Plasma Membranes Are Involved In Facilitated Diffusion Active Transport Other Processes

Transport systems can be described in a functional sense according to the number of molecules moved and the direction of movement (Figure 41-10) or according to whether movement is toward or away from equilibrium. A uniport system moves one type of molecule bidirectionally. In cotransport systems, the transfer of one solute depends upon the stoichiometric simultaneous or sequential transfer of another solute. A symport moves these solutes in the same direction. Examples are the proton-sugar transporter in bacteria and the Na+ -sugar transporters (for glucose and certain other sugars) and Na+-amino acid transporters in mammalian cells. Antiport systems move two molecules in opposite directions (eg, Na+ in and Ca2+ out).

Molecules that cannot pass freely through the lipid bilayer membrane by themselves do so in association with carrier proteins. This involves two processes— facilitated diffusion and active transport—and highly specific transport systems.

Facilitated diffusion and active transport share many features. Both appear to involve carrier proteins, and both show specificity for ions, sugars, and amino acids.

Mutations in bacteria and mammalian cells (including some that result in human disease) have supported these conclusions. Facilitated diffusion and active transport resemble a substrate-enzyme reaction except that no covalent interaction occurs. These points of resemblance are as follows: (1) There is a specific binding site for the solute. (2) The carrier is saturable, so it has a maximum rate of transport (Vmax; Figure 41-11). (3) There is a binding constant (Km) for the solute, and

Solute concentration

Figure 41-11. A comparison of the kinetics of carrier-mediated (facilitated) diffusion with passive diffusion. The rate of movement in the latter is directly proportionate to solute concentration, whereas the process is saturable when carriers are involved. The concentration at half-maximal velocity is equal to the binding constant (Km) of the carrier for the solute. (Vmax maximal rate.)

Solute concentration

Figure 41-11. A comparison of the kinetics of carrier-mediated (facilitated) diffusion with passive diffusion. The rate of movement in the latter is directly proportionate to solute concentration, whereas the process is saturable when carriers are involved. The concentration at half-maximal velocity is equal to the binding constant (Km) of the carrier for the solute. (Vmax maximal rate.)

so the whole system has a Km (Figure 41-11). (4) Structurally similar competitive inhibitors block transport.

Major differences are the following: (1) Facilitated diffUsion can operate bidirectionally, whereas active transport is usually unidirectional. (2) Active transport always occurs against an electrical or chemical gradient, and so it requires energy.

Facilitated Diffusion

Some specific solutes diffuse down electrochemical gradients across membranes more rapidly than might be expected from their size, charge, or partition coefficients. This facilitated diffusion exhibits properties distinct from those of simple diffusion. The rate of facilitated diffusion, a uniport system, can be saturated; ie, the number of sites involved in diffusion of the specific solutes appears finite. Many facilitated diffusion systems are stereospecific but, like simple diffusion, require no metabolic energy.

As described earlier, the inside-outside asymmetry of membrane proteins is stable, and mobility of proteins across (rather than in) the membrane is rare; therefore, transverse mobility of specific carrier proteins is not likely to account for facilitated diffusion processes except in a few unusual cases.

A "Ping-Pong" mechanism (Figure 41-12) explains facilitated diffusion. In this model, the carrier protein exists in two principal conformations. In the "pong" state, it is exposed to high concentrations of solute, and molecules of the solute bind to specific sites on the carrier protein. Transport occurs when a confor-mational change exposes the carrier to a lower concentration of solute ("ping" state). This process is completely reversible, and net flux across the membrane depends upon the concentration gradient. The rate at which solutes enter a cell by facilitated diffusion is de termined by the following factors: (1) The concentration gradient across the membrane. (2) The amount of carrier available (this is a key control step). (3) The rapidity of the solute-carrier interaction. (4) The rapidity of the conformational change for both the loaded and the unloaded carrier.

