12.5 A Major Role of Membrane Proteins Is to Function as Transporters

Transporter proteins are a specific class of pump or channel that facilitates the movement of molecules across a membrane. Each cell type expresses a specific set of transporters in its plasma membrane. The set of transporters expressed is crucial because these transporters largely determine the ionic composition inside a cell and the compounds that can be taken up from the cell’s environment. In some senses, the array of transporters expressed by a cell determines its characteristics because a cell can execute only those biochemical reactions for which it has taken up the substrates.

Two factors determine whether a small molecule will cross a membrane: (1) the concentration gradient of the molecule across the membrane and (2) the molecule’s solubility in the hydrophobic environment of the membrane. In accord with the Second Law of Thermodynamics, molecules spontaneously move from a region of higher concentration to one of lower concentration. For many molecules, the cell membrane is an obstacle to this movement, but, as discussed earlier, some molecules can pass through the membrane because they dissolve in the lipid bilayer. Such molecules are called lipophilic molecules. The steroid hormones provide a physiological example. These cholesterol relatives can pass through a membrane in their path of movement in a process called simple diffusion.

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Matters become more complicated when the molecule is highly polar. For example, sodium ions are present at 143 mM outside a typical cell and at 14 mM inside the cell, yet sodium does not freely enter the cell, because the charged ion cannot pass through the hydrophobic membrane interior. In some circumstances, such as in a nerve impulse, sodium ions must enter the cell. How are these ions able to cross the hydrophobic membrane interior? Sodium ions pass through specific channels in the hydrophobic barrier—channels that are formed by membrane proteins. This means of crossing the membrane is called facilitated diffusion because the diffusion across the membrane is facilitated by the channel. It is also called passive transport because the energy driving the ion movement originates in the ion gradient itself, without any contribution by the transport system. Channels, like enzymes, display substrate specificity in that they facilitate the transport of some ions but not other ions, not even those that are closely related.

How is the sodium gradient established in the first place? In this case, sodium must move, or be pumped, against a concentration gradient. Because moving the ion from a low concentration to a higher concentration results in a decrease in entropy, it requires an input of free energy. Protein pumps embedded in the membrane are capable of using an energy source to move the molecule up a concentration gradient in a process called active transport.

The Na+–K+ ATPase Is an Important Pump in Many Cells

Figure 12.16: Energy transduction by membrane proteins. The Na+–K+ ATPase converts the free energy of phosphoryl transfer into the free energy of a Na+ ion gradient.

Cells must control their intracellular salt concentrations to prevent unfavorable interactions with high concentrations of ions and to facilitate specific processes such as a nerve impulse. In particular, most animal cells contain a high concentration of K+ and a low concentration of Na+ relative to the extracellular fluid. These ionic gradients are generated by a specific transport system, an enzyme called the Na+–K+ pump or the Na+K+ ATPase. The hydrolysis of ATP by the pump provides the energy needed for the active transport of three Na+ ions out of the cell and two K+ ions into the cell, generating the gradients (Figure 12.16). In other words, the Na+–K+ ATPase is an ATP-driven pump.

The active transport of Na+ and K+ is of great physiological significance. Indeed, more than a third of the ATP consumed by a resting animal is used to pump these ions. The Na+–K+ gradient in animal cells controls cell volume, renders neurons and muscle cells electrically excitable, and drives the active transport of sugars and amino acids. This third phenomenon is called secondary active transport because the sodium gradient generated by the Na+–K+ ATPase (the primary instance of active transport) can be used to power active transport of other molecules (the secondary instance of active transport) when the sodium flows down its gradient.

The pump is called the Na+–K+ ATPase because the hydrolysis of ATP takes place only when Na+ and K+ are present. The Na+–K+ ATPase is referred to as a P-type ATPase because it forms a key phosphorylated intermediate. In the formation of this intermediate, a phosphoryl group obtained from ATP is linked to the side chain of a specific conserved aspartate residue in the pump to form phosphorylaspartate. Other examples of P-type ATPases include the Ca2+ ATPase, which transports calcium ions out of the cytoplasm and into the extracellular fluid, mitochondria, and sarcoplasmic reticulum of muscle cells, a key process in muscle contraction; the gastric H+K+ ATPase, which is responsible for pumping protons into the stomach to lower the pH to near 1.0; and P4-ATPase, a flippase that moves membrane lipids from the outer leaflet to the inner leaflet. Indeed, P-type ATPases are found in all the kingdoms of life.

