Objectives
- 1.
Explain how the distribution of lipids and proteins in the cell membrane influences the membrane permeability to hydrophobic and hydrophilic solutes and ions.
- 2.
Differentiate the following mechanisms based on the source of energy driving the process and the necessity for an integral cell membrane protein: diffusion, mediated (facilitated) transport, and secondary active transport.
- 3.
Explain how the transport rates of certain molecules and ions are accelerated by specific integral membrane proteins (“carrier” and “channel” molecules).
- 4.
Explain how coupling of solute transport enables one solute to be transported against its electrochemical gradient by using energy stored in the electrochemical gradient of the coupled solute.
- 5.
Explain the two-step process involved in the net transport of certain solutes across epithelia.
Diffusion across biological membranes is limited by lipid solubility
We have just learned that all transport processes are driven by electrical or chemical gradients ( Chapter 9 ). The question is, “How are substances (solutes and the solvent, water) actually transported across biological membranes?” Bear in mind that some substances must be concentrated in cells, or rapidly taken up, because they are essential for cell function. Other substances must be excluded or extruded from cells. For example, too much Na + (or any other solute) in cells would be an excessive osmotic burden and cause cells to swell (see the discussion of the Donnan effect in Chapter 4 ).
Biological membranes are primarily lipid bilayers ( Chapter 1 ), which are poorly permeable to polar and hydrophilic solutes; these substances bear a net charge or have internal charge separation (i.e., they are uncharged molecules that behave like electric dipoles in which the positive and negative charge are separated). The ability of polar substances to pass across lipid membranes tends to be inversely proportional to molecular weight: the larger the molecule, the lower the permeability. Also, pure phospholipid bilayers are modestly permeable to water, a polar solvent ( Chapter 1 ), but the presence of cholesterol greatly reduces water permeability. Consequently, the phospholipid-cholesterol bilayer that constitutes the PM in many types of cells is not sufficiently permeable to water for physiological needs.
Consider the problem of moving a polar solute, such as glucose, across a biological membrane. Glucose crosses lipid bilayers extremely slowly ( Table 2.1 ). In other words, the bilayer is an effective barrier to the transport of these substances. Yet glucose is an essential fuel for cells.
Channel, carrier, and pump proteins mediate transport across biological membranes
To mediate and regulate the transfer of water and polar solutes, biological membranes contain integral proteins called channels, carriers, and “pumps” ( Fig. 10.1 ). More than one third of all the genes in the human genome code for membrane proteins, and approximately half of these genes (i.e., one sixth of the genome) code for transport proteins.
Transport through channels is relatively fast
Channels in biological membranes ( Chapter 8 ) are proteins with central pores that open to both the extracellular fluid and the cytoplasm simultaneously ( Fig. 10.1 A). The narrowest region within the pore, called the selectivity filter, determines which substance(s) (ions, water, etc.) may pass through the channel. For example, channels in the family called aquaporins a
a Peter Agre shared the 2003 Nobel Prize in Chemistry for his discovery of aquaporins and his determination of their structure and function.
are selectively permeable to water. Some other channels ( Chapters 7 and 8 ) are highly selective for Na + ions, for K + ions, or for Ca 2+ ions, whereas still others are less selective and may, for example, be permeable to both Na + and K + (e.g., the nicotinic acetylcholine channel; Chapter 13 ).Channels characteristically have high permeability. Typical turnover numbers for ion channels (i.e., the maximum number of ions that can pass through a channel in 1 second) are approximately 10 6 to 10 8 . For example, approximately 30 million K + ions can pass through a single K + channel in 1 second ( Table 10.1 ).
| Transporter | Turnover Number a (Per Sec) |
|---|---|
| K + channel | 30,000,000 |
| Valinomycin (carrier) | 30,000 |
| Glucose carrier (GLUT-1) | 3,000 |
| Na + /Ca 2+ exchanger | 2,000 |
| Ca 2+ pump (SERCA) | 200 |
| Na + pump | 150 |
a Rate of cycling of the transporter molecule, except for ion channels such as the K + channel, where the turnover number indicates the maximum number of ions transported in 1 second under physiological conditions. Thus each Na + pump molecule cycles 150 times per second and transports 450 Na + (and 300 K + ) per second.
