Supplemental Lecture (98/03/28 update) by Stephen T. Abedon (abedon.1@osu.edu)

  1. Chapter title: Transport Across Membranes
    1. A list of vocabulary words is found toward the end of this document
    2. Lipid bilayers sans membrane proteins are great barriers to polar molecules and ions, as well as to large molecules and molecular complexes. This is wonderful to the extent that polar molecules, ions, and various larger substances constitute cell poisons. More often than not, however, substances answering to this description can serve as cell nutrients. In fact, for the most part the transporting of large, polar, or ionic substances across lipid bilayers is how cells "eat" (i.e., obtain nutrients). Consequently, and as might be expected, organisms have evolved a variety of mechanisms by which substances are recognized and then transported across lipid bilayers by specialized membrane proteins.
    3. Transport mechanisms may be divided into those that transport smaller substances and those that transport larger substances, as well as those that require energy and those that do not. In addition to transport into cells, a large variety of substances are transported out of cells utilizing similar transport mechanisms.
    4. Control of movement of substances that cannot cross lipid bilayers spontaneously play varied and very important roles in cell metabolism. In this lecture we will concentrate on the transport of relatively small substances directly across lipid bilayers. In the lecture titled endomembrane system we will consider the movement of larger substances into and out of cells, though not exactly directly across lipid bilayers.
  2. Lipid bilayer excluded substance
    1. The following discussion is considerably easier to present if we come up with an inclusive phrase/term to describe those polar, ionic, and large molecule that are generally incapable (or show low probability) of spontaneously crossing lipid bilayers.
    2. For better or for worse, I propose we employ the terribly inelegant term, lipid bilayer-excluded substance.
    3. Keep in mind that this is a phrase/term that I have invented on the spot simply to improve the flow of my writing/lecturing and not something which deserves memorization beyond an understanding of the underlying concepts.
  3. Concentration gradient
    1. An environment consisting of two or more adjacent regions differing in the concentration of a solute.
    2. Movement with a concentration gradient is from a region of high solute concentration to a region of low solute concentration.
    3. This occurs spontaneously with rate dependent on rates of diffusion. The probability that it occurs is dependent upon the degree to which the low concentration and high concentration environments differ in terms of solute concentration.
    4. Against a gradient:
      1. Movement against a concentration gradient describes the movement of a solute from a region of low solute concentration to a region of high solute concentration. Such movement is not forbidden, just unlikely.
      2. Net movement against a concentration gradient can be accomplished only via pumping, an energy-requiring process.
  4. Diffusion
    1. Random movement:
      1. Diffusion is the random movement of dissolved substances and solvent molecules.
      2. Diffusion is driven by thermal energy, i.e., the random bouncing around and shaking displayed by all substances existing above absolute 0 degrees Kelvin. Diffusion also results in an increase in entropy and therefore is energetically favored.
    2. Diffusion across lipid bilayers:
      1. Substances such as water are able to cross lipid bilayers without protein assistance. These substances are said to move from one side of a membrane to the other by the process of diffusion.
      2. This basically means that (see illustration below):
        1. Substances begin their journey dissolved in extracellular water.
        2. They bounce around randomly until they collide with a lipid bilayer.
        3. They then can become dissolved in the lipid bilayer.
        4. They then may bounce around randomly within the lipid bilayer until they collide with the other (intracellular) side of the lipid bilayer.
        5. They then can exit the lipid bilayer.
        6. And become dissolved in intracellular water.
        7. They could also fail to leave the lipid bilayer or exit on the same side they entered it.
      3. Alternatively, they could start on the inside and work their way out.
    3. The key here is that since movement is random:
      1. the substance doesn't have to ever find its way into a lipid bilayer
      2. once inside a lipid bilayer never has to find its way out again
      3. could just as easily find its way out back on the extracellular side as on the intracellular side of the lipid bilayer
    4. Flow from higher concentration:
      1. Nevertheless, there is high statistical probability that the net flow of a substance by diffusion will be from a region of high concentration to a region of low concentration.
      2. If such regions correspond to the extracellular and intracellular (or vice versa) sides of a lipid bilayer, that spontaneous net movement across the lipid bilayer is expected.
  5. Illustration, diffusion across membrane
  6. Selective permeability
    1. Non-random control:
      1. To a large extent the passage of lipid bilayer-excluded substances across an intact cell membrane is predetermined (i.e., evolutionarily) by whether such passage is beneficial to the organism whose cell membrane is being crossed.
      2. Exceptions to this rule generally are either inert (good) or poisons (bad).
    2. Membrane protein gates:
      1. Both the movement of lipid bilayer-excluded substances across a membrane and the control of which lipid bilayer-excluded substances may pass, and when, is a function of membrane proteins.
