MEMBRANE TRANSPORT:

MEMBRANE TRANSPORT:
The Composition and Architecture of Membranes
 Biological membranes define cellular boundaries, divide cells into discrete compartments, organize complex reaction sequences, and act in signal reception and energy transformations
 The fluid mosaic model describes features common to all biological membranes. The lipid bilayer is the basic structural unit. Fatty acyl chains of phospholipids and the steroid nucleus of sterols are oriented toward the interior of the bilayer; their hydrophobic interactions stabilize the bilayer but give it flexibility.
 Peripheral proteins are loosely associated with the membrane through electrostatic interactions and hydrogen bonds or by covalently attached lipid anchors. Integral proteins associate firmly with membranes by hydrophobic interactions between the lipid bilayer and their nonpolar amino acid side chains, which are oriented toward the outside of the protein molecule.

MEMBRANE DYNAMICS::

 Lipids in a biological membrane can exist in liquid-ordered or liquid-disordered states; in the latter state, thermal motion of acyl chains makes the interior of the bilayer fluid. Fluidity is affected by temperature, fatty acid composition, and sterol content.
 Flip-flop diffusion of lipids between the inner and outer leaflets of a membrane is very slow except when specifically catalyzed by flippases.

 Caveolin is an integral membrane protein that associates with the inner leaflet of the plasma membrane, forcing it to curve inward to form caveolae, probably involved in membrane transport and signaling.

 Integrins are transmembrane proteins of the plasma membrane that act both to attach cells to each other and to carry messages between the extracellular matrix and the cytoplasm.

 Specific proteins mediate the fusion of two membranes, which accompanies processes such as viral invasion and endocytosis and exocytosis.

SOLUTE TRANSPORT ACROSS MEMBRANES:
 A few nonpolar compounds can dissolve in the lipid bilayer and cross the membrane unassisted, but for polar or charged compounds or ions, a membrane protein is essential for transmembrane movement.
Passive Transport Is Facilitated by Membrane Proteins:
 When two aqueous compartments containing unequal concentrations of a soluble compound or ion are separated by a permeable divider (membrane), the solute moves by simple diffusion from the region of higher concentration, through the membrane, to the region of lower concentration, until the two compartments have equal solute concentrations.
 When ions of opposite charge are separated by a permeable membrane, there is a transmembrane electrical gradient, a membrane potential, Vm (expressed in volts or millivolts).
 This membrane potential produces a force opposing ion movements that increase Vm and driving ion movements that reduce Vm.
 Together, these two factors are referred to as the electrochemical gradient or electrochemical potential.
o This behavior of solutes is in accord with the second law of thermodynamics: molecules tend to spontaneously assume the distribution of greatest randomness and lowest energy.

 Membrane proteins lower the activation energy for transport of polar compounds and ions by providing an alternative path through the bilayer for specific solutes.

 Proteins that bring about this facilitated diffusion, or passive transport, are not enzymes in the usual sense; their “substrates” are moved from one compartment to another, but are not chemically altered.

 Membrane proteins that speed the movement of a solute across a membrane by facilitating diffusion are called transporters or permeases.

 Like enzymes, transporters bind their substrates with stereochemical specificity through multiple weak, noncovalent interactions. The negative free-energy change associated with these weak interactions, ∆Gbinding, counterbalances the positive free-energy change that accompanies loss of the water of hydration from the substrate, ∆Gdehydration, thereby lowering ∆G‡ for transmembrane passage.






FIGURE 11–28 Energy changes accompanying passage of a hydrophilic solute through the lipid bilayer of a biological membrane. (a) In simple diffusion, removal of the hydration shell is highly endergonic, and the energy of activation (∆G‡) for diffusion through the bilayer is very high. (b) A transporter protein reduces the ∆G‡ for transmembrane diffusion of the solute. It does this by forming noncovalent interactions with the dehydrated solute to replace the hydrogen bonding with water and by providing a hydrophilic transmembrane passageway.




Transporters Can Be Grouped into Superfamilies Based on Their Structures::
 There are two very broad categories of transporters: carriers and channels.
Carriers bind their substrates with high stereospecificity, catalyze transport at rates well below the limits of free diffusion, and are saturable in the same sense as are enzymes: there is some substrate concentration above which further increases will not produce a greater rate of activity.

Channels generally allow transmembrane movement at rates several orders of magnitude greater than those typical of carriers, rates approaching the limit of unhindered diffusion. Channels typically show less stereospecificity than carriers and are usually not saturable.

Most channels are oligomeric complexes of several, often identical, subunits, whereas many carriers function as monomeric proteins.





