C2006/F2402 '10 OUTLINE FOR LECTURE #5  Last updated 02/03/10 06:00 PM

(c) 2010 Dr. Deborah Mowshowitz , Columbia University, New York, NY

Handouts:   5A-- Measurement & Classification of Transport & 5B -- Models for Active Transport 

I. How is Transport of Small Molecules Measured?

    A. Need a suitable experimental set up. A common method: using RBC ghosts.  How is it done?

• Put ghosts in solution with some concentration of X on the outside and none on the inside.

• At appropriate time intervals, you take a sample, centrifuge out the ghosts, and measure the amount of X in the ghosts.

• You repeat with different starting concentrations of [X]_out

    B. What do you learn from doing this?

• You can tell active transport from passive.

• You can tell if a carrier or pump protein is required for transport (as vs. none or a channel)

• You can measure the properties of the transport protein -- equivalents of K_m and V_max.

    C. Details of experimental set up -- See handout 5A.

• Put ghosts in solution with some concentration of X inside and outside:
  
      C_o = concentration outside = [X]_out = some fixed value to start
  
      C_i = concentration inside = [X]_{in ;} usually = 0 to start.
   

• You measure C_i as a function of time.  This generates 'curve #1' -- the top set of three curves on 5A.

• You repeat with different starting values of C_o.  This generates the data plotted in 'curve #2' -- the second set of three curves on 5A.

II. What do Results of Measurement Look Like? What do they mean?

    A. Curve # 1 -- Uptake of X vs time: Measure  [X]_in at increasing times at some (outside, essentially fixed) concentration of X; plot conc. of X inside vs. time. This allows you to distinguish active and passive transport.

1. For active transport of neutral molecules, [X]_in at equilibrium will exceed [X]_out.

2. For passive transport of neutral molecules, [X]_in at equilibrium will equal [X]_out.

(If X is charged, the situation is more complicated, as explained below.)

Question: If you measure uptake a second time, using a higher concentration of X, will the slope of curve #1 be the same? Will it level off at the same value?

    B. Curve #2 -- Uptake of X vs concentration: Measure initial rate of uptake of X (from curve #1) at varying concentrations of added (outside) X; plot rate of uptake (flux) vs. initial concentration of [X]_out.  See handout or Sadava 5.15 (5.11) or Becker fig. 8-6. This allows you to find out what sort of protein (if any) is involved in transport. 

1. If an enzyme-like protein (carrier or pump) is involved in transport, curve will be hyperbolic -- carrier or pump protein will saturate at high [X] just as an enzyme does. Why? If [X] is high enough, all protein molecules will be "busy" or engaged, and transport reaches a max. value. Adding more X won't increase the rate of transport. (Same as reaching  V_max with a V vs [S] curve for an enzyme.)

2. If no protein, or a channel-like protein, is involved in transport, curve will be linear (at physiological, that is reasonable, concentrations of X.). There is no time consuming event such as the binding of X or a major conformational change in the protein that limits the rate of the reaction at high [X].

Note: for a channel the curve will saturate at extremely high levels of X. These saturating levels are not usually reached in practice.

    C.   Curve #1 vs Curve #2.  For both curves, you are considering the reaction X_out ↔ X_in. So what's the big difference?

1. In Curve #1, you are looking at how the concentration of X_in varies with time (starting with a fixed  concentration of X_out, and no X inside_.).

a. Curve shows uptake as a function of time.

b. (Initial) Slope of the curve = rate of uptake (with time as the variable) |

c. Plateau value  =  yield =  final value of [X]_in when curve #1 levels off.

d. Note that curve #1 ALWAYS levels off.

e. Same idea as plotting P formed (or S used up) vs time for an enzyme catalyzed reaction.

2. In Curve #2, you are looking at how the rate of uptake (flux) -- initial slope of curve #1 -- varies for different starting concentrations of [X]_out. 

a. Curve shows uptake as a function of concentration of X added (outside).

b. (Initial) Slope of the curve = rate of change of uptake (with [X]_out as the variable)

c. This curve levels off * only if a protein must bind to X and/or change conformation significantly in order to move X.

d. Same idea as a plotting V vs S for an enzyme catalyzed reaction.

*at physiological values of [X]_out

III. Kinetics and Properties of each type of Transport -- How you tell the cases apart.

All the cases below refer to the reaction [X]_in ↔ [X]_out.  All the important features are summarized in the table on handout 5A.

