Uptake Of Glucose And Lactose In Epithelial Cells By A Gradient

FIGURE 11-42 Lactose uptake in E. coli. (a) The primary transport of H+ out of the cell, driven by the oxidation of a variety of fuels, establishes both a proton gradient and an electrical potential (inside negative) across the membrane. Secondary active transport of lactose into the cell involves symport of H+ and lactose by the lactose transporter. The uptake of lactose against its concentration gradient is entirely dependent on this inflow of H+, driven by the electrochemical gradient.

CN inhibition of fuel oxidation

CN inhibition of fuel oxidation

[Lactose]m

Lactose Via Passive Transporter

Time

(b) When the energy-yielding oxidation reactions of metabolism are blocked by cyanide (CN-), the lactose transporter allows equilibration of lactose inside and outside the cell via passive transport. Mutations that affect Glu325 or Arg302 have the same effect as cyanide. The dashed line represents the concentration of lactose in the surrounding medium.

[Lactose]m

Time

(b) When the energy-yielding oxidation reactions of metabolism are blocked by cyanide (CN-), the lactose transporter allows equilibration of lactose inside and outside the cell via passive transport. Mutations that affect Glu325 or Arg302 have the same effect as cyanide. The dashed line represents the concentration of lactose in the surrounding medium.

Lactose Permease

FIGURE 11-43 Structure of the lactose transporter (lactose permease) of E. coli. (a) Ribbon representation viewed parallel to the plane of the membrane shows the 12 transmembrane helices arranged in two nearly symmetrical domains shown in different shades of blue. In the form of the protein for which the crystal structure was determined, the substrate sugar (red) is bound near the middle of the membrane where it is exposed to the cytoplasm (derived from PDB ID 1PV7). (b) The structural changes postulated to take place during one transport

FIGURE 11-43 Structure of the lactose transporter (lactose permease) of E. coli. (a) Ribbon representation viewed parallel to the plane of the membrane shows the 12 transmembrane helices arranged in two nearly symmetrical domains shown in different shades of blue. In the form of the protein for which the crystal structure was determined, the substrate sugar (red) is bound near the middle of the membrane where it is exposed to the cytoplasm (derived from PDB ID 1PV7). (b) The structural changes postulated to take place during one transport cycle. The two halves of the transporter undergo a large, reversible conformational change in which the two domains tilt relative to each other, exposing the substrate-binding site first to the periplasm (structure on the right), where lactose is picked up, then to the cytoplasm (left), where the lactose is released. The interconversion of the two forms is driven by changes in the pairing of charged (protonatable) side chains such as those of Glu325 and Arg302 (green), which is affected by the transmembrane proton gradient.

nism for transmembrane passage of the substrate (Fig. 11—43b) involves a rocking motion between the two domains, driven by substrate binding and proton movement, alternately exposing the substrate-binding domain to the cytoplasm and to the periplasm. This so-called rocking banana model is similar to that shown in Figure 11-32 for GLUT1.

How is proton movement into the cell coupled with lactose uptake? Extensive genetic studies of the lactose transporter have established that of the 417 residues in the protein, only 6 are absolutely essential for cotrans-port of H+ and lactose—some for lactose binding, others for proton transport. Mutation in either of two residues (Glu325 and Arg302; Fig. 11-43) results in a protein still able to catalyze facilitated diffusion of lactose but incapable of coupling H+ flow to uphill lactose transport. A similar effect is seen in wild-type (unmutated) cells when their ability to generate a proton gradient is blocked with CN~: the transporter carries out facilitated diffusion normally, but it cannot pump lactose against a concentration gradient (Fig. 11-42b). The balance between the two conformations of the lactose transporter is affected by changes in charge pairing between side chains.

In intestinal epithelial cells, glucose and certain amino acids are accumulated by symport with Na+, down the Na+ gradient established by the Na+K+ ATPase of the plasma membrane (Fig. 11-44). The apical surface of the intestinal epithelial cell is covered with microvilli, long thin projections of the plasma membrane

Apical surface

Basal surface

Intestinal lumen

Microvilli

Apical surface

Basal surface

Intestinal lumen

Microvilli

2 Na

Na4 - glucose symporter (driven by high extracellular [Na4])

Glut2 Uniport

Na4K4 ATPase

Glucose

Glucose uniporter GLUT2 (facilitates downhill efflux)

2 Na

Glucose o

Na4 - glucose symporter (driven by high extracellular [Na4])

Na4K4 ATPase

Glucose

Glucose uniporter GLUT2 (facilitates downhill efflux)

FIGURE 11-44 Glucose transport in intestinal epithelial cells. Glucose is cotransported with Na4 across the apical plasma membrane into the epithelial cell. It moves through the cell to the basal surface, where it passes into the blood via GLUT2, a passive glucose transporter. The Na4K4 ATPase continues to pump Na4 outward to maintain the Na4 gradient that drives glucose uptake.