 Among the carriers, some simply facilitate diffusion down a concentration gradient; they are the uniporter superfamily.
 Others (active transporters) can drive substrates across the membrane against a concentration gradient, some using energy provided directly by a chemical reaction (primary active transporters) and some coupling uphill transport of one substrate with the downhill transport of another (secondary active transporters).

The Glucose Transporter of Erythrocytes Mediates Passive Transport:
 Glucose enters the erythrocyte by facilitated diffusion via a specific glucose transporter.
 The glucose transporter of erythrocytes (called GLUT1 to distinguish it from related glucose transporters in other tissues) is a type III integral protein with 12 hydrophobic segments, each of which is believed to form a membrane-spanning helix.
 GLUT1 is specific for D-glucose & it is ubiquitous.



 Twelve glucose transporters are encoded in the human genome, each with unique kinetic properties, patterns of tissue distribution, and function.

The Chloride-Bicarbonate Exchanger Catalyzes Electroneutral Cotransport of Anions across the Plasma Membrane::
 The erythrocyte contains another facilitated diffusion system, an anion exchanger that is essential in CO2 transport to the lungs from tissues such as skeletal muscle and liver.
 Waste CO2 released from respiring tissues into the blood plasma enters the erythrocyte, where it is converted to bicarbonate (HCO3 -) by the enzyme carbonic anhydrase.
 The HCO3 _ reenters the blood plasma for transport to the lungs (Fig. 11–33). Because HCO3 _ is much more soluble in blood plasma than is CO2, this roundabout route increases the capacity of the blood to carry carbon dioxide from the tissues to the lungs.

 In the lungs, HCO3 _ reenters the erythrocyte and is converted to CO2, which is eventually released into the lung space and exhaled. To be effective, this shuttle requires very rapid movement of HCO3 _ across the erythrocyte membrane.
 (Recall that HCO3 _ is the primary buffer of blood pH)
 The chloride-bicarbonate exchanger, also called the anion exchange (AE) protein, increases the permeability of the erythrocyte membrane to HCO3 _ more than a millionfold.
 Like the glucose transporter, it is an integral protein.
 This protein mediates the simultaneous movement of two anions: for each HCO3 _ ion that moves in one direction, one Cl- ion moves in the opposite direction , with no net transfer of charge; the exchange is electroneutral.


 The coupling of Cl- and HCO3 - movements is obligatory; in the absence of chloride, bicarbonate transport stops. In this respect, the anion exchanger is typical of all systems, called cotransport systems, that simultaneously carry two solutes across a membrane.
 When, as in this case, the two substrates move in opposite directions, the process is antiport.
 In symport, two substrates are moved simultaneously in the same direction.
 As we noted earlier, transporters that carry only one substrate, such as the erythrocyte glucose transporter, are uniport systems.

 Erythrocytes contain the AE1 transporter, AE2 is prominent in liver, and AE3 is present in plasma membranes of the brain, heart, and retina.

Active Transport Results in Solute Movement against a Concentration or Electrochemical Gradient::
Active transport, by contrast, results in the accumulation of a solute above the equilibrium point.
Active transport is thermodynamically unfavorable (endergonic) and takes place only when coupled (directly or indirectly) to an exergonic process such as the absorption of sunlight, an oxidation reaction, the breakdown of ATP, or the concomitant flow of some other chemical species down its electrochemical gradient.
In primary active transport, solute accumulation is coupled directly to an exergonic chemical reaction, such as conversion of ATP to ADP + Pi.
Secondary active transport occurs when endergonic (uphill) transport of one solute is coupled to the exergonic (downhill) flow of a different solute that was originally pumped uphill by primary active transport.

The amount of energy needed for the transport of a solute against a gradient can be calculated from the initial concentration gradient. The general equation for the free-energy change in the chemical process that converts S to P is-------


When the “reaction” is simply transport of a solute from a region where its concentration is C1 to a region where its concentration is C2, no bonds are made or broken and the standard free-energy change, ∆G’◦, is zero. The free-energy change for transport, ∆Gt, is then




When the solute is an ion, its movement without an accompanying counterion results in the endergonic separation of positive and negative charges, producing an electrical potential; such a transport process is said to be electrogenic.

The energetic cost of moving an ion depends on the electrochemical potential , the sum of the chemical and electrical gradients:


o where Z is the charge on the ion, is the Faraday constant (96,480 J/V . mol), and ∆Ψ is the transmembrane electrical potential (in volts).

o Remember that ∆G for flow down an electrochemical gradient has a negative value, and ∆G for transport of ions against an electrochemical gradient has a positive value.