Channel density controls the membrane permeability to a substance
Aquaporins facilitate water flow between the extracellular fluid and the cytoplasm to maintain osmotic balance (equilibrium) in cells that require high water permeability (e.g., skeletal muscle and some epithelia). The relative permeability to water depends on the number of channels (aquaporin tetramers) present, per unit area, in the PM (i.e., the channel density), and hormones may regulate this. The hormone vasopressin increases water permeability in, for example, the principal cells of the renal cortical collecting duct, by stimulating cAMP production. In turn, cAMP promotes the insertion of aquaporin-2 (AQP-2) molecules into the apical membrane of the epithelial cells. As a result, the osmotic driving force can speed water reabsorption from the renal tubule lumen. Defects in this hormonal mechanism, such as loss-of-function mutations in the vasopressin receptor or the AQP-2 molecule, lead to diabetes insipidus (excretion of a high volume of dilute urine). This occurs because AQP-2 molecules cannot be inserted into the apical membrane of the renal cortical collecting duct. Therefore even though the osmotic force may favor water reabsorption, the intrinsically low water permeability in the kidney cortical collecting ducts minimizes water reabsorption.
The rate of transport through open channels depends on the net driving force
In contrast to the aforementioned situation, impaired insulin secretion or reduced sensitivity to insulin leads to high glucose content and high osmotic pressure in the renal proximal tubule fluid. Water reabsorption is thereby reduced even though the epithelial cell apical membranes may contain a large number of AQP-2 molecules. This causes diabetes mellitus (excretion of a large volume of sweet urine with an osmolality approaching that of plasma). In this case the abnormally high osmotic pressure in the kidney tubule lumen reduces the osmotic driving force for water reabsorption from the lumen even though apical membrane water permeability may be high ( Chapter 11 ).
Transport of substances through some channels is controlled by “gating” the opening and closing of the channels
Another mechanism of regulating channel permeability is by channel gating ( Fig. 10.1 B), which may be regulated by voltage or by ligands such as Ca 2+ or ATP ( Chapters 7 and 8 ). Still, other channels are gated by pressure and membrane deformation, as exemplified by the mechanosensitive monovalent cation channels in some sensory nerve terminals.
Carriers are integral membrane proteins that open to only one side of the membrane at a time
In contrast to channels, the solute binding sites in carriers undergo spontaneous conformational changes and thus have alternating access to the two sides of the membrane. A solute binds to the carrier at one side of the membrane; then, as a result of a conformational change, a “gate” closes and the solute is transiently occluded (the transition state ; Fig. 10.1 C). Then, through a further conformational change in the protein, the “gate” on the opposite side opens so that the solute can dissociate from the carrier at this side of the membrane ( Fig. 10.1 C, c). The terms exofacial and endofacial denote the transporter conformations in which the solute binding sites face the extracellular fluid and cytoplasm, respectively.
Carriers facilitate transport through membranes
Simple carriers are integral membrane proteins that bind and transport a single solute species across a membrane. They move the bound solute down its electrochemical gradient (i.e., down a concentration gradient and, in the case of charged solutes, a voltage gradient). The rate of transport is substantially slower than that mediated by channels ( Table 10.1 ) because substrate binding and carrier conformation changes take time. Nevertheless, transport is much faster than would be expected for diffusion in the absence of such carriers. In other words, carriers simply speed up (facilitate) processes that would normally occur, albeit much more slowly, by diffusion; hence this process is sometimes called facilitated diffusion ( Box 10.1 ). Thus transport by the simple carrier cannot be used to generate a steady-state electrochemical gradient, because the generation of such a gradient requires the expenditure of energy. This distinguishes passive transport from active transport , which is discussed later and in Chapter 11 .