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Figure 12.17: ABC transporters. The multidrug-resistance protein (MDR) and the cystic fibrosis transmembrane regulator (CFTR) are homologous proteins composed of two transmembrane domains and two ATP-binding domains, called ATP-binding cassettes (ABCs).

!clinic! CLINICAL INSIGHT: Multidrug Resistance Highlights a Family of Membrane Pumps with ATP-Binding Domains

Studies of human disease revealed another large family of active-transport proteins, with structures and mechanisms quite different from those of the P-type ATPase family. Tumor cells often become resistant to drugs that were initially quite toxic to the cells. Remarkably, the development of resistance to one drug also makes the cells less sensitive to a range of other compounds. This phenomenon is known as multidrug resistance. In a significant discovery, the onset of multidrug resistance was found to correlate with the expression of a protein that acts as an ATP-dependent pump that extrudes a wide range of small molecules from cells that express it. The protein is called the multidrug-resistance (MDR) protein or P-glycoprotein (“glyco” because it includes a carbohydrate moiety). When cells are exposed to a drug, the MDR protein pumps the drug out of the cell before the drug can exert its effects. The MDR protein comprises four domains: two membrane-spanning domains and two ATP-binding domains (Figure 12.17A). The ATP-binding domains of these proteins are called ATP-binding cassettes (ABCs). Transporters that include these domains are called ABC transporters. ABC transporters are one of the largest protein superfamilies and are found in all forms of life.

Another example of an ABC transporter is the cystic fibrosis transmembrane conductance regulator (CFTR; Figure 12.17B). CFTR acts as an ATP-regulated chloride channel in the plasma membranes of epithelial cells. Mutations in the gene for CFTR cause a decrease in fluid and salt secretion by CFTR and result in cystic fibrosis. As a consequence of the malfunctioning CFTR, secretion from the pancreas is blocked and heavy, dehydrated mucus accumulates in the lungs, leading to chronic lung infections. Certain ABC transporters also function as flippases, but in contrast to the P-type flippase, these enzymes transport lipids from the inner leaflet to the outer leaflet of the cell membrane.

!clinic! CLINICAL INSIGHT: Harlequin Ichthyosis Is a Dramatic Result of a Mutation in an ABC Transporter Protein

A number of human diseases in addition to cystic fibrosis result from defects in ABC transporter proteins. One especially startling disorder is harlequin ichthyosis, which results from a defective ABC transporter for lipids in keratinocytes, a common type of skin cell. Babies suffering from this very rare disorder are born encased in thick skin, which restricts their movement. As the skin dries out, hard diamond shaped plaques form, severely distorting facial features. The newborns usually die within a few days because of feeding difficulties, respiratory distress, or infections that are likely due to cracks in the skin.

Secondary Transporters Use One Concentration Gradient to Power the Formation of Another

Figure 12.18: Antiporters and symporters. Secondary transporters can transport two substrates in opposite directions (antiporters) or two substrates in the same direction (symporters).

Many active-transport processes are not directly driven by the hydrolysis of ATP. Instead, the thermodynamically uphill flow of one species of ion or molecule is coupled to the downhill flow of a different species. Membrane proteins that move ions or molecules uphill by this means are termed secondary transporters or cotransporters. These proteins can be classified as either antiporters or symporters. Antiporters couple the downhill flow of one species to the uphill flow of another in the opposite direction across the membrane; symporters use the flow of one species to drive the flow of a different species in the same direction across the membrane (Figure 12.18).

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Glucose is moved into some animal cells by the sodium-glucose linked transporter (SGLT), a symporter powered by the simultaneous entry of Na+. This free-energy input of Na+ flowing down its concentration gradient is sufficient to generate a 66-fold concentration gradient of an uncharged molecule such as glucose (Figure 12.19). Recall that the sodium ion gradient was initially generated by the Na+–K+ ATPase, demonstrating that the action of the secondary active transporter depends on the primary active transporter.

Figure 12.19: Secondary transport. The ion gradient set up by the Na+–K+ ATPase can be used to move materials into the cell, through the action of a secondary transporter such as the Na+–glucose linked transporter, a symporter.
Figure 12.20: Foxglove. Foxglove (Digitalis purpurea) is a highly poisonous plant due to the high concentration of potent cardiotonic steroids. Digitalis, one of the most widely used drugs, is obtained from foxglove.

