Protein-Mediated Transport

In the body, simple diffusion across membranes is limited to lipophilic molecules. The majority of molecules in the body are either lipophobic or electrically charged and therefore cannot cross membranes by simple diffusion. Instead, the vast majority of solutes cross membranes with the help of membrane proteins, a process we call mediated transport.

If mediated transport is passive and moves molecules down their concentration gradient, and if net transport stops when concentrations are equal on both sides of the membrane, the process is facilitated diffusion (Fig. 5.5). If protein-mediated transport requires energy from ATP or another outside source and moves a substance against its concentration gradient, the process is known as active transport.

Membrane Proteins Have Four Major Functions

Protein-mediated transport across a membrane is carried out by membrane-spanning transport proteins. For physiologists, classifying membrane proteins by their function is more useful than classifying them by their structure. Our functional classification scheme recognizes four broad categories of membrane proteins: (1) structural proteins, (2) enzymes, (3) receptors, and (4) transport proteins. Figure 5.8 is a map comparing the structural and functional classifications of membrane proteins. These groupings are not completely distinct, and as you will learn, some membrane proteins have more than one function, such as receptor-channels and receptor-enzymes.

FIG. 5.8 Map of membrane proteins

Structural Proteins

The structural proteins of membranes have three major roles.

  1. They help create cell junctions that hold tissues together, such as tight junctions and gap junctions [Fig. 3.8].

  2. They connect the membrane to the cytoskeleton to maintain the shape of the cell [Fig. 3.2]. The microvilli of transporting epithelia are one example of membrane shaping by the cytoskeleton [Fig. 3.4b].

  3. They attach cells to the extracellular matrix by linking cytoskeleton fibers to extracellular collagen and other protein fibers [here].

Enzymes

Membrane enzymes catalyze chemical reactions that take place either on the cell’s external surface or just inside the cell. For example, enzymes on the external surface of cells lining the small intestine are responsible for digesting peptides and carbohydrates. Enzymes attached to the intracellular surface of many cell membranes play an important role in transferring signals from the extracellular environment to the cytoplasm [see Chapter 6].

Receptors

Membrane receptor proteins are part of the body’s chemical signaling system. The binding of a receptor with its ligand usually triggers another event at the membrane (Fig. 5.9). Sometimes the ligand remains on the cell surface, and the receptor-ligand complex triggers an intracellular response. In other instances, the receptor-ligand complex is brought into the cell in a vesicle [here]. Membrane receptors also play an important role in some forms of vesicular transport, as you will learn later in this chapter.

FIG. 5.9 Membrane receptors bind extracellular ligands

Transport Proteins

The fourth group of membrane proteins—transport proteins—moves molecules across membranes. There are several different ways to classify transport proteins. Scientists have discovered that the genes for most membrane transport proteins belong to one of two gene “superfamilies”: the ATP-binding cassette (ABC) superfamily or the solute carrier (SLC) superfamily. The ABC family proteins use ATP’s energy to transport small molecules or ions across membranes. The 52 families of the SLC superfamily include most facilitated diffusion transporters as well as some active transporters.

A second way to classify transport* recognizes two main types of transport proteins: channels and carriers (Fig. 5.10). Channel proteins create water-filled passageways that directly link the intracellular and extracellular compartments. Carrier proteins, also just called transporters, bind to the substrates that they carry but never form a direct connection between the intracellular fluid and extracellular fluid. As Figure 5.10 shows, carriers are open to one side of the membrane or the other, but not to both at once the way channel proteins are.

*The Transporter Classification System, www.tcdb.org

Why do cells need both channels and carriers? The answer lies in the different properties of the two transport proteins. Channel proteins allow more rapid transport across the membrane but generally are limited to moving small ions and water. Carriers, while slower, can move larger molecules than channels can. There is some overlap between the two types, both structurally and functionally. For example, the aquaporin protein AQP has been shown to act both as a water channel and as a carrier for certain small organic molecules.