      2. The rate of movement of lipid bilayer-excluded substances across cell membranes can be controlled by both limiting the number of specific membrane proteins present and to specifically modify (enhance or inhibit) the function of those membrane proteins already present.
    3. There are a variety of mechanisms by which lipid bilayer-excluded substances substances cross lipid bilayers via protein action. These include:
      1. diffusion through channels
      2. facilitated diffusion
      3. active transport
      4. cotransport
      5. group translocation
  7. Facilitated diffusion
    1. Facilitated random movement:
      1. All dissolved molecules, including those that can readily pass through lipid bilayers and those that cannot (lipid bilayer-excluded substances), are capable of diffusion.
      2. The big difference is that for lipid bilayer-excluded substances, diffusion tends to be limited to regions not co-occupied with a lipid bilayer.
    2. Protein facilitators:
      1. For lipid bilayer-excluded substances, diffusion across cell membranes may nevertheless be highly probable but such diffusion must be facilitated by lipid bilayer spanning proteins.
      2. Such proteins essentially form channels or conveyors:
      3. which accept specific lipid bilayer-excluded substances at one end
      4. allow the substance to enter the lipid bilayer without dissolving in it
      5. then deliver the substance to either end randomly
      6. Net movement therefore occurs away from whichever side collides with the faciliating protein more, that side being whichever possesses with the solute in greater concentrations.
      7. See text figure 1055.1.
    3. Some of these proteins are highly specific to the transport of certain substances while other facilitating proteins are much less selective (for example, consisting of a pore that blocks the passage only of substances larger than the pore).
    4. Only thermal motion required:
      1. Regardless of the mechanism, no energy is used in facilitating this movement (other than the thermal energy/motion responsible for diffusion).
      2. Consequently, net movement by facilitated diffusion cannot be achieved against a concentration gradient.
    5. Only with concentration gradient:
      1. Facilitated diffusion thus constitutes a cheap way of transporting lipid bilayer-excluded substances into or out of a cell in the direction of a concentration gradient.
      2. Essentially, once the membrane protein apparatus is manufactured and inserted into the membrane, facilitated diffusion transports lipid bilayer-excluded substances into or out of cells for free, so long as movement is with a concentration gradient.
  8. Active transport
    1. Movement against concentration gradients:
    2. Movement of substances against concentration gradients requires expenditure of energy.
    3. Such movement across lipid bilayers is called active transport.
    4. Active transport allows lipid bilayer-excluded substance concentrations to be maintained intracellularly artificially high, or artificially low, relative to the extracellular concentration.
  9. ATP-driven ion transport [sodium-potassium pump]
    1. A specific membrane protein (sodium-potassium pump) pumps sodium out of cells and potassium into cells against a concentration gradient in a manner stoichiometrically balanced as follows:
    2. 3Na (intracell) + 2K (extracell) + ATP ---
      
      3Na (extracell) + 2K (intracell) + ADP + Pi
    3. Mechanism overview:
      1. This is a thumb-nail sketch of how the sodium-potassium pump functions:
        1. intracellularly the pump presumably has a relatively low affinity for potassium ions but high affinity for sodium ions.
        2. Sodium and potassium ions move to or into the pump via diffusion but only sodium ions can bind.
        3. Sodium ion binding stimulates ATP hydrolysis.
        4. This ATP hydrolysis drives a pump conformational change.
        5. As a result of this conformational change sodium ions, as well as the section of protein bound to these ions, is presented extracellularly.
        6. The pump thus is no longer shaped in a manner which will allow attachment of intracellular sodium atoms.
        7. Pump conformational change with ATP hydrolysis also results in a change in pump affinity (lowered) for sodium ions.
        8. Sodium ions are consequently free to diffuse away from the pump .
        9. Since the sodium ions are now presented extracellularly, they diffuse into the extracellular environment (thus raising the concentration of sodium ions in the extracellular environment).
        10. Pump conformational change with ATP hydrolysis results in a increased pump affinity for potassium ions. Recall that the region of the pump capable of binding sodium or potassium ions is now found extracellularly.
        11. extracellular potassium and sodium ions diffuse to or into the pump but only potassium ions bind.
        12. Potassium ion binding stimulates a relaxation of the above ATP hydrolysis-driven conformational change.
        13. Upon this second, relaxing conformational change, bound potassium ions are carried across the membrane and thus presented intracellularly (i.e., as were the sodium ions prior to ATP hydrolysis).
        14. Relaxation of conformational change-driven change in pump affinity results in lowered potassium affinity and raised sodium affinity.
        15. Potassium ions are free to diffuse into the intracellular environment (thus raising the concentration of potassium ions in the intracellular environment).