    A. Simple Diffusion (Case 1)

1. Curve #1 (concentration of substance X inside plotted vs. time) plateaus at [X]_in = [X]_out.

2. Curve #2 (rate of uptake of X plotted vs concentration of X added outside) does not saturate.

3. Energy:

a. Reversibility: Rxn ( X _in ↔ X _out) is strictly reversible.

b. K_eq = 1; Standard free energy change (ΔG^o) = 0; at equil. [X]_in = [X]_out

c. ΔG. Actual free energy change (ΔG) and direction of transport depends on concentration of X. If [X] is higher outside, X will go in and vice versa.

4. Importance. Used by steroid hormones, some small molecules, gases. Only things that are very small or nonpolar can use this mechanism to cross membranes. Materials (usually small molecules) can diffuse into capillaries by diffusing through the liquid in the spaces between the cells. (The cells surrounding capillaries do not have tight junctions, except in the brain.) 

    B. Carrier mediated Transport = Facilitated Diffusion using a carrier protein (Case 3). Note we are deferring case 2.

1. Curve #1 same as above (case 1)

2. Curve #2 saturates. See Becker fig. 8-6, or Sadava fig. 5.12 (5.11)

3. Mechanism: Carrier acts like enzyme or permease, with V_max, K_m etc. See Becker fig. 8-8. 

        Carrier can be considered an enzyme that catalyzes:

X_out   ↔  X_in

        Carrier is specific, just like an enzyme. Will only catalyze movement of X and closely related compounds.

4. Energy as above (case 1) -- substance flows down its gradient, so transport is reversible, depending on relative concentrations in and out.

5. Regulation: Activity of a transport protein (or any transmembrane protein) can be regulated at least 3 ways -- methods a-c below. Methods a & b are common to many proteins and are primarily listed here for comparison (more details elsewhere). Method c is unique to transmembrane proteins.

a. allosteric feedback -- inhibition/activation of carrier proteins

b. covalent modification (reversible) of the carrier proteins  --  common modifications are

(1). Phosphorylation  -- addition of phosphate groups -- catalyzed by kinases.

    Kinases catalyze: X + ATP → X-P + ADP

(2). Dephosphorylation -- removal of phosphate groups --  catalyzed by phosphatases.
   
    Phosphatases catalyze: X-P + H₂O → X + P_i

P (bold) = phosphate group attached to molecule; 
P_i = inorganic phosphate group (in solution)

(3). Reactions carried out by phosphatases and kinases are generally irreversible. However the covalent modifications of the carrier proteins are reversible -- by using both types of enzymes.

c. removal/insertion of carrier into membranes.

(1). Newly made membrane proteins are inserted into the membrane of a vesicle, by a mechanism to be discussed later.

(2). Vesicle can fuse with plasma membrane; process is reversible.

(a). Fusion of the vesicle with the plasma membrane (exocytosis) inserts transport protein into plasma membrane where it can promote transport.

(b). Budding (endocytosis) of a vesicle back into the cytoplasm removes the transport protein and stops transport. 

(3). Some channels and/or carrier proteins are regulated in this way -- channel or carrier proteins can be inserted into the membrane (or removed) in response to the appropriate hormonal signals. Examples --

        (a). GLUT4 -- the insulin sensitive glucose transporter. Insulin is the signal for insertion of the transporter into the plasma membrane of some cells, allowing increased glucose uptake. Details next time.

        (b). Water channels (aquaporins) in kidney cells.  The hormone ADH (anti-diuretic hormone) is the signal to promote insertion of the channels into the plasma membrane. Therefore water is recovered, not lost. More details when we get to kidney.

Notes: (1). This discussion is about the regulation of the activity of pre-existing protein molecules. Regulation of the amount of protein by adjusting rates of synthesis, degradation, etc., will be discussed later.
            (2). Method c applies to cases 2 & 3. Could apply to cases 4 & 5, but I don't know of any examples.

        6. Example -- How glucose enters and/or exits most cells. Transport of glucose across a membrane (down its gradient) requires a GLUT (glucose transporter/carrier) protein. Either GLUT4, which is regulated as above, or a different GLUT that is constitutively present in the membrane. More details on the GLUT family of proteins next time.

To see how you analyze uptake, try problem 2-1. To summarize everything so far, try 2-4.

    C. Active Transport (Cases 4 & 5)

1. What's the same? Curve #2 saturates as in previous case.

2. What's different? Curve #1: when it plateaus, [X]_in greater than [X]_out  -- because movement of substance linked to some other energy releasing reaction. (This assumes we are following the reaction X_out → X _in).

