TRANSPORT MECHANISMS

 

  1. Membrane transport - introduction

    1. Several forms of transport.

      1. Passive-requires no chemical energy input (although there is energy in the gradient).

        1. Free Diffusion

        2. Facilitated diffusion

        3. Osmosis

        4. Ion transport

      2. Active-requires chemical energy

        1. Active transport.

        2. Endocytosis-exocytosis.

    2. These once well defined terms are not so well defined anymore, now that we better understand what is going on.

  2. Free Diffusion (Simple Diffusion): the movement of a molecule from an area of high concentration of that molecule to an area of low concentration.

    1. It costs the cell no chemical energy (other than that in the gradient), but it is also typically slow. If diffusion will get the job done, biological systems will often go to elaborate mechanisms to utilize the free cost of diffusion (e.g., lungs, intestines, gills, roots ... with large surface areas and thin membrane diffusional barriers, counter current exchange).

    2. Diffusion can be easily and fully explained by just the fact that molecules move independently and randomly. (Think of flies randomly moving in and out of a room with a window cracked open; eventually the concentration of flies in and out will be the same.)

    3. Note that this is a process where, when looking at one molecule, the probability of the molecule to go in one direction is equal to the probability of the same kind of molecule going in the opposite direction.

      4. To consider the overall diffusional rate we need to consider the

      rateoverall = rate of flow from out to in - rate of flow from in to out

      The rate of flow from out to in depends on frequency at which molecules collide with the membrane (which directly relates to the concentration: [X]) times the probability that they get through (diffusional constant = kD). The same is true for the flow out. Therefore, the overall rate of flow (Fick's Law) becomes:

      rateoverall = kD [X]out - kD [X]in

      rateoverall = kD * ([X]out - [X]in)

      rateoverall = kD * Æ [X]

      The difference in overall flow is merely due to the difference in molecular concentration on each side. (show computer diffusion simulation). This difference of the concentration of a solute on one side of the membrane minus the concentration on the other side is called the gradient (Æ [X]):

    4. 'Diffusional equilibrium' can be defined in four equivalent ways:

      1. The rate of movement in one direction across a membrane equals the rate of movement in the opposite direction. Therefore:

      2. ...the overall rate is zero. The net flow is zero.

      3. The concentration on one side of the membrane equals the concentration on the other. Therefore:

      4. ...the concentration of the solute on one side minus the other is zero; i.e. the gradient is zero :Æ X = 0.

    5. The kinds of molecules that can freely diffuse through the lipid bilayer biological membrane are small and low in charge. The ones that seem to diffuse rather readily and are of major biological importance are CO2, O2 and H2O. The larger the molecule, and the more charge that it has, means that has less chance of freely diffusing across a lipid bilayer (this means essentially all other molecules of biological interest are naturally impermeable such as sugars, amino acids, DNA, ATP, ions, phosphate, proteins, etc.).

    6. Factors built into the diffusional 'constant' that affect rate are:

      1. the size of the diffusing molecule (the rate is faster for smaller molecules).

      2. the hydrophobicity of the diffusing molecule (the rate is faster for more hydrophobic molecules).

      3. the membrane thickness (the rate is inversely proportional to the thickness).

      4. the number of pores, if pores exist (the rate is directly proportional to the number of pores)

      5. the membrane surface area (the rate is directly proportional to the surface area).

    7. A typical problem using Fick's law:

    If it is known that the glycine diffuses at a rate of 25 nanomoles per minute when the outside concentration is 30 mM and the inside concentration is 5 mM, then what would be the rate if the outside concentration is decreased to 15 mM.

    In the first case:

    25 nanomoles/min = const (30mM - 5mM)

    Now that we know the permeability coefficient, we can figure out the rate in the second case:

     

    (Note that another way to look at the problem is that in the first case the gradient was 25 mM and in the second case was 10 mM, therefore you would expect the rate to drop 2.5 times.)

  3. Osmosis is the movement of water from an area of low concentration of particles to an area of high concentration of particles (The particles are usually solute molecules dissolved in the water).

    1. The rate of water movement (osmosis) is directly proportional to the osmotic gradient. The osmotic gradient can be approximated as the sum of all solute particle concentrations on one side of a membrane minus the sum on the other.