        16. At this point the pump has essentially returned to the state it was when we began this sequence.
    4. Consequence:
      1. Thus, with ATP hydrolysis coupled to sodium and potassium ion pumping a cell may maintain the following:
        1. high intracellular concentration of potassium ions.
        2. lower extracellular concentration of potassium ions.
        3. higher extracellular concentration of sodium ions.
        4. low intracellular concentration of sodium ions.
      2. Note that the continued existence of these gradients demands that the cell membrane be intact.
    5. Crucial but expensive:
      1. A great deal of energy is cumulatively expended by the sodium-potassium pumps but the maintenance of the above noted concentration gradients is key to a number of processes including:
        1. nerve function.
        2. muscle function
        3. active transport of many additional substances (i.e., cotransport).
  10. Cotransport
    1. Ion concentration driven transport:
      1. Once sodium, potassium, or other ion gradients have been produced by ATP-driven transport pumps they represent potential energies that may be utilized to perform the work.
      2. The capturing of the energy to drive transport against a concentration gradient is a kind of active transport known as cotransport.
    2. That is, the movement of an ion from a high concentration to a low concentration is analogous to the movement of water (or some other liquid) from a high point to a lower point. In both cases a machine can be placed between the high point and the low point and capture some of the potential energy lost. (ATP similarly represents a higher potential energy state than ADP and the potential energy lost may be utilized by biological machinery.)
    3. A great many cotransport systems have been described whereby an extracellular ion such as sodium is allowed back into a cell at the same time a second desired molecule is let in.
    4. Mechanism overview:
      1. A typical cotransport mechanism might look like the following:
        1. Both glucose and sodium bind to a specific membrane protein.
        2. This triggers the protein to carry both the glucose molecule and the sodium molecule into the cell.
        3. Once in the cell the protein releases both the sodium ion and glucose molecule which are then free to diffuse away from the protein.
        4. The membrane protein at this point shows a lowered affinity for both glucose and sodium ions.
        5. It also employs energy stored upon glucose and sodium ion movement into the cell to spring back to its original conformation waiting for a glucose molecule and sodium ion on the extracellular side of the membrane.
      2. Plausible ideas about how a cotransport pump might work ought to be derivable from your knowledge of the mechanisms of sodium-potassium pumping , above; you ought to be able to derive the above or something like it without actually memorizing it!
  11. Group translocation
    1. Bacteria mechanism:
      1. Historically group translocation is considered to be a bacterial transport mechanism.
      2. However, group translocation-like transport mechanisms have also been demonstrated in eukaryotes.
    2. Part facilitated diffusion:
      1. Group translocation is a mechanism that blends facilitated diffusion essentially with active transport.
      2. The facilitated diffusion part is due to a dependence on movement only with the concentration gradient and a mechanism of passage through the membrane which employs membrane proteins.
    3. The active transport part is due to the requirement of energy expenditure in order to create the favorable concentration gradient.
    4. Changed upon entry:
      1. Specifically, lipid bilayer-excluded substances are modified (given a charge, for example) upon entry into the cell.
      2. Consequently, the concentration of the original lipid bilayer-excluded substance inside the cell remains very low and the favorable gradient is maintained.
    5. No way home:
      1. A gradient favorable to the removal of the modified lipid bilayer-excluded substance by the same transport mechanism is also created.
      2. Problems associated with this are avoided by disallowing the transport protein from recognizing (i.e., having affinity for) the modified lipid bilayer-excluded substance.
  12. Vocabulary
    1. Active transport
    2. ATP-driven ion transport
    3. Concentration gradient
    4. Cotransport
    5. Diffusion
    6. Diffusion across membrane
    7. Facilitated diffusion
    8. Group translocation
    9. lipid bilayer excluded substance
    10. Movement against a concentration gradient
    11. Movement with a concentration gradient
    12. Selective permeability
    13. Sodium-potassium pump.
  13. Practice questions
    1. The movement of a material through the membrane toward a region of lower concentration by means of specific membrane proteins, is called _________. (circle correct answer) [PEEK]
      1. active transport
      2. movement against a concentration gradient
      3. facilitated diffusion
      4. group translocation
      5. none of the above
    2. Describe with words and/or diagrams a plausible cotransport system employing hydrogen ions and the sugar galactose. [PEEK]
    3. What information do you need to predict in which direction net movement of an amino acid across a membrane (via facilitated diffusion) might occur (in answering this question, assume that you know that the amino acid is capable of crossing the membrane--i.e., that is not one of the pieces of information you need)? [PEEK]
    4. Which is the correct stoichiometry for the sodium-potassium pump we discussed? [PEEK]
      1. three sodium, two potassium, one ATP
      2. two sodium, two potassium, one ATP
      3. two sodium, two potassium, two ATP
      4. one sodium, one potassium, one ATP
      5. all of the above
      6. none of the above
    5. Discuss the mechanism and consequences of ATP-driven sodium-potassium pumping. Be as thorough and as detailed as you can. Place any figures or diagrams on the reverse of this or the second sheet. Properly direct attention to these from text (e.g., ". . . figure 1 . . .") or else I won't know they are there. Relative points will be gained for any and all relevant detail. Relative points will be lost for incorrect information, so don't BS! In addition, do try to write both neatly and well. (essay question) [PEEK]
    6. An example of transport across membranes which does not require energy input is __________. (circle one correct answer) [PEEK]
      1. facilitated diffusion.