3. Mechanism -- An enzyme-like transport protein is involved as in previous case.

a. X moves up its gradient. Protein acts as transporter or pump catalyzing movement of X up its gradient. Therefore transporter action must be powered, directly, or indirectly, by breakdown of ATP.

b. Most primary active transport involves movement of cations. (But see ABC transporters below.) Gradient of cations can then be used to do work, such as secondary active transport.

4. Energy relationships:

a. Reversibility: Reaction is not readily reversible. Although all reactions are micro-reversible, X is virtually always transported in the same direction in living cells (which is either in or out for any particular active transporter).

b. K_eq not = 1 and standard free energy (ΔG^o)  not = zero. At equil. [X]_in is not equal to [X]_out

c. Coupling: Overall reaction usually has large, negative ΔG^o (& ΔG) because in overall reaction, transport of X (uphill, against the gradient) is coupled to a very downhill reaction. The downhill reaction is either

(1). Splitting of ATP (in primary active transport), or

(2). Running of some ion (say Y) down its gradient (in secondary active transport).

5.  Secondary (Indirect) Active Transport -- How does ATP fit in? Process occurs in 2 steps:

a. Step 1. Preparatory stage: Splitting of ATP sets up a gradient of some ion (say Y), usually a cation (Na⁺ or H⁺). 

b. Step 2. Secondary Active Transport Proper: Y runs down its gradient, and the energy obtained is used to drive X up its gradient. See Becker fig. 8-10.

c. Overall: Step (1) is primary active transport; step (2) is secondary and can go on (in the absence of ATP) until the Y gradient is dissipated. Note that step (1) cannot occur at all without ATP but step (2) can continue without any ATP (for a while).

Try problem 2-2 & 2-10.

6. Some Examples  & Possible mechanisms (models are discussed below). Click on links for animations.

  *This is how glucose enters the epithelial cells lining the intestine or kidney tubule. The glucose exits those cells by facilitated diffusion using a different transporter -- GLUT2 protein.

    D. Channels (Case #2)

1. Curve #1

a. Very high rate of transport -- Initial slope of Curve #1 very steep. 

b. Transport is passive

    (1). If X is neutral, the only force that drives X through the channel is the concentration of X.

    (2). If X is charged (an ion) you have to take the electrical forces into account as well as the concentration. (Details below on how to do this.)

    (3). In summary, Curve #1 plateaus with  [X]_in = [X]_out only if X is neutral or there is no electric potential. 

2. Curve #2: Shape like simple diffusion (linear, no saturation) at physiological concentrations. Curve plateaus only at extraordinarily high concentrations, so we are assuming no saturation.

3. Gating/regulation

a.  Most Channels are gated = % time any particular channel/gate is open is controlled (but each individual gate is either open all the way or shut).

(1). Ligand gated -- opens or shuts in response to ligands (= chemicals that bind to substance under discussion). Typical substances that open ligand gated channels are  hormones, neurotransmitters, etc. For a picture see Sadava fig. 5-10 (5.9).

(2). Voltage gated -- opens or shuts in response to changes in voltage. Allows transmission of electrical signals as in muscle and nerve -- see Becker figs. 13-8 & 13-9.

(3). Mechanically gated -- opens or shuts in response to pressure. Important in touch, hearing and balance. 

b. Other means of regulation -- some channels are regulated by insertion into the membrane or removal from the membrane as explained above for aquaporins in kidney.

c. Some channels are open all the time (ungated); An example = K⁺ leak channels. These allow a little K⁺ to leave or "leak out" of cells, causing cells to have a slight overall negative charge. This is critical to conduction of impulses by nerve and muscle as will be explained in detail later. Why do leak channels only allow "a little" K⁺ to leave? Why isn't the concentration of K⁺ on both sides of the membrane the same? See below.

4. Curve #1 for ions. Most channels are ion channels -- transport charged particles, not neutral molecules. This raises new energy considerations:

a.  Role of charge: If X is charged, need to consider both chemical gradient & voltage (charge gradient or electrical potential). Concentration & voltage can both "push" ions the same way or push in opposite directions.

b. Result of charge: K_eq not usually equal to 1 here --  Curve #1 plateaus when chemical gradient and voltage are balanced (not necessarily at [X]_out = [X]_in). Example:  K⁺ ions stop leaking out of the cell and you reach equilibrium for K⁺ when the charge difference across the cell membrane (which pushes K⁺  in) balances out the concentration difference across the membrane (which pushes K⁺ out).