Carriers such as the GLUT-1 sugar transporter spontaneously change conformation, whether or not glucose is bound, so that the glucose binding site is open (accessible) either to the extracellular fluid or to the cytoplasm. Consider, for example, the situation in which the extracellular fluid glucose concentration is high and its concentration in the cytoplasm is low. Glucose molecules will then bind to the GLUT-1 binding sites with higher probability (because of the higher glucose concentration) when the sites are facing the extracellular fluid than when the sites are facing the cytoplasm. Glucose that is bound at the outside will tend to dissociate when the carrier conformation changes and the sites face the cytoplasm with its low glucose concentration. The result will be a net transport of glucose from the extracellular fluid to the cytoplasm. When the concentrations of glucose are equal on the two sides of the membrane, no net transport will occur, because the probability of glucose binding at the internal and at the external faces of the membrane will then be the same. Large movements of glucose (fluxes) in both directions may take place under the latter circumstances, but they will be equal in magnitude. Thus in the steady state, the simple carrier cannot be used to transport a solute against a concentration gradient.
Sugars are transported by carriers
Several solutes are transported by solute-selective simple carriers. A good example is the glucose carrier that mediates glucose transport across the human red blood cell (RBC) PM. This protein, GLUT-1, belongs to a family of sugar transporters ( Box 10.2 ). GLUT-1 has 12 membrane-spanning helices, 5 of which are amphiphilic : each has a hydrophobic and a hydrophilic surface. The hydrophobic regions interact with the surrounding lipids in the bilayer. In contrast, the hydrophilic surfaces of the five helices face one another and form a central, water-filled transmembrane channel or pore ( Fig. 10.2 ). This is the general structure of many transporter molecules.
GLUT-1 is one of five homologous human sugar transporters (GLUT-1 to GLUT-5). Each is the product of a different gene, and each has a different tissue distribution and is regulated differently. Moreover, these transporters are members of one family of a superfamily of solute transporters, the major facilitator superfamily , with more than 1000 members and some well-conserved sequence motifs. The superfamily already includes as many as 34 separate families. Each family has a family-specific signature sequence and specificity for a single class of substrates. Some other families within this superfamily include the monocarboxylic acid transporters that transport pyruvate and lactate, for example, and the anion/cation cotransporters that transport sodium and phosphate simultaneously.
The need for carrier-mediated glucose transport is exemplified by the consequences of mutations in the human GLUT-1 gene. GLUT-1 also is expressed in the brain, where it mediates glucose transport across the blood-brain barrier and glucose uptake by glial cells. Individuals with a mutant GLUT-1 gene have a cerebrospinal fluid glucose concentration that is approximately 50% of normal despite a normal blood glucose level. The low brain glucose causes a devastating neurological syndrome ( Box 10.3 ).
Patients with a rare defect in GLUT-1 have infantile seizures (convulsions), starting at age 3–4 months. They also have microcephaly (small head size) and developmental delays. Laboratory examination reveals hypoglycorrhachia (i.e., a very low glucose concentration, approximately half normal) in the cerebrospinal fluid (CSF) and a low CSF/blood glucose ratio (< 0.5). The rate of glucose uptake by the red blood cells (RBCs) from these patients is also less than 50% of normal; this can be used as a diagnostic test. These manifestations can be explained by mutations in the GLUT-1 gene that cause malfunction of the expressed protein (a reduced turnover rate or maximum velocity of transport). GLUT-1 normally is expressed in many cells, including RBCs and epithelial cells of the choroid plexus and ependyma, as well as in blood vessel endothelial cells; it facilitates the transport of glucose from the blood to the CSF. GLUT-1 is also expressed in glia, where the transporter is concentrated in foot processes that surround neuronal synapses. Thus in patients with defective GLUT-1, the glucose concentration within glial cells may be particularly low. This may be rate limiting for cellular energy metabolism and brain function.