Channel Proteins Form Open, Water-Filled Passageways

Channel proteins are made of membrane-spanning protein subunits that create a cluster of cylinders with a tunnel or pore through the center. Nuclear pore complexes [here] and gap junctions [Fig. 3.8b] can be considered very large forms of channels. In this book, we restrict use of the term channel to smaller channels whose centers are narrow, water-filled pores (Fig. 5.11). Movement through these smaller channels is mostly restricted to water and ions. When water-filled ion channels are open, tens of millions of ions per second can whisk through them unimpeded.

FIG. 5.11 The structure of channel proteins

Channel proteins are named according to the substances that they allow to pass. Most cells have water channels made from a protein called aquaporin. In addition, more than 100 types of ion channels have been identified. Ion channels may be specific for one ion or may allow ions of similar size and charge to pass. For example, there are Na+ channels, K+ channels, and nonspecific monovalent (“one-charge”) cation channels that transport Na+, K+ and lithium ions Li+. Other ion channels you will encounter frequently in this text are Ca2+ channels and Cl channels. Ion channels come in many subtypes, or isoforms.

The selectivity of a channel is determined by the diameter of its central pore and by the electrical charge of the amino acids that line the channel. If the channel amino acids are positively charged, positive ions are repelled and negative ions can pass through the channel. On the other hand, a cation channel must have a negative charge that attracts cations but prevents the passage of Cl or other anions.

Channel proteins are like narrow doorways into the cell. If the door is closed, nothing can go through. If the door is open, there is a continuous passage between the two rooms connected by the doorway. The open or closed state of a channel is determined by regions of the protein molecule that act like swinging “gates.”

According to current models, channel “gates” take several forms. Some channel proteins have gates in the middle of the protein’s pore. Other gates are part of the cytoplasmic side of the membrane protein. Such a gate can be envisioned as a ball on a chain that swings up and blocks the mouth of the channel (Fig.  5.10a). One type of channel in neurons has two different gates.

Channels can be classified according to whether their gates are usually open or usually closed. Open channels spend most of their time with their gate open, allowing ions to move back and forth across the membrane without regulation. These gates may occasionally flicker closed, but for the most part these channels behave as if they have no gates. Open channels are sometimes called either leak channels or pores, as in water pores.

Gated channels spend most of their time in a closed state, which allows these channels to regulate the movement of ions through them. When a gated channel opens, ions move through the channel just as they move through open channels. When a gated channel is closed, which it may be much of the time, it allows no ion movement between the intracellular and extracellular fluid.

What controls the opening and closing of gated channels? For chemically gated channels, the gating is controlled by intracellular messenger molecules or extracellular ligands that bind to the channel protein. Voltage-gated channels open and close when the electrical state of the cell changes. Finally, mechanically gated channels respond to physical forces, such as increased temperature or pressure that puts tension on the membrane and pops the channel gate open. You will encounter many variations of these channel types as you study physiology.

Concept Check

  1. Positively charged ions are called       , and negatively charged ions are called          .

Carrier Proteins Change Conformation to Move Molecules

The second type of transport protein is the carrier protein (Fig. 5.10b). Carrier proteins bind with specific substrates and carry them across the membrane by changing conformation. Small organic molecules (such as glucose and amino acids) that are too large to pass through channels cross membranes using carriers. Ions such as Na+ and K+ may move by carriers as well as through channels. Carrier proteins move solutes and ions into and out of cells as well as into and out of intracellular organelles, such as the mitochondria.

Some carrier proteins move only one kind of molecule and are known as uniport carriers. However, it is common to find carriers that move two or even three kinds of molecules. A carrier that moves more than one kind of molecule at one time is called a cotransporter. If the molecules being transported are moving in the same direction, whether into or out of the cell, the carrier proteins are symport carriers {sym−, together + portare, to carry}. (Sometimes, the term cotransport is used in place of symport.) If the molecules are being carried in opposite directions, the carrier proteins are antiport carriers {anti, opposite + portare, to carry}, also called exchangers. Symport and antiport carriers are shown in Figure 5.10b.