P-Type ATPases Undergo Phosphorylation during Their Catalytic Cycles::

The family of active transporters called P-type ATPases are ATP-driven cation transporters that are reversibly phosphorylated by ATP as part of the transport cycle; phosphorylation forces a conformational change that is central to moving the cation across the membrane.

Each P-type ATPase transporter is an integral protein.

All P-type transport ATPases have similarities in amino acid sequence, especially near the Asp residue that undergoes phosphorylation, and all are sensitive to inhibition by the phosphate analog vanadate.



In animal tissues, the Na+K+ATPase (an antiporter for Na+ and K+) and the Ca2+ ATPase (a uniporter for Ca2+) are ubiquitous P-type ATPases that maintain differences in the ionic composition of the cytosol and the extracellular medium.
Parietal cells in the lining of the mammalian stomach have a P-type ATPase that pumps H+ and K+across the plasma membrane, thereby acidifying the stomach contents.

In vascular plants, a P-type ATPase pumps protons out of the cell, establishing an electrochemical difference of as much as 2 pH units and 250 mV across the plasma membrane.
A similar P-type ATPase in the bread mold Neurospora pumps protons out of cells to establish an inside-negative membrane potential, which is used to drive the uptake of substrates and ions from the surrounding medium by secondary active transport.
 Bacteria use P-type ATPases to pump out toxic heavy metal ions such as Cd2+and Cu2+.

In virtually every animal cell type, the concentration of Na+ is lower in the cell than in the surrounding medium, and the concentration of K+ is higher. This imbalance is maintained by a primary active transport system in the plasma membrane.

The enzyme Na+K+ ATPase couples breakdown of ATP to the simultaneous movement of both Na+and K+ against their electrochemical gradients.
For each molecule of ATP converted to ADP and Pi, the transporter moves two K+ ions inward and three Na+ ions outward across the plasma membrane. The Na+K+ ATPase is an integral protein.

Because three Na+ions move outward for every two K+ ions that move inward, the process is electrogenic—it creates a net separation of charge across the membrane. The result is a transmembrane potential of -50 to -70 mV (inside negative relative to outside), which is characteristic of most animal cells and essential to the conduction of action potentials in neurons.

 The steroid derivative ouabain is a potent and specific inhibitor of the Na+K+ ATPase.
 Ouabain and another steroid derivative, digitoxigenin, are the active ingredients of digitalis, an extract of the leaves of the foxglove plant.

 Digitalis has been used to treat congestive heart failure since its introduction for that purpose (treatment of “dropsy”) . It strengthens heart muscle contractions without increasing the heart rate and thus increases the efficiency of the heart.


 Digitalis inhibits the efflux of Na+ , raising the Intracellular[Na+] enough to activate a Na+ Ca2+ antiporter in cardiac muscle. The increased influx of Ca+through this antiporter produces elevated cytosolic [Ca2+], which strengthens the contractions of the heart.




PUMPS
1. P-Type Ca2+ Pumps Maintain a Low Concentration of Calcium in the Cytosol::
The cytosolic concentration of free Ca2+ is generally at or below 100 nM, far lower than that in the surrounding medium, whether pond water or blood plasma.
The ubiquitous occurrence of inorganic phosphates (Pi and PPi) at millimolar concentrations in the cytosol necessitates a low cytosolic Ca2+concentration, because inorganic phosphate combines with calcium to form relatively insoluble calcium phosphates.
Calcium ions are pumped out of the cytosol by a P-type ATPase, the plasma membrane Ca2+ pump.

Another P-type Ca2+ pump in the endoplasmic reticulum moves Ca2+ into the ER lumen, a compartment separate from the cytosol.

In myocytes, Ca2+ is normally sequestered in a specialized form of endoplasmic reticulum called the sarcoplasmic reticulum.
 The sarcoplasmic and endoplasmic reticulum calcium (SERCA) pumps are closely related in structure and mechanism, and both are inhibited by the tumor-promoting agent thapsigargin, which does not affect the plasma membrane Ca2+ pump.

The plasma membrane Ca2+ pump and SERCA pumps are integral proteins that cycle between phosphorylated and dephosphorylated conformations in a mechanism similar to that for Na+K+ ATPase


Phosphorylation favors a conformation with a high-affinity Ca2+ binding site exposed on the cytoplasmic side, and dephosphorylation favors one with a low-affinity Ca2+ binding site on the lumenal side.

By this mechanism, the energy released by hydrolysis of ATP during one phosphorylation-dephosphorylation cycle drives Ca2+ across the membrane against a large electrochemical gradient.

The amino acid sequences of the SERCA pumps and the Na+K+ ATPase share 30% identity and 65% sequence similarity, and their topology relative to the membrane is also the same. Thus it seems likely that the Na+K+ ATPase structure is similar to that of the SERCA pumps and that all P-type ATPase transporters share the same basic structure.