Transport by carriers exhibits kinetic properties similar to those of enzyme catalysis
Carrier-mediated transport is saturable and vectorial
Because limited numbers of carrier molecules are present in the membrane, raising the solute (e.g., sugar) concentration sufficiently will saturate all the carrier molecules so that no more sugar can be bound. This limits the maximal rate of transport ( Fig. 10.3 ). The transport follows Michaelis-Menten (saturation) kinetics , which also governs the rate of enzymatic reactions. Indeed, carriers are comparable to enzymes because both of them speed up spontaneous processes. The product of a carrier’s reaction, however, is the vectorial (directional) movement of the substrate from one side of the membrane to the other, rather than the chemical alteration of the substrate.
Carrier-mediated transport exhibits substrate specificity
The binding site on a transporter protein recognizes certain solutes, but not others. GLUT-1, for example, transports d -glucose and d -galactose, but not l -glucose (the transporter is stereospecific ) or maltose or ribose.
Carrier-mediated transport can be inhibited either competitively or noncompetitively
GLUT-1 transports both d -galactose and d -glucose. By competing for sugar binding sites on the carriers, d -galactose can be expected to inhibit the transport of d -glucose and vice versa (competitive inhibition). The presence of galactose reduces the apparent affinity of GLUT-1 for glucose but does not affect the maximum velocity of glucose transport at saturating glucose concentrations ( Fig. 10.3 ). In contrast, certain molecules such as phloretin and dinitrofluorobenzene are noncompetitive inhibitors of GLUT-1. These inhibitors do not affect the affinity of the carrier for glucose, but reduce the maximum transport velocity ( Fig. 10.3 ).
Simple carriers exhibit reversibility and countertransport
Carriers facilitate the movement of solutes in both directions across the membrane (i.e., they are “reversible”). The direction of net movement is, in general, determined by the electrochemical gradient of the transported substance. Consider, however, a situation in which RBCs are equilibrated with glucose (i.e., equilibrium is achieved, in which external and internal concentrations are equal and there is no net driving force). If a high concentration of galactose is added to the extracellular solution, the carriers will mediate a net efflux of glucose (i.e., they will move glucose outward). Initially, carriers in the endofacial conformation bind only glucose (the only solute available). When these carriers change to the exofacial conformation, some of the bound glucose will be displaced by galactose, which will then be transported inward—this is glucose-galactose exchange. The glucose concentration inside the RBCs will temporarily fall below that outside the cells until the galactose concentration inside the cells rises to equal that outside (i.e., when the galactose concentration gradient falls to zero). During this brief period (i.e., under non–steady-state conditions ), a countertransport of glucose in exchange for galactose takes place. This demonstrates that the carriers alternately open to the two membrane surfaces.
Carrier-mediated transport can be regulated
The glucose carrier isoform found in adipocytes (fat cells) and in skeletal and cardiac muscle cells, GLUT-4, is regulated by insulin. In the absence of insulin stimulation, most GLUT-4 molecules reside in intracellular vesicular membranes. Insulin stimulation promotes the fusion of these vesicles with the PM, thereby increasing the density of GLUT-4 molecules in the PM and accelerating glucose transport. As a result, insulin shortens the time required for the intracellular glucose concentration to reach that in the extracellular fluid. This is advantageous after a meal, when the blood sugar concentration rises and insulin secretion is increased. The enhanced rate of glucose entry enables faster glycogen synthesis in muscle and adipocytes. Thus insulin facilitates glucose storage (as glycogen) but does not enable GLUT-4 to concentrate free glucose in the cells.
β-Adrenergic agonists such as epinephrine and isoproterenol inhibit GLUT-4-mediated glucose uptake in skeletal muscle, apparently by stimulating glycogenolysis. Glycogenolysis causes the glucose concentration within the cells to rise, inhibiting further net entry of glucose. In other words, this inhibition is the result of a change in the glucose concentration gradient rather than a change in the GLUT-4 turnover (or cycling rate).