Carriers are large, complex proteins with multiple subunits. The conformation change required of a carrier protein makes this mode of transmembrane transport much slower than movement through channel proteins. A carrier protein can move only 1,000 to 1,000,000 molecules per second, in contrast to tens of millions of ions per second that move through a channel protein.

Carrier proteins differ from channel proteins in another way: carriers never create a continuous passage between the inside and outside of the cell. If channels are like doorways, then carriers are like revolving doors that allow movement between inside and outside without ever creating an open hole. Carrier proteins can transport molecules across a membrane in both directions, like a revolving door at a hotel, or they can restrict their transport to one direction, like the turnstile at an amusement park that allows you out of the park but not back in.

One side of the carrier protein always creates a barrier that prevents free exchange across the membrane. In this respect, carrier proteins function like the Panama Canal (Fig. 5.12). Picture the canal with only two gates, one on the Atlantic side and one on the Pacific side. Only one gate at a time is open.

FIG. 5.12 Carrier proteins

When the Atlantic gate is closed, the canal opens into the Pacific. A ship enters the canal from the Pacific, and the gate closes behind it. Now the canal is isolated from both oceans with the ship trapped in the middle. Then the Atlantic gate opens, making the canal continuous with the Atlantic Ocean. The ship sails out of the gate and off into the Atlantic, having crossed the barrier of the land without the canal ever forming a continuous connection between the two oceans.

Movement across the membrane through a carrier protein is similar (Fig. 5.12b). The molecule being transported binds to the carrier on one side of the membrane (the extracellular side in our example). This binding changes the conformation of the carrier protein so that the opening closes. After a brief transition in which both sides are closed, the opposite side of the carrier opens to the other side of the membrane. The carrier then releases the transported molecule into the opposite compartment, having brought it through the membrane without creating a continuous connection between the extracellular and intracellular compartments.

Carrier proteins can be divided into two categories according to the energy source that powers the transport. As noted earlier, facilitated diffusion is protein-mediated transport in which no outside source of energy except a concentration gradient is needed to move molecules across the cell membrane. Active transport is protein-mediated transport that requires an outside energy source, either ATP or the potential energy stored in a concentration gradient that was created using ATP. We will look first at facilitated diffusion.

Concept Check

  1. Name four functions of membrane proteins.

  2. Which kinds of particles pass through open channels?

  3. Name two ways channels differ from carriers.

  4. If a channel is lined with amino acids that have a net positive charge, which of the following ions is/are likely to move freely through the channel? Na+, Cl, K+, Ca2+

  5. Why can’t glucose cross the cell membrane through open channels?

Facilitated Diffusion Uses Carrier Proteins

Some polar molecules appear to move into and out of cells by diffusion, even though we know from their chemical properties that they are unable to pass easily through the lipid core of the cell membrane. The solution to this seeming contradiction is that these polar molecules cross the cell membrane by facilitated diffusion, with the aid of specific carriers. Sugars and amino acids are examples of molecules that enter or leave cells using facilitated diffusion. For example, the family of carrier proteins known as GLUT transporters move glucose and related hexose sugars across membranes.

Facilitated diffusion has the same properties as simple diffusion (see TBL. 5.6). The transported molecules move down their concentration gradient, the process requires no input of outside energy, and net movement stops at equilibrium, when the concentration inside the cell equals the concentration outside the cell (Fig. 5.13):

[ glucose ] ECF   =   [ glucose ] ICF

FIG. 5.13 Facilitated diffusion of glucose into cells

*

Facilitated diffusion carriers always transport molecules down their concentration gradient. If the gradient reverses, so does the direction of transport.