2. F-Type ATPases Are Reversible, ATP-Driven Proton Pumps::
The F-type ATPase active transporters play a central role in energy-conserving reactions in mitochondria, bacteria, and chloroplasts.
The F-type ATPases catalyze the uphill transmembrane passage of protons driven by ATP hydrolysis (“F-type” originated in the identification of these ATPases as energy-coupling factors).
The Fo integral membrane protein complex subscript o denoting its inhibition by the drug oligomycin) provides a transmembrane pore for protons, and the peripheral protein F1 (subscript 1 indicating that it was the first of several factors isolated from mitochondria) is a molecular machine that uses the energy of ATP to drive protons uphill (into a region of higher H+ concentration).
 Eubacteria such as E. coli use an FoF1 ATPase complex in their plasma membrane to pump protons outward, and archaebacteria have a closely homologous proton pump, the AoA1 ATPase.

The reaction catalyzed by F-type ATPases is reversible, so a proton gradient can supply the energy to drive the reverse reaction, ATP synthesis.
The proton gradient needed to drive ATP synthesis is produced by other types of proton pumps powered by substrate oxidation or sunlight.

V-type ATPases, a class of proton-transporting ATPases structurally related to the F-type ATPases, are responsible for acidifying intracellular compartments in many organisms (thus V for vacuolar).
Proton pumps of this type maintain the vacuoles of fungi and higher plants at a pH between 3 and 6, well below that of the surrounding cytosol (pH 7.5).
V-type ATPases are also responsible for the acidification of lysosomes, endosomes, the Golgi complex, and secretory vesicles in animal cells.

All V-type ATPases have a similar complex structure, with an integral (transmembrane) domain (Vo) that serves as a proton channel and a peripheral domain (V1) that contains the ATP-binding site and the ATPase activity.

SUMMARY -Solute Transport across Membranes
■ Movement of polar compounds and ions across biological membranes requires protein transporters. Some transporters simply facilitate passive diffusion across the membrane from the side with higher concentration to the side with lower. Others bring about active movement of solutes against an electrochemical gradient; such transport must be coupled to a source of metabolic energy.
■ Carriers, like enzymes, show saturation and stereospecificity for their substrates. Transport via these systems may be passive or active. Primary active transport is driven by ATP or electron-transfer reactions; secondary active transport, by coupled flow of two solutes, one of which (often H+ or Na+) flows down its electrochemical gradient as the other is pulled up its gradient.
■ The GLUT transporters, such as GLUT1 of erythrocytes, carry glucose into cells by facilitated diffusion. These transporters are uniporters, carrying only one substrate. Symporters permit simultaneous passage of two substances in the same direction; examples are the lactose transporter of E. coli, driven by the energy of a proton gradient (lactose-H+ symport), and the glucose transporter of intestinal epithelial cells, driven by a Na+ gradient (glucose Na+symport). Antiporters mediate simultaneous passage of two substances in opposite directions; examples are the chloride-bicarbonate exchanger of erythrocytes and the ubiquitous Na+K+ ATPase.
■ In animal cells, Na+K+ATPase maintains the differences in cytosolic and extracellular concentrations of Na+ and K+, and the resulting Na+ gradient is used as the energy source for a variety of secondary active transport processes.
■ The Na+K+ ATPase of the plasma membrane and the Ca2+ transporters of the sarcoplasmic and endoplasmic reticulums (the SERCA pumps) are examples of P-type ATPases; they undergo reversible phosphorylation during their catalytic cycle and are inhibited by the phosphate analog vanadate. F-type ATPase proton pumps (ATP synthases) are central to energy-conserving mechanisms in mitochondria and chloroplasts. V-type ATPases produce gradients of protons across some intracellular membranes, including plant vacuolar membranes.
■ ABC transporters carry a variety of substrates, including many drugs, out of cells, using ATP as energy source.
■ Ionophores are lipid-soluble molecules that bind specific ions and carry them passively across membranes, dissipating the energy of electrochemical ion gradients.
■ Water moves across membranes through aquaporins.
■ Ion channels provide hydrophilic pores through which select ions can diffuse, moving down their electrical or chemical concentration gradients; they are characteristically unsaturable and have very high flux rates. Many ion channels are highly specific for one ion, and most are gated by either voltage or a ligand. In bacterial K+ channels, a selectivity filter provides ligands with the right geometry to replace the water of hydration of a K+ ion as it crosses the membrane. Some K+ channels are voltage gated. The acetylcholine receptor/channel is gated by acetylcholine, which triggers subtle conformational changes that open and close the transmembrane path.