      1. If you have 0.3 M glucose and 0.4 M sucrose on the outside of a micell, and 0.6 M fructose on the inside , then the osmotic gradient is approximately 0.1 osmolar:

        2. [glucose] [sucrose] [fructose]

        osmotic gradient = ( 0.3 M + 0.4 M ) - (0.6 M) = 0.1 osM

      2. If you had 0.4 M fructose and 0.4 M sucrose on the outside and 0.5 M sucrose and 0.1 M starch on the inside, then the osmotic gradient is approximately 0.2 M osmolar:

        3. [fructose] [sucrose] [sucrose] [starch]

        osmotic gradient=(0.4 M + 0.4 M) - (0.5 M + 0.1 M) = 0.2 osM

        If this was the same micell as in 1), then the rate would of water movement would be twice as fast because the osmotic gradient is twice as large.

      3. If you had 0.4 M NaCl on the outside and 0.5 M sucrose on the inside, then the osmotic gradient is approximately 0.3 M osmolar. In this case, one has to realize that NaCl disassociates into 0.4M Na+ and 0.4 M Cl-:

      [Na+] [Cl-] [sucrose]

      osmotic gradient = ( 0.4 M + 0.4 M ) - (0.5 M) = 0.3 M

    2. If this was the same micell as in 1), then the rate would of water movement would be three times as fast because the osmotic gradient is three times as large.

    3. Osmosis is not free diffusion. The diffusion of water would be the movement of water from an area of high water concentration to an area of low water concentration. Free diffusion of water does occur, but is usually of lower magnitude than osmosis.

    4. Although the rate of osmosis is well defined, the mechanism of osmosis is not well understood. Apparently the mechanism is intimately involved with disruption and stability of the hydrogen bonding structure of water.

  4. Facilitated diffusion - the situation where the diffusion of a molecule across a membrane is aided by a carrier molecule.

    1. In effect, this process is a phenomenon whereby the permeability coefficient is raised.

    2. Obeys all of the laws of diffusion.

    3. Often facilitated diffusion is all that a cell needs, since frequently the diffusion gradient is in a favorable direction anyway:

      1. Internal consumption of needed molecules keeps a favorable gradient and the molecules will continue to diffuse in.

      2. Internal production of waste molecules will cause a favorable gradient such that unwanted molecules will continue to diffuse out.

    4. Carriers in membranes are proteins (transport proteins):

      1. Carrier proteins are numerous in all biological membranes and are found dissolved in the lipid bilayer of the membrane.

        1. A carrier protein always has an uncharged (non-polar) outer surface which allows it to be soluble within the lipid of the membrane.

        2. A carrier protein always has a binding site to specifically bind the molecule that is going to be transported (i.e., lock and key and induced fit models apply here). This binding site will usually have charges to correspond to the molecule that is being bound. These charges will be buried in the protein so that they will be away from the lipid part of the membrane.

        3. The carriers have no directionality; they work as well in one direction as the other.

        4. Saturation kinetics, similar to that for enzymes, applies to carriers as well.

      2. The mechanisms of these carriers tends to follow the 'tunnel mechanism': The carrier protein spans from one side of the membrane to the other. Through the center of the protein can flow the molecule to be transported (i.e., like a tunnel). This pore through the center is a specific binding site that is very selective for the molecule that would be allowed to pass.

      3. The carriers can be controlled ('turned on and off')

        1. Control can come from ions, second binding sites (as in allosteric control), membrane fluidity, etc. (e.g. Na+-K+ gates of nerve cells)

        2. Indirectly, control can come from DNA induction and/or repression (i.e. the production or nonproduction of the proteins).

  5. Ion transport (for the most part ion transport is under the same principles as discussed for facilitated diffusion and the to be discussed phenomenon of active transport)

    1. Charges play a role in forcing ions across a membrane (usually via carriers)

    2. Most eukaryotic cells have a positive charge outside and a negative charge inside.

    3. The Nernst equation can be used to determine the force due to a charge gradient. (n is the charge of the ion, T absolute temperature, R is the gas constant, and F is the Faraday constant.) The membrane voltage potential is proportional to the natural log of the ion concentration gradient .

    4. An ion will always be under the influence of two forces: 1) a diffusional gradient (Fick's law) and 2) an ion gradient (Nernst equation). The eventual equilibrium will balance off these two forces - at equilibrium (Donnan equilibrium) where there is no further net flow of the ion, there is usually a nonzero concentration gradient and a nonzero charge gradient whose two forces on the ion exactly offset each other.