Cells in which facilitated diffusion takes place can avoid reaching equilibrium by keeping the concentration of substrate in the cell low. With glucose, for example, this is accomplished by phosphorylation (Fig. 5.13c). As soon as a glucose molecule enters the cell on the GLUT carrier, it is phosphorylated to glucose 6-phosphate, the first step of glycolysis [here]. Addition of the phosphate group prevents build-up of glucose inside the cell and also prevents glucose from leaving the cell.

Concept Check

  1. Liver cells (hepatocytes) are able to convert glycogen to glucose, thereby making the intracellular glucose concentration higher than the extracellular glucose concentration. In what direction do the hepatic GLUT2 transporters carry glucose when this occurs?

Active Transport Moves Substances against Their Concentration Gradients

Active transport is a process that moves molecules against their concentration gradient—that is, from areas of lower concentration to areas of higher concentration. Rather than creating an equilibrium state, where the concentration of the molecule is equal throughout the system, active transport creates a state of disequilibrium by making concentration differences more pronounced. Moving molecules against their concentration gradient requires the input of outside energy, just as pushing a ball up a hill requires energy [see Fig. 4.2, here]. The energy for active transport comes either directly or indirectly from the high-energy phosphate bond of ATP.

Active transport can be divided into two types. In primary (direct) active transport, the energy to push molecules against their concentration gradient comes directly from the high-energy phosphate bond of ATP. Secondary (indirect) active transport uses potential energy [here] stored in the concentration gradient of one molecule to push other molecules against their concentration gradient. All secondary active transport ultimately depends on primary active transport because the concentration gradients that drive secondary transport are created using energy from ATP.

The mechanism for both types of active transport appears to be similar to that for facilitated diffusion. A substrate to be transported binds to a membrane carrier and the carrier then changes conformation, releasing the substrate into the opposite compartment. Active transport differs from facilitated diffusion because the conformation change in the carrier protein requires energy input.

Primary Active Transport

Because primary active transport uses ATP as its energy source, many primary active transporters are known as ATPases. You may recall that the suffix -ase signifies an enzyme, and the stem (ATP) is the substrate upon which the enzyme is acting [here]. These enzymes hydrolyze ATP to ADP and inorganic phosphate (Pi), releasing usable energy in the process. Most of the ATPases you will encounter in your study of physiology are listed in Table 5.7. ATPases are sometimes called pumps, as in the sodium-potassium pump, or Na+-K+-ATPase, mentioned earlier in this chapter.

Table 5.7 Primary Active Transporters

Names Type of Transport
Na+-K+-ATPase or sodium-potassium pump Antiport
Ca2+-ATPase Uniport
H+-ATPase or proton pump Uniport
H+-K+-ATPase Antiport

The sodium-potassium pump is probably the single most important transport protein in animal cells because it maintains the concentration gradients of Na+ and K+ across the cell membrane (Fig. 5.14). The transporter is arranged in the cell membrane so that it pumps 3 Na+ out of the cell and 2 K+ into the cell for each ATP consumed. In some cells, the energy needed to move these ions uses 30% of all the ATP produced by the cell. Figure 5.15 illustrates the current model of how the Na+-K+-ATPase works.

FIG. 5.14 The sodium-potassium pump, Na+-K+-ATPase

FIG. 5.15 Mechanism of the Na+-K+-ATPase

Secondary Active Transport

The sodium concentration gradient, with Na+ concentration high in the extracellular fluid and low inside the cell, is a source of potential energy that the cell can harness for other functions. For example, nerve cells use the sodium gradient to transmit electrical signals, and epithelial cells use it to drive the uptake of nutrients, ions, and water. Membrane transporters that use potential energy stored in concentration gradients to move molecules are called secondary active transporters.

Secondary active transport uses the kinetic energy of one molecule moving down its concentration gradient to push other molecules against their concentration gradient. The cotransported molecules may go in the same direction across the membrane (symport) or in opposite directions (antiport). The most common secondary active transport systems are driven by the sodium concentration gradient.