Hormones regulate facilitated diffusion by changing the number of transporters available. Insulin increases glucose transport in fat and muscle by recruiting transporters from an intracellular reservoir. Insulin also enhances amino acid transport in liver and other tissues. One of the coordinated actions of glucocorticoid hormones is to enhance transport of amino acids into liver, where the amino acids then serve as a substrate for glu-coneogenesis. Growth hormone increases amino acid transport in all cells, and estrogens do this in the uterus. There are at least five different carrier systems for amino acids in animal cells. Each is specific for a group of closely related amino acids, and most operate as Na+-symport systems (Figure 41-10).

Active Transport

The process of active transport differs from diffusion in that molecules are transported away from thermody-namic equilibrium; hence, energy is required. This energy can come from the hydrolysis of ATP, from electron movement, or from light. The maintenance of electrochemical gradients in biologic systems is so important that it consumes perhaps 30-40% of the total energy expenditure in a cell.

In general, cells maintain a low intracellular Na+ concentration and a high intracellular K+ concentration (Table 41-1), along with a net negative electrical potential inside. The pump that maintains these gradients is an ATPase that is activated by Na+ and K+ (Na+-K+ ATPase; see Figure 41-13). The ATPase is an integral

Figure 41-12. The "Ping-Pong" model of facilitated diffusion. A protein carrier (gray structure) in the lipid bi-layer associates with a solute in high concentration on one side of the membrane. A conformational change ensues ("pong" to "ping"), and the solute is discharged on the side favoring the new equilibrium. The empty carrier then reverts to the original conformation ("ping" to "pong") to complete the cycle.

Figure 41-12. The "Ping-Pong" model of facilitated diffusion. A protein carrier (gray structure) in the lipid bi-layer associates with a solute in high concentration on one side of the membrane. A conformational change ensues ("pong" to "ping"), and the solute is discharged on the side favoring the new equilibrium. The empty carrier then reverts to the original conformation ("ping" to "pong") to complete the cycle.

INSIDE I I OUTSIDE

Membrane

INSIDE I I OUTSIDE

Membrane

Figure 41-13. Stoichiometry of the Na+-K+ ATPase pump. This pump moves three Na+ ions from inside the cell to the outside and brings two K+ ions from the outside to the inside for every molecule of ATP hydrolyzed to ADP by the membrane-associated ATPase. Ouabain and other cardiac glycosides inhibit this pump by acting on the extracellular surface of the membrane. (Courtesy of R Post.)

Figure 41-13. Stoichiometry of the Na+-K+ ATPase pump. This pump moves three Na+ ions from inside the cell to the outside and brings two K+ ions from the outside to the inside for every molecule of ATP hydrolyzed to ADP by the membrane-associated ATPase. Ouabain and other cardiac glycosides inhibit this pump by acting on the extracellular surface of the membrane. (Courtesy of R Post.)

membrane protein and requires phospholipids for activity. The ATPase has catalytic centers for both ATP and Na+ on the cytoplasmic side of the membrane, but the K+ binding site is located on the extracellular side of the membrane. Ouabain or digitalis inhibits this ATP-ase by binding to the extracellular domain. Inhibition of the ATPase by ouabain can be antagonized by extracellular K+.

Nerve Impulses Are Transmitted Up & Down Membranes

The membrane forming the surface of neuronal cells maintains an asymmetry of inside-outside voltage (electrical potential) and is electrically excitable. When appropriately stimulated by a chemical signal mediated by a specific synaptic membrane receptor (see discussion of the transmission of biochemical signals, below), gates in the membrane are opened to allow the rapid influx of Na+ or Ca2+ (with or without the efflux of K+), so that the voltage difference rapidly collapses and that segment of the membrane is depolarized. However, as a result of the action of the ion pumps in the membrane, the gradient is quickly restored.