DID YOU KNOW?

Interestingly, digitalis was used effectively long before the discovery of the Na+–K+ ATPase. In 1785, William Withering, a British physician, heard tales of an elderly woman, known as “the old woman of Shropshire,” who cured people of “dropsy” (which today would be recognized as congestive heart failure) with an extract of foxglove. Withering conducted the first scientific study of the effects of foxglove on congestive heart failure and documented its effectiveness.

Digitalis Inhibits the Na+–K+ Pump by Blocking Its Dephosphorylation

The interplay between active transport and secondary active transport is especially well illustrated by the action of the cardiotonic steroids. Heart failure can result if the muscles in the heart are not able to contract with sufficient strength to effectively pump blood. Certain steroids derived from plants, such as digitalis and ouabain, are known as cardiotonic steroids because of their ability to strengthen heart contractions. Interestingly, cardiotonic steroids exert their effect by inhibiting the Na+–K+ pump.

Digitalis is a mixture of cardiotonic steroids derived from the dried leaf of the foxglove plant Digitalis purpurea (Figure 12.20). The compound increases the force of contraction of heart muscle and is consequently a choice drug in the treatment of congestive heart failure. Inhibition of the Na+–K+ pump by digitalis means that Na+ is not pumped out of the cell, diminishing the Na+ gradient. The reduced Na+ gradient in turn affects the sodium–calcium exchanger. This exchanger, an example of secondary active transport, relies on Na+ influx to simultaneously power the expulsion of Ca+ from the cell. The diminished Na+ gradient results in slower extrusion of Ca2+ by the sodium–calcium exchanger. The subsequent increase in the intracellular level of Ca2+ enhances the ability of cardiac muscle to contract.

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Specific Channels Can Rapidly Transport Ions Across Membranes

Pumps can transport ions at rates approaching several thousand ions per second. Other membrane proteins, the passive-transport systems called ion channels, are capable of ion-transport rates that are more than 1000 times as fast. These rates of transport through ion channels are close to rates expected for ions diffusing freely through aqueous solution. Yet ion channels are not simply tubes that span membranes through which ions can rapidly flow. Rather, they are highly sophisticated molecular machines that respond to chemical and physical changes in their environments and undergo precisely timed conformational changes to regulate when ions can flow into and out of the cell. Channels can be classified on the basis of the environmental signals that activate them. Voltage-gated channels are opened in response to changes in membrane potential, whereas ligand-gated channels are opened in response to the binding of small molecules (ligands), such as neurotransmitters.

Visual image (left) and thermal image (right) of a mouse.

Among the most important manifestations of ion-channel action is the nerve impulse, which is the fundamental means of communication in the nervous system. A nerve impulse, or action potential, is an electrical signal produced by the flow of ions across the plasma membrane of a neuron. In particular, Na+ transiently flows into the cell and K+ flows out. This ion traffic is through protein channels that are both specific and rapid. The importance of ion channels is illustrated by the effect of tetrodotoxin, which is produced by the pufferfish (Figure 12.21). Tetrodotoxin inhibits the Na+ channel, and the lethal dose for human beings is just 10 ng.

Figure 12.21: Pufferfish. The pufferfish is regarded as a culinary delicacy in Japan. Tetrodotoxin is produced by the pufferfish. Several people die every year in Japan from eating poorly prepared pufferfish.

!bio! BIOLOGICAL INSIGHT: Venomous Pit Vipers Use Ion Channels to Generate a Thermal Image

A large family of cation channels comprises the TRP (transient receptor potential) channels. These channels serve a host of functions in vertebrates including detecting taste, pain, and temperature. Venomous pit vipers, such as the western diamondback rattlesnake (Crotalus atrox), possess TRP channels that are activated by infrared (750 nm–1 mm) radiation, thus enabling the snakes to create a thermal landscape that can be overlain by their visual landscape. This dual-image view enables the pit vipers to locate and track prey with great speed and accuracy, much to the consternation of a host of rodents.