As one Na+ moves into the cell, it either brings one or more molecules with it or trades places with molecules exiting the cell. The major Na+-dependent transporters are listed in Table 5.8. Notice that the cotransported substances may be either other ions or uncharged molecules, such as glucose. As you study the different systems of the body, you will find these secondary active transporters taking part in many physiological processes.

Table 5.8 Examples of Secondary Active Transporters

Symport Carriers Antiport Carriers
Sodium-Dependent Transporters
Na+-K+-2Cl (NKCC) Na+-H+ (NHE)
Na+-glucose (SGLT) Na+-Ca2+ (NCX)
Na+-Cl
Na+-HCO3
Na+-amino acids (several types)
Na+-bile salts (small intestine)
Na+-choline uptake (nerve cells)
Na+-neurotransmitter uptake (nerve cells)
Nonsodium-Dependent Transporters
H+-peptide symporter (pepT) HCO3 -Cl

The mechanism of the Na+-glucose secondary active transporter (SGLT) is illustrated in Figure 5.16. Both Na+ and glucose bind to the SGLT protein on the extracellular fluid side. Sodium binds first and causes a conformational change in the protein that creates a high-affinity binding site for glucose . When glucose binds to SGLT , the protein changes conformation again and opens its channel to the intracellular fluid side . Sodium is released to the ICF as it moves down its concentration gradient. The loss of Na+ from the protein changes the binding site for glucose back to a low-affinity site, so glucose is released and follows Na+ into the cytoplasm . The net result is the entry of glucose into the cell against its concentration gradient, coupled to the movement of Na+ into the cell down its concentration gradient. The SGLT transporter can only move glucose into cells because glucose must follow the Na+ gradient.

FIG. 5.16 Sodium-glucose cotransport

In contrast, GLUT transporters are reversible and can move glucose into or out of cells depending on the concentration gradient. For example, when blood glucose levels are high, GLUT transporters on liver cells bring glucose into those cells. During times of fasting, when blood glucose levels fall, liver cells convert their glycogen stores to glucose. When the glucose concentration inside the liver cells builds up and exceeds the glucose concentration in the plasma, glucose leaves the cells on the reversible GLUT transporters. GLUT transporters are found on all cells of the body.

If GLUT transporters are everywhere, then why does the body need the SGLT Na+-glucose symporter? The simple answer is that both SGLT and GLUT are needed to move glucose from one side of an epithelium to the other. Consequently, SGLT transporters are found on certain epithelial cells, such as intestinal and kidney cells, that bring glucose into the body from the external environment. We discuss the process of transepithelial transport of glucose later in this chapter.

Concept Check

  1. Name two ways active transport by the Na+-K+-ATPase (Fig. 5.15) differs from secondary transport by the SGLT (Fig. 5.16) .

Carrier-Mediated Transport Exhibits Specificity, Competition, and Saturation

Both passive and active forms of carrier-mediated transport demonstrate specificity, competition, and saturation—three properties that result from the binding of a substrate to a protein [here].

Specificity

Specificity refers to the ability of a transporter to move only one molecule or only a group of closely related molecules [here]. One example of specificity is found in the GLUT family of transporters, which move 6-carbon sugars (hexoses), such as glucose, mannose, galactose, and fructose [here], across cell membranes. GLUT transporters have binding sites that recognize and transport hexoses, but they will not transport the disaccharide maltose or any form of glucose that is not found in nature (FIG. 5.17b). For this reason we can say that GLUT transporters are specific for naturally occurring 6-carbon monosaccharides.

FIG. 5.17 Transporter saturation and competition

Graph Question: How could the cell increase its transport rate in this example?

Graph Question: Can you tell from this graph if galactose is being transported?