Coupling transport of one solute to “downhill” transport of another solute enables carriers to move the cotransported or countertransported solute “uphill” against an electrochemical gradient
Coupled transport that does not directly use ATP hydrolysis is sometimes called secondary active transport . The reason is that the movement of one substance down its electrochemical energy gradient (i.e., “downhill” movement; Chapter 9 ) can be used to concentrate another substance (i.e., move it “uphill” against a concentration or voltage gradient). For example, the carrier-mediated transport of various solutes is coupled to Na + transport, using energy from the Na + electrochemical gradient that is maintained by the sodium pump ( Chapter 11 ). The Na + may be either cotransported or countertransported with the coupled solute. Cotransport is also referred to as symport , because both solutes move simultaneously in the same direction; countertransport is also called antiport or exchange , because the two coupled solutes move in opposite directions. The availability of a variety of coupled solute transporters reduces the need for many ATP-driven solute “pumps” which cycle much more slowly than co- and counter-transporters ( Table 10.1 ).
Na + /H + exchange is an example of Na + -coupled countertransport
Intracellular pH regulation in all cells depends on several transport mechanisms. Among these are the Cl − /base (e.g., Cl − /HCO 3 − and Cl − /OH − ) exchangers, which can mediate net proton (H + ) influx or efflux, and the Na + -HCO 3 − cotransporter, which mediates acid efflux. Another H + transporter, present in virtually all cells, is the Na + /H + exchanger ( sodium/proton exchanger or NHE) that mediates the electroneutral exchange of 1 Na + for 1 H + (i.e., there is no net charge transfer). Five molecular isoforms of NHE are expressed in mammalian cells in a tissue-specific manner. NHE1, for example, is expressed in cardiac muscle and in kidney and intestinal epithelia. NHE1 has 12 transmembrane segments and a large C-terminal cytoplasmic tail containing several sites involved in regulating exchanger activity (e.g., by phosphorylation and by Ca 2+ -calmodulin).
NHE rapidly extrudes protons from cells when intracellular pH (pH i ) falls to less than the normal value of approximately 7.2. Enough energy is available in the Na + electrochemical gradient to transport sufficient H + out of most cells to raise pH i to more than 8.0 ( Box 10.4 ), but this pH is nonphysiological. To prevent pH i from rising too much, intracellular H + regulates the NHE at an H + binding site that is distinct from the H + transport site. This H + regulatory site has a steep pH dependence and activates transport when protons are produced and pH i falls. Then, as protons are extruded and pH i approaches 7.2, NHE activity is rapidly downregulated. This behavior is different from that of many other Na + -coupled transport systems (discussed later), which operate close to electrochemical equilibrium.
The transport process mediated by the Na + /H + exchanger (NHE) with a 1 Na + :1 H + coupling ratio is described by the following equation:
Naout++Hin+⇌Nain++Hout+
To determine how the Na + and H + concentrations and the V m are related at equilibrium, we equate the total electrochemical potentials of Na + and H + on the two sides of the equation ( Chapter 9 ):
μNa+,out+μH+,in=μNa+,in+μH+,out
Expansion of this equation ( Chapter 9 ) yields
μNa+0+RTIn[Na+]o+(+1)(0)F+μH+0+RTIn[H+]i+(+1)VmF=μNa+0+RTIn[Na+]i+(+1)VmF+μH+0+RTIn[H+]o+(+1)(0)F
Note that the electrical terms drop out of this equation because the coupled exchange of 1 Na + for 1 H + is electroneutral, as is also the case for Cl − /HCO 3 − exchange ( Chapter 9 ).