      2. passive diffusion.
      3. movement directly through the phospholipid bilayer of the membrane.
      4. movement through pores (channel proteins).
      5. all of the above (are examples).
      6. none of the above (are not examples).
    7. Which of the following results in the substance becoming immediately present in the cytoplasm (i.e., actual movement across membranes rather than rendering the substance into a membrane-bound organelle floating in the cytoplasm)? (circle one correct answer) [PEEK]
      1. pinocytosis.
      2. endocytosis.
      3. facilitated diffusion.
      4. phagocytosis.
      5. all of the above.
      6. none of the above.
    8. Name a specific biomolecule (i.e., not class of) that you would expect a lipid bilayer to be impermeable to. [PEEK]
    9. What kind of structure allows relatively large, polar molecules to pass nonspecifically through membranes.[PEEK]
    10. True or false, ATP is expended to drive substances across membranes, with the concentration gradient, via a mechanism called facilitated diffusion. (circle one correct answer) [PEEK]
    11. Name two ways that facilitated diffusion differs from active transport. [PEEK]
    12. A hydrophobic molecule which enters a lipid bilayer has __________ probability of exiting on one aqueous side (a side that sports a lower concentration of that molecule) than/as on the other aqueous side (one that sports a greater concentration of that molecule). (circle best answer) [PEEK]
      1. a greater
      2. a lower
      3. the same
      4. a much lower
      5. a much greater
  14. Practice question answers
    1. iii, facilitated diffusion
    2. (microbiology 509 students, don't worry about having to know this) A proton concentration gradient is maintained by active transport. That is, a high concentration outside of the cell and a lower concentration inside of the cell. Galactose exists in higher concentrations inside of the cell and relatively low concentrations outside of the cell. A protein exists which spans the plasma membrane. The protein contains two active sites found, while in its ground state, on the outside of the cell. Protons and galactose are free to diffuse into these active sites and are captured if they do. When sufficient protons along with a galactose molecule have bound, the protein undergoes a conformational change which results in both the galactose molecule and the protons now being located on a face of the protein that is presented to the inside of the cell. These now cytoplasmically located protons and galactose are free to diffuse away from this cotransport protein. Diffusion away from the protein is promoted by protein affinity being lower on the inside of the cell for these substrates than it is when binding extracellular protons and galactose. The movement of these substances out of the active sites of the cotransport protein results in the protein relaxing to its ground state with its various active sites pointing out toward the cytoplasm. The cotransport protein is now ready to undergo an additional round of cotransport.
    3. On which side of the membrane is concentration highest?
    4. i, three sodium, two potassium, one ATP (microbiology 509 students, don't worry about having to know this)
    5. See ATP-driven ion transport (sodium-potassium pump) (microbiology 509 students, don't worry about having to know this).
    6. v, all of the above.
    7. iii, facilitated diffusion. Note that the rest of these mechansism involve pulling the substance into the cell, but not across the cell membrane. That is, the substance ends up inside of the cell, but separated from the cytoplasm by a membrane. Facilitated diffusion, on the other hand, is movement directly across a membrane and directly into the cytoplasm if that membrane is a plasma membrane.
    8. glucose, maltose, sucrose, starch, and other carbohydrates, proline or other amino acids, ATP, but not various lipids such as triglycerides.
    9. protein holes/pores.
    10. False
    11. facilitated diffusion does not require energy, active transport does; facilitated diffusion cannot concentrate solutes, active transport can.
    12. iii, the same. The difference in net movement is a consequence of differential rates of entry, not differential rates of exit.
  15. References
    1. Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 96-99.
    2. Mathews, C.K., van Holde, K.E. (1990). Biochemistry. The Benjamin/Cummings publishing co., inc. Redwood City, CA. pp. 317-331.
    3. Raven, P.H., Johnson, G.B. (1995). Biology (updated version). Third Edition. Wm. C. Brown publishers, Dubuque, Iowa. pp. 118-132.
    4. Tortora, G.J., Funke, B.R., Case, C.L. (1995). Microbiology. An Introduction. Fifth Edition. The Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA, pp. 118-132.