406 Chapter 11 Biological Membranes and Transport that greatly increase the surface area exposed to the intestinal contents. Na+-glucose symporters in the apical plasma membrane take up glucose from the intestine in a process driven by the downhill flow of Na+:

2Na0ut + glucoseout-> 2Na^n + glucosein

The energy required for this process comes from two sources: the greater concentration of Na+ outside than inside (the chemical potential) and the transmembrane potential (the electrical potential), which is inside-negative and therefore draws Na+ inward. The electrochemical potential of Na+ is

[Na+]0ut where n = 2, the number of Na+ ions cotransported with each glucose molecule. Given the typical membrane potential of —50 mV, an intracellular [Na+] of 12 mM, and an extracellular [Na+] of 145 mM, the energy, AG, made available as two Na+ ions reenter the cell is 22.5 kJ, enough to pump glucose against a large concentration gradient:

[glucose]in

[glucose]out and thus

[Glucose]in [Glucose]oUt

That is, the cotransporter can pump glucose inward until its concentration within the epithelial cell is about 9,000 times that in the intestine. As glucose is pumped from the intestine into the epithelial cell at the apical surface, it is simultaneously moved from the cell into the blood by passive transport through a glucose transporter (GLUT2) in the basal surface (Fig. 11-44). The crucial role of Na+ in symport and antiport systems such as these requires the continued outward pumping of Na+ to maintain the transmembrane Na+ gradient.

Because of the essential role of ion gradients in active transport and energy conservation, compounds that collapse ion gradients across cellular membranes are effective poisons, and those that are specific for infectious microorganisms can serve as antibiotics. One such substance is valinomycin, a small cyclic peptide that neutralizes the K+ charge by surrounding it with six carbonyl oxygens (Fig. 11-45). The hydrophobic peptide then acts as a shuttle, carrying K+ across membranes down its concentration gradient and deflating that gradient. Compounds that shuttle ions across membranes in this way are called ionophores ("ion bearers"). Both valinomycin and monensin (a Na+-carrying ionophore) are antibiotics; they kill microbial cells by disrupting secondary transport processes and energy-conserving reactions.

Aquaporins Form Hydrophilic Transmembrane Channels for the Passage of Water

A family of integral proteins discovered by Peter Agre, the aquaporins (AQPs), provide channels for rapid movement of water molecules across all plasma membranes (Table 11-6 lists a few examples). Ten aquaporins are known in humans, each with its specialized role. Erythrocytes, which swell or shrink rapidly in response to abrupt changes in extracellular os-molarity as blood travels through the renal medulla, have a high density of aqua-porin in their plasma membranes (2 X 105 copies of AQP-1 per cell). In the nephron (the functional unit of the kidney), the plasma membranes of proximal renal tubule cells have five different aquaporin types.

Peter Agre
Aquaporin Kidney

FIGURE 11-45 Valinomycin, a peptide ionophore that binds K+. In this image, the surface contours are shown as a transparent mesh, through which a stick structure of the peptide and a K+ atom (green) are visible. The oxygen atoms (red) that bind K+ are part of a central hydrophilic cavity. Hydrophobic amino acid side chains (yellow) coat the outside of the molecule. Because the exterior of the K+-valinomycin complex is hydrophobic, the complex readily diffuses through membranes, carrying K+ down its concentration gradient. The resulting dissipation of the transmembrane ion gradient kills microbial cells, making valinomycin a potent antibiotic.

These cells reabsorb water during urine formation, a process for which water movement across membranes is essential (Box 11-3). The plant Arabidopsis thaliana has 38 genes that encode various types of aquaporins, reflecting the critical roles of water movement in plant physiology. Changes in turgor pressure, for example, require rapid movement of water across a membrane.

Water molecules flow through an AQP-1 channel at the rate of about 109 s_1. For comparison, the highest known turnover number for an enzyme is that for catalase, 4 X 107 s_1, and many enzymes have turnover numbers between 1 s_1 and 104 s_1 (see Table 6-7). The low activation energy for passage of water through aquaporin channels (AG* < 15 kJ/mol) suggests that water moves through the channels in a continuous stream, in the direction dictated by the osmotic gradient. (For a discussion of osmosis, see p. 57.) It is essential that aquaporins not allow passage of protons (hydronium ions, H3O+), which would collapse membrane electrochemical potentials. And they do not. What is the basis for this extraordinary selectivity?

We find an answer in the structure of AQP-1, as determined by x-ray diffraction analysis (Fig. 11-46). AQP-1 has four monomers (each Mr 28,000) associated in a tetramer, each monomer forming a transmembrane pore with a diameter (2 to 3 A) sufficient to allow passage of water molecules in single file. Each monomer consists of six transmembrane helical segments and two shorter helices, each of which contains the sequence Asn-Pro-Ala (NPA). The NPA-containing short helices extend toward the middle of the bilayer from opposite

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