Algebraic rearrangement ( Box 9.4 ) then yields the expression:
RTIn[H+]i-RTIn[H+]o=RTIn[Na+]i-RTIn[Na+]o
and division by RT gives:
[H+]i[H+]o=[Na+]i[Na+]o
Now, solving for [H + ] i , we have:
[H+]i=[Na+]i[Na+]o×[H+]o
Then, if [Na + ] i = 15 mM and [Na + ] o = 150 mM, at equilibrium the [H + ] i should be 10-fold lower than [H + ] o (= 10 −7.4 M). Alternatively, if [H + ] is expressed as pH (the negative logarithm of [H + ]), intracellular pH (pH i ) should be 1.0 pH unit larger than extracellular pH (pH o = 7.4). In other words, there is sufficient potential energy in the Na + electrochemical gradient to lower [H + ] i by a factor of 10, to approximately 10 −8.4 M (i.e., raise pH i to 8.4), through NHE. However, the fact that pH i is normally approximately 7.2 and not 8.4 indicates that NHE-mediated proton extrusion is inactivated (as a result of the dissociation of H + from the regulatory site) before pH i reaches the equilibrium value predicted by Equation B10.6 .
Na + is cotransported with a variety of solutes such as glucose and amino acids
Na + -glucose cotransporters, SGLTs, some isoforms of which are found in the brush border (apical or luminal) membranes of intestinal and renal epithelial cells, are good examples of carriers that are obliged to cotransport two solutes simultaneously. These transporters ( Fig. 10.4 ) are members of a family of more than 35 Na + -coupled cotransporters. Other members include several Na + -amino acid cotransporters (e.g., the Na + -alanine cotransporter), the Na + -K + -2Cl − cotransporter, various neurotransmitter transporters (e.g., the Na + -norepinephrine, Na + -dopamine, and Na + -serotonin cotransporters), and the Na + – I − cotransporter .
Several widely prescribed antidepressant drugs such as fluoxetine (Prozac) selectively inhibit presynaptic serotonin (5-hydroxytryptamine [5-HT]) reuptake by Na + -serotonin cotransport. This enhances and prolongs activation of postsynaptic serotonergic neurons by 5-HT.
In thyroid glands, the Na + -I − cotransporter (sometimes called the Na + -I − symporter) is used to concentrate I − , a trace element (i.e., one that is present at very low abundance). This process promotes the iodination of tyrosine; iodinated tyrosine is then used to form the thyroid hormones, which are iodothyronines ( Box 10.5 ).
The sodium-iodide (Na + -I − ) cotransporter (NIC), which is expressed in thyroid follicular cells, the stomach, lactating mammary gland, and several other cell types, cotransports 1 I − with 2 Na + ions. Thus I − , an ion normally present in trace amounts, may be concentrated as much as 1000-fold within these cells ( Boxes 10.6 and 10.7 ). The I − trapped in the thyroid is used for the synthesis of the thyroid hormones thyroxine and triiodothyronine . The ability of the NIC to concentrate I − in the thyroid is used to concentrate radioactive 131 I in cancerous thyroid cells, both to detect the spread of the cancer and, after surgery, to destroy the remaining cancer cells by radiation. Moreover, inherited defects in the NIC result in defective I − trapping and thus in congenital hypothyroidism (low thyroid hormone levels).
The transport of 1 molecule of glucose (G) is tightly coupled to the transport of 1 Na + ion through the SGLT-2 cotransporter across the apical membrane of a renal proximal tubule cell. Accordingly, for this transporter, the coupled transport reaction is:
Gin+Nain+⇌Gout+Naout+
This transport reaction is somewhat more complex than NHE ( Box 10.4 ) because Na + -G cotransport involves the net movement of charge when 1 Na + and 1 G move across the membrane. The driving forces for this coupled transport are the electrochemical potential energies for Na + on the two sides of the membrane and the chemical potential energies for G (because z = 0; Chapter 9 ). Equating the potential energies on the two sides of transport equation B10.7 , we have:
μG,in+μNa+,in=μG,out+μNa+,out
Expanding this equation ( Chapter 9 ), with z = +1 for Na + , gives:
μG0+RTIn[G]i+μNa+0+RTIn[Na+]i+(+1)VmF=μG0+RTIn[G]o+μNa+0+RTIn[Na+]o+(+1)(0)F
Rearrangement then yields:
RTIn[G]i-RTIn[G]o=RTIn[Na+]o-RTIn[Na+]i-VmF
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