile Carrier Ionophore

Valinomycin (isolated from Streptomyces fulvissimus ) is a cyclic structure containing 12 units made from four different residues. Two are amino acids ( L-valine and D-valine ); the other two residues, L-lactate and D-hydroxyisovalerate, contribute ester linkages. Valinomycin is a depsipeptide, that is, a molecule with both peptide and ester bonds. (Considering the 12 units in the structure, valinomycin is called a dodecadepsipeptide.) Valinomycin consists of the 4-unit sequence (D-valine, L-lactate, L-valine, D-hydroxyisovalerate), repeated three times to form the cyclic structure in Figure 10.39. The structures of uncomplexed valinomycin and the K⁺-valinomycin complex have been studied by X-ray crystallography (Figure 10.40). The structure places K⁺ at the center of the valinomycin ring, coordinated with the carbonyl oxygens of the 6 valines. The polar groups of the valinomycin structure are positioned toward the center of the ring, whereas the nonpolar groups (the