The following features are so widely characteristic of carrier-mediated transport processes that they are generally considered sufficient and often necessary criteria for the implication of carriers in the transport of a given solute:
1. Virtually all carriers appear to display a high degree of structural specificity with regard to the substances they will bind and transport. For example, the carriers responsible for the transport of glucose into animal cells are highly stereospecific; they will rapidly bind and transport the dextrorotary form (D-glucose) but have little affinity for the levorotary form (L-glucose). Conversely, the carriers responsible for the transport of amino acids into animal cells possess a high degree of selectivity in favor of the L-stereoisomer and little affinity for the D-stereoisomer.
2. All carrier-mediated transport processes exhibit saturation kinetics; that is, the rate of transport gradually approaches a maximum as the concentration of the solute transported by the carrier increases. Once this maximum rate is achieved, a further increase in the solute concentration has no effect on the transport rate. Plots of the rate of transport against concentration often closely resemble the hyperbolic plots characteristic of MichaelisMenten enzyme kinetics, and, under these conditions, the kinetics of the transport process can be described by defining the maximum transport rate (Jmax) and the substrate concentration at which the transport rate is half-maximum (Kt). Thus, Jt = [Jmax(C()/(Kt + Ci)], where Ct is the concentration of the solute.
A graph illustrating the saturation kinetics characteristic of carrier-mediated transport is shown in Fig. 15b. In contrast, as illustrated by Fig. 15a, transport due to simple diffusion is usually (but not always) characterized by a linear relation between transport rate and solute concentration as predicted by Eq. 4. It should be emphasized that ionic diffusion through channels may exhibit saturation but usually only when concentrations are well beyond the physiologic range.
The saturation phenomena observed in carrier-mediated transport processes reflect the presence of a fixed and limited number of carrier molecules or binding sites in the membrane. When the solute concentration is sufficiently high so that all of the carrier sites are occupied (or complexed), a further increase in concentration cannot elicit a further increase in transport rate.
One consequence of the presence of a limited number of carrier molecules for a given class of transported solutes is the phenomenon of competitive inhibition. This is observed when two or more solutes that are capable of being transported by the same carrier are present simultaneously, competing with one another for the limited number of available binding sites. This phenomenon is closely analogous to competitive inhibition in enzyme-substrate interactions and often may also be described by classic Michaelis-Menten kinetics.
In addition to providing evidence for carrier-mediated transport, the phenomenon of competitive inhibition has proved to be extremely useful for the purpose of defining the transport specificity of a given carrier mechanism. Thus, if two solutes A and B are each transported by carriers and exhibit mutual, classic competitive inhibition, one may conclude that the same carrier mechanism is involved; if they in no way compete with each other, at least two distinct carrier mechanisms must be involved. For example, all of the D-hexoses that are absorbed by the small intestine mutually compete with one another for the same limited transport system.
When glucose and galactose are separately present in the intestinal lumen at high concentrations, they are each absorbed at approximately the same maximum rate. On the other hand, if the same concentrations of glucose and galactose are instilled into the intestinal lumen in the form of a mixture, each will be absorbed at a rate significantly lower than that observed when they were present separately. The total rate of sugar transport will be equal to the maximum rates observed when each sugar was present separately, indicating that the two sugars are competing for the same carrier system and are sharing in the saturation of the total number of available sites. On the other hand, the transport of glucose is not inhibited by the presence of hexoses that are not subject to carrier-mediated transport.
The characteristics of carrier-mediated transport processes that we have just described—the high degree of structural specificity and saturation kinetics and competitive inhibition—strongly resemble the characteristics of enzyme-substrate interactions. After the introduction of the carrier hypothesis, it was suspected that carriers were enzyme-like molecules that comprise part of the protein portion of the lipoprotein membrane. However, for many years, these carriers defied isolation and characterization, and until recently there was a relatively large group of investigators who doubted, and even denied, their existence, but the results of numerous studies during the past few decades have dispelled these doubts. The development of techniques for isolating cell membranes and gently detaching their protein components has led to the isolation of integral proteins that are capable of specifically binding transported solutes. In many instances, these purified proteins have been reinserted into artificial lipid membranes, and these artificial (reconstituted) systems are capable of mediating the transport of specific solutes.