The Structure of the Potassium Ion Channel Reveals the Basis of Ion Specificity

The K+ channel is one of the most extensively studied ion channels and thus provides us with a clear example of how a channel function can be both specific and rapid. Beginning from the inside of the cell, the pore starts with a diameter of approximately 10 Å and then constricts to a smaller cavity with a diameter of 8 Å (Figure 12.22). Both the opening to the outside and the central cavity of the pore are filled with water, and a K+ ion can fit in the pore without losing its shell of bound water molecules. Approximately two-thirds of the way through the membrane, the pore becomes more constricted (3-Å diameter). At that point, any K+ ions must give up their water molecules and interact directly with groups from the protein. The channel structure effectively reduces the thickness of the membrane from 34 Å to 12 Å by allowing the solvated ions to penetrate into the membrane before the ions must directly interact with the channel.

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Figure 12.22: A path through a channel. A potassium ion entering the K+ channel can pass a distance of 22 Å into the membrane while remaining solvated with water (blue). At this point, the pore diameter narrows to 3 Å (yellow), and potassium ions must shed their water and interact with carbonyl groups (red) of the pore amino acids.

The restricted part of the pore is built from residues contributed by the two transmembrane a helices. In particular, a stretch of five amino acids within this region function as the selectivity filter that determines the preference for K+ over other ions (Figure 12.23). This region of the strand lies in an extended conformation and is oriented such that the peptide carbonyl groups are directed into the channel, in a good position to interact with the potassium ions. The potassium ion relinquishes its associated water molecules because it can bind with the oxygen atoms of the carbonyl groups of the selectivity filter.

Figure 12.23: The selectivity filter of the potassium ion channel. Potassium ions interact with the carbonyl groups of the selectivity filter, located at the 3-Å-diameter pore of the K+ channel. Only two of the four channel subunits are shown.

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Potassium ion channels are 100-fold more permeable to K+ than to Na+. How is this high degree of selectivity achieved? Ions having a radius larger than 1.5 Å cannot pass into the narrow diameter (3 Å) of the selectivity filter of the K+ channel. However, a bare Na+ is small enough to pass through the pore. Indeed, the ionic radius of Na+ (0.95 Å) is substantially smaller than that of K+ (1.33 Å). How, then, is Na+ rejected?

Sodium ions are too small to react with the selectivity filter. For ions to relinquish their water molecules, other polar interactions—such as those that take place between the K+ ion and the selectivity filter’s carbonyl groups—must replace the interactions with water. The key point is that the free-energy costs of dehydrating these ions are considerable. The channel pays the cost of dehydrating potassium ions by providing compensating interactions with the carbonyl oxygen atoms lining the selectivity filter (Figure 12.24). Sodium ions are rejected because the energy required to dehydrate them would not be recovered. The K+ channel does not closely interact with sodium ions, which must stay hydrated and, hence, cannot pass through the channel.

Figure 12.24: The energetic basis of ion selectivity. The energy cost of dehydrating a potassium ion is compensated by favorable interactions with the selectivity filter. Because a sodium ion is too small to interact favorably with the selectivity filter, the free energy of desolvation cannot be compensated, and the sodium ion does not pass through the channel.

The Structure of the Potassium Ion Channel Explains Its Rapid Rate of Transport

!quickquiz! QUICK QUIZ 2

What determines the direction of flow through an ion channel?

The tight binding sites required for ion selectivity should slow the progress of ions through a channel, yet ion channels achieve rapid rates of ion transport. How is this apparent paradox resolved? A structural analysis of the K+ channel at high resolution provides an appealing explanation. Four K+-binding sites crucial for rapid ion flow are present in the constricted region of the K+ channel. Consider the process of ion conductance starting from inside the cell (Figure 12.25). A hydrated potassium ion proceeds into the channel and through the relatively unrestricted part of the channel. The ion then gives up its coordinated water molecules and binds to a site within the selectivity-filter region. The ion can move between the four sites within the selectivity filter because they have similar energy levels and thus ion affinities. As each subsequent potassium ion moves into the selectivity filter, its positive charge will repel the potassium ion at the nearest site, causing it to shift to a site farther up the channel and in turn push upward any potassium ion already bound to a site farther up. Thus, each ion that binds anew favors the release of an ion from the other side of the channel. This multiple-binding-site mechanism solves the apparent paradox of high ion selectivity and rapid flow.

Figure 12.25: A model for K+-channel transport. The selectivity filter has four binding sites (white circles). hydrated potassium ions can enter these sites, one at a time, losing their hydration shells (red lines). When two ions occupy adjacent sites, electrostatic repulsion forces them apart. Thus, as ions enter the channel from one side, other ions are pushed out the other side.

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