When large areas of the membrane are depolarized in this manner, the electrochemical disturbance propagates in wave-like form down the membrane, generating a nerve impulse. Myelin sheets, formed by Schwann cells, wrap around nerve fibers and provide an electrical insulator that surrounds most of the nerve and greatly speeds up the propagation of the wave (signal) by allowing ions to flow in and out of the membrane only where the membrane is free of the insulation. The myelin membrane is composed of phospholipids, cholesterol, proteins, and GSLs. Relatively few proteins are found in the myelin membrane; those present appear to hold together multiple membrane bilayers to form the hydrophobic insulating structure that is impermeable to ions and water. Certain diseases, eg, multiple sclerosis and the Guillain-Barré syndrome, are characterized by demyelination and impaired nerve conduction.

Glucose Transport Involves Several Mechanisms

A discussion of the transport of glucose summarizes many of the points made in this chapter. Glucose must enter cells as the first step in energy utilization. In adipocytes and muscle, glucose enters by a specific transport system that is enhanced by insulin. Changes in transport are primarily due to alterations of Vmax (presumably from more or fewer active transporters), but changes in Km may also be involved. Glucose transport involves different aspects of the principles of transport discussed above. Glucose and Na+ bind to different sites on the glucose transporter. Na+ moves into the cell down its electrochemical gradient and "drags" glucose with it (Figure 41-14). Therefore, the greater the Na+ gradient, the more glucose enters; and if Na+ in extracellular fluid is low, glucose transport stops. To maintain a steep Na+ gradient, this Na+-glucose symport is dependent on gradients generated by an Na+-K+ pump that maintains a low intracellular Na+ concentration. Similar mechanisms are used to transport other sugars as well as amino acids.

The transcellular movement of sugars involves one additional component: a uniport that allows the glucose accumulated within the cell to move across a different surface toward a new equilibrium; this occurs in intestinal and renal cells, for example.

Cells Transport Certain Macromolecules Across the Plasma Membrane

The process by which cells take up large molecules is called "endocytosis." Some of these molecules (eg, polysaccharides, proteins, and polynucleotides), when hydrolyzed inside the cell, yield nutrients. Endocytosis provides a mechanism for regulating the content of certain membrane components, hormone receptors being a case in point. Endocytosis can be used to learn more about how cells function. DNA from one cell type can be used to transfect a different cell and alter the latter's function or phenotype. A specific gene is often employed in these experiments, and this provides a unique way to study and analyze the regulation of that gene. DNA transfection depends upon endocytosis; endocy-

LUMEN

LUMEN

Glucose

EXTRACELLULAR FLUID

Figure 41-14. The transcellular movement of glucose in an intestinal cell. Glucose follows Na+ across the luminal epithelial membrane. The Na+ gradient that drives this symport is established by Na+ -K+ exchange, which occurs at the basal membrane facing the extracellular fluid compartment. Glucose at high concentration within the cell moves "downhill" into the extracellular fluid by facilitated diffusion (a uniport mechanism).

Glucose

EXTRACELLULAR FLUID

Figure 41-14. The transcellular movement of glucose in an intestinal cell. Glucose follows Na+ across the luminal epithelial membrane. The Na+ gradient that drives this symport is established by Na+ -K+ exchange, which occurs at the basal membrane facing the extracellular fluid compartment. Glucose at high concentration within the cell moves "downhill" into the extracellular fluid by facilitated diffusion (a uniport mechanism).

tosis is responsible for the entry of DNA into the cell. Such experiments commonly use calcium phosphate, since Ca2+ stimulates endocytosis and precipitates DNA, which makes the DNA a better object for endocytosis. Cells also release macromolecules by exocyto-sis. Endocytosis and exocytosis both involve vesicle formation with or from the plasma membrane.