In short, considerable progress has been made toward defining the biochemical and/or molecular basis of carrier-mediated transport. The precise mechanism(s) by which the transported solutes are translocated across the membrane after binding, however, remains a mystery. But, in light of our current understanding of the assembly of proteins in biologic membranes, it is certain that the notions that carriers are ferry boats or that integral proteins flip-flop across the lipid bilayer are incorrect. It is more likely that carriers are integral proteins that in many respects resemble channels and that binding and translocation of solutes from one side of a gate to the other takes place within these channels.
As discussed above, the two functions that membrane carriers must fulfill are (1) to provide a mechanism by which otherwise impermeant solutes can enter or leave cells across membranes, and (2) to provide a mechanism by which substances can be actively transported into or out of cells. The two classes of carrier-mediated transport processes that fulfill these functions are referred to as facilitated diffusion and active transport, respectively.
Facilitated diffusion is the term reserved for carrier-mediated processes that are only capable of transferring a substance from a region of higher concentration to one of lower concentration. These processes are sometimes also referred to as equilibrating carrier systems, inasmuch as net transport ceases when the concentrations of the transported solute are the same on the two sides of the membrane—that is, when the system is equilibrated with respect to the solute in question. Thus, facilitated diffusion resembles noncarrier-mediated diffusional processes in that the direction of net flow is always downhill. It differs from them in that it exhibits all of the characteristics of carrier-mediated processes and often results in the transmembrane transfer of a solute that could not otherwise permeate the membrane; indeed, the latter is its sole function.
The classic example of facilitated diffusion is glucose transport across the membranes surrounding many animal cells, such as erythrocytes, striated muscle, and adipocytes. The glucose concentrations in these cells are much lower than the glucose concentrations in the extracellular fluid because glucose is rapidly metabolized by these cells after gaining entry. The only well-documented exceptions to this statement are renal proximal tubular cells, small intestinal epithelial cells, and the cells of the choroid plexus. These cells are responsible for transepithelial glucose absorption, and their intracellular glucose concentrations may exceed those in the extracellular fluid; as is discussed below, mechanisms other than facilitated diffusion are responsible for the uptake of hexoses by these cells. Thus, for most cells, the problem is not that of transporting glucose against a concentration difference but of transporting glucose rapidly across an essentially imper-meant barrier. This is accomplished by a carrier mechanism, illustrated in Fig. 16. Glucose (represented by the small circle labeled S) combines with the carrier from one side of the membrane to form a glucose-carrier complex. This is followed by a change in conformation of the complex that permits glucose to dissociate from the carrier and enter the solution on the other side of the membrane. The free carrier site is then available for another passenger. Because there are a limited number of carriers, the process is saturable and subject to competitive inhibition.
One of the important features of the carrier model for facilitated diffusion, as illustrated in Fig. 16, is that the carrier itself is unaltered during the translocation process, and only thermal energy is required for the conformational change that exposes the binding site to one or the other side of the membrane. Thus, the transport process is symmetrical, and it is just as easy for the solute (S) to move from the extracellular fluid into the cell as in the opposite direction. Consequently, when the concentrations of solute on the two sides of the membrane are equal, the system is entirely symmetrical, and the carrier-mediated flows in both directions will be equal. This is the reason why this transport process is not capable of bringing about net transport from a region of lower concentration to one of higher concentration and why net transport ceases when the solute distribution is equilibrated.
To date, five carrier proteins capable of mediating the facilitated diffusion of glucose have been identified; in the jargon of molecular biology, they are referred to as GLUT1-5. All five consist of a polypeptide chain composed of approximately 500 amino acids and possess a high degree of homology, including 12 putative transmembrane-spanning segments. Their specific properties and differences are pointed out in other chapters.
Active transport is the term reserved for carrier-mediated transport processes that are capable of bringing about the net transfer of an uncharged solute from a region of lower concentration to one of higher concentration or the transfer of a charged solute against combined chemical and electrical driving forces. Thus, active transport processes are capable of counteracting or reversing the direction of diffusion, a spontaneous process, and therefore are capable of performing work. The concept that active transport processes perform work may be difficult to grasp for those who have not had some acquaintance with thermodynamics. Because this is an extremely important concept, we digress for a moment and attempt to provide it an intuitive basis.