For many years, scientists assumed that there must be different isoforms of the glucose-facilitated diffusion carrier because they had observed that glucose transport was regulated by hormones in some cells but not in others. However, it was not until the 1980s that the first glucose transporter was isolated. To date, 14 SCL2A (GLUT) genes have been identified. The important GLUT proteins you will encounter in this book include GLUT1, found in most cells of the body; GLUT2, found in liver and in kidney and intestinal epithelia; GLUT3, found in neurons; GLUT4, the insulin-regulated transporter of skeletal muscle; and GLUT5, the intestinal fructose transporter. The restriction of different GLUT transporters to different tissues is an important feature in the metabolism and homeostasis of glucose.

Competition

The property of competition is closely related to specificity. A transporter may move several members of a related group of substrates, but those substrates compete with one another for binding sites on the transporter. For example, GLUT transporters move the family of hexose sugars, but each different GLUT transporter has a “preference” for one or more hexoses, based on its binding affinity.

The results of an experiment demonstrating competition are shown in Figure 5.17d. The graph shows glucose transport rate as a function of glucose concentration. The top line (red) shows transport when only glucose is present. The lower line (black) shows that glucose transport decreases if galactose is also present. Galactose competes for binding sites on the GLUT transporters and displaces some glucose molecules. With fewer glucose molecules able to bind to the GLUT protein, the rate of glucose transport into the cell decreases.

Sometimes, the competing molecule is not transported but merely blocks the transport of another substrate. In this case, the competing molecule is a competitive inhibitor [here]. In the glucose transport system, the disaccharide maltose is a competitive inhibitor (Fig. 5.17b). It competes with glucose for the binding site, but once bound, it is too large to be moved across the membrane.

Competition between transported substrates has been put to good use in medicine. An example involves gout, a disease caused by elevated levels of uric acid in the plasma. One method of decreasing uric acid in plasma is to enhance its excretion in the urine. Normally, the kidney’s organic anion transporter (OAT) reclaims urate (the anion form of uric acid) from the urine and returns the acid to the plasma. However, if an organic acid called probenecid is administered to the patient, OAT binds to probenecid instead of to uric acid, preventing the reabsorption of urate. As a result, more urate leaves the body in the urine, lowering the uric acid concentration in the plasma.

Saturation

The rate of substrate transport depends on the substrate concentration and the number of carrier molecules, a property that is shared by enzymes and other binding proteins [here]. For a fixed number of carriers, however, as substrate concentration increases, the transport rate increases up to a maximum, the point at which all carrier binding sites are filled with substrate. At this point, the carriers are said to have reached saturation. At saturation, the carriers are working at their maximum rate, and a further increase in substrate concentration has no effect. Figure 5.17c represents saturation graphically.

As an analogy, think of the carriers as doors into a concert hall. Each door has a maximum number of people that it can allow to enter the hall in a given period of time. Suppose that all the doors together can allow a maximum of 100 people per minute to enter the hall. This is the maximum transport rate, also called the transport maximum. When the concert hall is empty, three maintenance people enter the doors every hour. The transport rate is 3 people/60 minutes, or 0.05 people/minute, well under the maximum. For a local dance recital, about 50 people per minute go through the doors, still well under the maximum. When the most popular rock group of the day appears in concert, however, thousands of people gather outside. When the doors open, thousands of people are clamoring to get in, but the doors allow only 100 people/minute into the hall. The doors are working at the maximum rate, so it does not matter whether there are 1,000 or 3,000 people trying to get in. The transport rate saturates at 100 people/minute.

How can cells increase their transport capacity and avoid saturation? One way is to increase the number of carriers in the membrane. This would be like opening more doors into the concert hall. Under some circumstances, cells are able to insert additional carriers into their membranes. Under other circumstances, a cell may withdraw carriers to decrease movement of a molecule into or out of the cell.

All forms of carrier-mediated transport show specificity, competition, and saturation, but as you learned earlier in the chapter, they also differ in one important way: passive mediated transport—better known as facilitated diffusion—requires no input of energy from an outside source. Active transport requires energy input from ATP, either directly or indirectly.