A. Endocytosis

All eukaryotic cells are continuously ingesting parts of their plasma membranes. Endocytotic vesicles are generated when segments of the plasma membrane invagi-nate, enclosing a minute volume of extracellular fluid and its contents. The vesicle then pinches off as the fusion of plasma membranes seals the neck of the vesicle at the original site of invagination (Figure 41-15). This vesicle fuses with other membrane structures and thus achieves the transport of its contents to other cellular compartments or even back to the cell exterior. Most endocytotic vesicles fuse with primary lysosomes to form secondary lysosomes, which contain hydrolytic enzymes and are therefore specialized organelles for in-tracellular disposal. The macromolecular contents are digested to yield amino acids, simple sugars, or nu-cleotides, and they diffuse out of the vesicles to be

Figure 41-15. Two types of endocytosis. An endocytotic vesicle (V) forms as a result of invagination of a portion of the plasma membrane. Fluid-phase endocytosis (A) is random and nondirected. Receptor-mediated endocytosis (B) is selective and occurs in coated pits (CP) lined with the protein clathrin (the fuzzy material). Targeting is provided by receptors (black symbols) specific for a variety of molecules. This results in the formation of a coated vesicle (CV).

Figure 41-15. Two types of endocytosis. An endocytotic vesicle (V) forms as a result of invagination of a portion of the plasma membrane. Fluid-phase endocytosis (A) is random and nondirected. Receptor-mediated endocytosis (B) is selective and occurs in coated pits (CP) lined with the protein clathrin (the fuzzy material). Targeting is provided by receptors (black symbols) specific for a variety of molecules. This results in the formation of a coated vesicle (CV).

reused in the cytoplasm. Endocytosis requires (1) energy, usually from the hydrolysis of ATP; (2) Ca2+ in extracellular fluid; and (3) contractile elements in the cell (probably the microfilament system) (Chapter 49).

There are two general types of endocytosis. Phagocytosis occurs only in specialized cells such as macrophages and granulocytes. Phagocytosis involves the ingestion of large particles such as viruses, bacteria, cells, or debris. Macrophages are extremely active in this regard and may ingest 25% of their volume per hour. In so doing, a macrophage may internalize 3% of its plasma membrane each minute or the entire membrane every 30 minutes.

Pinocytosis is a property of all cells and leads to the cellular uptake of fluid and fluid contents. There are two types. Fluid-phase pinocytosis is a nonselective process in which the uptake of a solute by formation of small vesicles is simply proportionate to its concentration in the surrounding extracellular fluid. The formation of these vesicles is an extremely active process. Fi-

broblasts, for example, internalize their plasma membrane at about one-third the rate of macrophages. This process occurs more rapidly than membranes are made. The surface area and volume of a cell do not change much, so membranes must be replaced by exocytosis or by being recycled as fast as they are removed by endocy-tosis.

The other type of pinocytosis, absorptive pinocyto-sis, is a receptor-mediated selective process primarily responsible for the uptake of macromolecules for which there are a finite number of binding sites on the plasma membrane. These high-affinity receptors permit the selective concentration of ligands from the medium, minimize the uptake of fluid or soluble unbound macro-molecules, and markedly increase the rate at which specific molecules enter the cell. The vesicles formed during absorptive pinocytosis are derived from invaginations (pits) that are coated on the cytoplasmic side with a filamentous material. In many systems, the protein clathrin is the filamentous material. It has a three-limbed structure (called a triskelion), with each limb being made up of one light and one heavy chain of clathrin. The polymerization of clathrin into a vesicle is directed by assembly particles, composed of four adapter proteins. These interact with certain amino acid sequences in the receptors that become cargo, ensuring selectivity of uptake. The lipid PIP2 also plays an important role in vesicle assembly. In addition, the protein dynamin, which both binds and hydrolyzes GTP, is necessary for the pinching off of clathrin-coated vesicles from the cell surface. Coated pits may constitute as much as 2% of the surface of some cells.