The direction of all natural change in the universe is for systems to move from a state of higher energy to one of lower energy. Thus, an unsupported weight will fall from a position of higher gravitational (or potential) energy to one of lower gravitational energy; electrons will flow through a conductor from a region of electronegativity (the cathode of a battery) to one that is electropositive (the anode); uncharged solutes will diffuse from a region of higher concentration to one of lower concentration. All of these processes are spontaneous inasmuch as they are accompanied by a decrease in the free energy of the system and do not require any external assistance or intervention; they will occur in a completely isolated system. It is a universal experience (and one of the basic tenets of thermodynamics) that once a spontaneous change has taken place, the initial conditions cannot be restored without an investment of energy; that is, the only way one can reverse a spontaneous process is by performing work. In the examples cited above, mechanical work is required to restore the weight to its original height, and electrical work is needed to recharge the battery. A thoroughly mixed solution can be unmixed by ultracentrifugation, ultrafiltration through an appropriate molecular sieve, or distillation, but, whatever means are chosen, it is clear that unmixing will never occur spontaneously and that the result of diffusion can only be reversed through the investment of energy. The means by which a biologic cell reverses diffusional flows is referred to as active transport; here, too, work is performed, and energy derived from metabolism must be invested.
It follows that the ability of a cell to carry out active transport processes is dependent on an intact supply of metabolic energy, and all active transport processes can be inhibited by deprivation of essential substrates or through the use of metabolic poisons. Indeed, the sine qua non of active transport is the presence of a direct or indirect linkup or coupling between the carrier mechanism and cell metabolism; when an active transport process is initiated or accelerated, there is a concomitant increase in the metabolic rate (as measured by glucose utilization, oxygen consumption, etc.), and inhibition of active transport results in a decrease in the metabolic rate. The two classes of active transport processes are primary active transport and secondary active transport, which differ in the ways they derive (or are coupled to) a supply of energy.
Primary active transport implies that the carrier mechanism responsible for the movement of a solute against a concentration difference or a combined concentration and electrical potential difference (for the case of ions) is directly coupled to metabolic energy. The best-studied primary active transport processes in animal cells include:
1. The carrier mechanism found in virtually all cells from higher animals that is responsible for maintaining their low cell Na+ and high cell K+ concentrations
2. Carrier mechanisms found in sarcoplasmic reticulum and many plasma membranes responsible for active transport of Ca2+
3. Carrier mechanisms capable of actively extruding protons from the cells present in the gastric mucosa and renal tubule and actively pumping protons into intracellular organelles (e.g., lysozomes)
These carrier proteins have been purified, and all possess adenosine triphosphatase (ATPase) activity; that is, the same protein that is involved in the binding and translocation of Na+ and K+ is also capable of hydrolyzing adenosine triphosphate (ATP) and using the chemical energy released to perform the work of transport. The same holds for the Ca2+ pumps and the proton pumps. The precise mechanisms whereby the chemical energy of the terminal phosphate bond of ATP is converted into transport work is not clear.
Secondary active transport refers to processes that mediate the uphill movements of solutes but are not directly coupled to metabolic energy; instead, the energy required is derived from coupling to the downhill movement of another solute. Let us illustrate such systems by considering Fig. 17. Figure 17A portrays a rotating carrier molecule (C) within a membrane that has two binding sites, one for Na+ and the other for a solute (S), which, at this instant, are shown facing compartment o. Let us assume that the carrier can rotate only when both binding sites are empty or filled and is immobile when only one site is filled. Thus, it can transport Na+ and S from compartment o to compartment i in a one-to-one fashion. Now let us assume that there is no (or very little) Na+ in compartment i and that every Na+ that enters from compartment o is removed.
Clearly, under these conditions, S can only move (to any appreciable extent) from compartment o to compartment i; because there is no Na+ in compartment i, S that enters this compartment cannot move back out and is trapped. In time, the concentration of S in
3. Membrane Transport
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