  6. Active transport - the use of energy in forcing a molecule across a membrane.

    1. Functions

      1. This transport is faster than diffusion.

      2. Can (and usually does) transport molecules from an area of low concentration to one of higher concentration.

        1. Such high concentrations may be beneficial to the chemical reactions that are occurring on that side of the membrane.

        2. Active transport can be used to develop a charge gradient if the molecule being actively transported is an ion.

    2. The best known example is sodium-potassium ATPase. The forced movement of Na+ in one direction and K+ in the opposite direction using ATP as the energy source.

      1. It must have specific binding sites for:

        1. Na+

        2. K+

        3. ATP

      2. It must catalyze the breakdown of ATP.

      3. It must utilize the energy of the breakdown to forcibly expel the Na+ on one side and the K+ on the other.

      4. Up to one third of an animals energy is used for this one protein alone!!! It is the essence of how nerves work, how the kidney works, and is important for every other cell in the animal body.

      5. It is a gigantic, complicated molecule probably made up of several polypeptides held together in quaternary structure.

      6. It has been purified and reconstituted. There is a catalytic subunit (MW 100,000) and a glycoprotein subunit (MW 45,000).

      7. It seems to pump out 3 Na+ ions for every 2 K+ ions pumped in, thus causing a net positive charge outside relative to the inside.

      8. Found in eukaryotic cell membranes only.

    3. Another well identified active transport molecule is H+ ATPase pump (now better known as ATP synthase), which will be discussed later. There also seems to be a Ca++ active transport enzyme.

    4. Development of a charge gradient using active transport.

    5. "Pseudo active transport" - Frequently what looks like the active transport of a molecule is really a coupling of carriers with a more standard active transport enzyme. For example glucose can be driven to high concentrations inside the cell by using Na+-K+ ATPase as a motor, ATP as the energy source, and a Na+-glucose carrier. As the Na+ is pumped out, a sodium gradient is built up. The concentration gradient and the charge gradient favors the diffusion of the Na+ back into the cell, but it can not pass through the phospholipid bilayer because of the charge on the Na+. However, it could attach to a carrier, and go across by facilitated diffusion. If the carrier is designed such that it will not bind Na+ unless a glucose molecule is also attached, then a Na+ can go across only if a glucose goes too! The sodium will move down its concentration and charge gradient, thus forcing the glucose molecule across the membrane. Now the sodium ion can be pumped out again (at the expense of ATP) and force in another glucose molecule ....

      6. Thus, in effect, glucose has been actively transported but the active transport enzyme was actually Na+-K+ ATPase. Using coupling of active transport enzymes with specifically designed carriers, essentially any molecule can be 'actively transported'.

    6. Active transport can be reversed too! A high concentration gradient can lead to the formation of ATP!! More on this subject later when we talk about mitochondria.

  7. Exocytosis-endocytosis

    1. Mass transport of material by infolding of the membrane (endocytosis) or by expulsion of a membrane vesicle from within (exocytosis)

    2. Requires energy (ATP) [despite what the book says], thus must be regarded as a form of active transport. The mechanism of these processes involves functioning microfilaments (actin filaments) (which use ATP to cause movement).

    3. Endocytosis is often broken down into three concepts:

      1. phagocytosis - intake of solid material

      2. pinocytosis - intake of liquid material.

      3. receptor mediated endocytosis involves recognition of a molecule before it is allowed to enter by endocytosis. This usually involves an indentation on the eukaryotic cell membrane called a coated pit and the protien that coats the pit is called clathrin.

    4. Cellular examples:

      1. Good at endocytosis: Amoebae, white blood cells, lymph cells(phagocytes)

      2. Good at exocytosis: hormone producing cells, amoebae(to rid body of wastes)

      3. Prokaryotes and organelles themselves are generally considered incapable of this phenomenon.

      4. Plant cells???

    Diffusion: The overall flow of a solute from the area in which the solute is high in concentration to an area in which the solute is low in concentration. The rate follows Fick's law:

    Rate of solute movement = (Permeability coefficient) (solute conc out - solute conc in)

    An example for glucose:

    Rate of movement of glucose = Pglucose ([glucose]out - [glucose]in)

    Pglucose depends on the probability of a glucose molecule getting across a membrane when it collides with the membrane. It also depends on temperature and membrane surface area.

    Osmosis: The overall flow of water from an area of low total solute concentration to an area of total high solute concentration.

    Rate of water movement = (Water Permeability ) (osmolarity out - osmolarity in)

    where the osmolarity is the tendency of water to move (yes, this is a circular definition); but osmolarity can be approximated by summing all of the concentrations of solute particles dissolved in the water

    9.