As an example, the low-density lipoprotein (LDL) molecule and its receptor (Chapter 25) are internalized by means of coated pits containing the LDL receptor. These endocytotic vesicles containing LDL and its receptor fuse to lysosomes in the cell. The receptor is released and recycled back to the cell surface membrane, but the apoprotein of LDL is degraded and the choles-teryl esters metabolized. Synthesis of the LDL receptor is regulated by secondary or tertiary consequences of pinocytosis, eg, by metabolic products—such as choles terol—released during the degradation of LDL. Disorders of the LDL receptor and its internalization are medically important and are discussed in Chapter 25.

Absorptive pinocytosis of extracellular glycopro-teins requires that the glycoproteins carry specific carbohydrate recognition signals. These recognition signals are bound by membrane receptor molecules, which play a role analogous to that of the LDL receptor. A galactosyl receptor on the surface of hepatocytes is instrumental in the absorptive pinocytosis of asialoglyco-proteins from the circulation (Chapter 47). Acid hydro-lases taken up by absorptive pinocytosis in fibroblasts are recognized by their mannose 6-phosphate moieties. Interestingly, the mannose 6-phosphate moiety also seems to play an important role in the intracellular targeting of the acid hydrolases to the lysosomes of the cells in which they are synthesized (Chapter 47).

There is a dark side to receptor-mediated endocyto-sis in that viruses which cause such diseases as hepatitis (affecting liver cells), poliomyelitis (affecting motor neurons), and AIDS (affecting T cells) initiate their damage by this mechanism. Iron toxicity also begins with excessive uptake due to endocytosis.

B. Exocytosis

Most cells release macromolecules to the exterior by ex-ocytosis. This process is also involved in membrane remodeling, when the components synthesized in the Golgi apparatus are carried in vesicles to the plasma membrane. The signal for exocytosis is often a hormone which, when it binds to a cell-surface receptor, induces a local and transient change in Ca2+ concentration. Ca2+ triggers exocytosis. Figure 41-16 provides a comparison of the mechanisms of exocytosis and endo-cytosis.

Molecules released by exocytosis fall into three categories: (1) They can attach to the cell surface and become peripheral proteins, eg, antigens. (2) They can become part of the extracellular matrix, eg, collagen and glycosaminoglycans. (3) They can enter extracellular fluid and signal other cells. Insulin, parathyroid hormone, and the catecholamines are all packaged in gran

Figure 41-16. A comparison of the mechanisms of endocytosis and exocytosis. Exocytosis involves the contact of two inside surface (cytoplasmic side) monolayers, whereas endocytosis results from the contact of two outer surface mono-layers.

Figure 41-16. A comparison of the mechanisms of endocytosis and exocytosis. Exocytosis involves the contact of two inside surface (cytoplasmic side) monolayers, whereas endocytosis results from the contact of two outer surface mono-layers.

ules and processed within cells, to be released upon appropriate stimulation.

Some Signals Are Transmitted Across Membranes

Specific biochemical signals such as neurotransmitters, hormones, and immunoglobulins bind to specific receptors (integral proteins) exposed to the outside of cellular membranes and transmit information through these membranes to the cytoplasm. This process, called transmembrane signaling, involves the generation of a number of signals, including cyclic nucleotides, calcium, phosphoinositides, and diacylglycerol. It is discussed in detail in Chapter 43.

Information Can Be Communicated by Intercellular Contact

There are many areas of intercellular contact in a meta-zoan organism. This necessitates contact between the plasma membranes of the individual cells. Cells have developed specialized regions on their membranes for intercellular communication in close proximity. Gap junctions mediate and regulate the passage of ions and small molecules (up to 1000-2000 MW) through a narrow hydrophilic core connecting the cytosol of adjacent cells. These structures are primarily composed of the protein connexin, which contains four membrane-spanning a helices. About a dozen genes encoding different connexins have been cloned. An assembly of 12 connexin molecules forms a structure (a connexon) with a central channel that forms bridges between adjacent cells. Ions and small molecules pass from the cy-tosol of one cell to that of another through the channels, which open and close in a regulated fashion.

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