5. Mechanism. 

a. High Capacity: Lack of saturation and high rate of transport indicate that max. capacity of channel is very large and is not easily reached. This is assumed to be because of one or both of the following:

(1). Binding of ion to channel protein is weak (Km >> 1), and/or

(2). No major conformational change of channel protein is required for ion to pass through.

b. Specificity: Channels are very specific -- each channel transports only one or a very small # of related substances.

c. One model (FYI). How to explain the combination of high speed (& capacity) and high specificity? Mechanism of specificity has been recently figured out for one channel. For pictures see Sadava fig. 5.11 (5.10) & Becker fig. 13-8 & 13-9. For more, see Nobel Prize in Chemistry for 2003, or an interview with Rod MacKinnon about the channel. This is a current hot topic of research, and may be discussed again when we get to nerve function.

See Sadava fig. 44.6 (44.5) for comparison of ion pumps and ion channels;  Becker p. 199 (203)  for comparison of carrier and channel proteins. 

6. Terminology. (Reminder from last time)

        a. Facilitated diffusion? Diffusion through a channel is usually called "facilitated diffusion" because a protein is needed (as a "facilitator" to form the channel) for transport across the membrane. (As in your texts, and on handout 4B.)

        b. Simple diffusion? Diffusion though a channel is also sometimes called "simple diffusion," because the rate of transport as a function of [X] is generally linear, as for simple diffusion, for physiological concentrations of X. (See above and handout 5A, case 2.) In other words, the kinetics of passage through a channel are linear (at physiological concentrations of X), like simple diffusion --  not hyperbolic, as in carrier mediated transport or standard enzyme catalyzed reactions.

        c. Better Terminology: Therefore, for clarity, transport through a channel is often called "channel mediated diffusion," or "facilitated diffusion through a channel."

See problem 2-6, A. Can you rule out transport through a channel? (In this problem 'facilitated diffusion' = 'carrier mediated transport.')

IV. Active Transport -- How does it work? See above for links to animations & references to pictures in texts.

    A. Examples of Models

1.  An example of primary active transport: Na⁺/K⁺ pump found in all eukaryotic cells (See handout 5B).

a. Pump/enzyme has 2 major forms: one faces in (E₁), one faces out (E₂).

b. Forms have different affinities for K⁺ and Na⁺. (See handout)

c. Role of Phosphorylation:  addition/removal of phosphate switches the pump from one form to the other.

d. Role of kinase & phosphatase activities of pump

(1). This is an example of the use of kinases & phosphatases (for addition/removal of phosphates) to regulate protein activity by reversible covalent modification.

(2). Result of enzyme action in this case:

• Kinase activity (catalyzes phosphorylation) flips pump "out" ( E₁ → E₂)

• Phosphatase activity (catalyzes removal of phosphate) flips pump "in" (E₂ → E₁)

e. Role of Ion binding: activates appropriate enzyme, kinase or phosphatase

f. Location of Enzymes: kinase and phosphatase are part of the pump itself. Not separate proteins.

g. How one cycle goes:

• E₁ → E₂: Binding of Na⁺ on inside activates kinase. Kinase phosphorylates pump. Flips pump out, dumps Na⁺, picks up K⁺.

• E₂ → E₁: Binding of K⁺ on outside activates phosphatase. Phosphatase de-phosphorylates pump. Flips pump in, dumps K⁺, picks up Na⁺.

g. Stochiometry of antiport: 3 Na⁺ out per 2 K⁺ in per ATP hydrolyzed. Some of the charge differential is balanced by anion transport. Cells are negative on inside relative to outside, but most of the charge imbalance is NOT due to the pump. (Most charge imbalance is due to K⁺ leak channels as explained above.)

h. Reversibility -- this pump is reversible in the sense that all reactions are reversible: high Na⁺ and K⁺ gradients can be used to make ATP from ADP and P_i , although this never happens in vivo (see 4 below). However it is irreversible in that ATP cannot be used to pump Na⁺ in and K⁺ out. It can only be used to pump K⁺ in and Na⁺ out. (In contrast to the situation with Na⁺/Glucose co-transport.)

2.  An example of secondary active transport: Na⁺/Glucose co-transport

a. Transporter Protein has 2 forms with different affinities for glucose. Either form can face in or out.

b. Role of Na⁺: Binding of Na⁺ switches the protein from one form to the other; alters affinity for glucose.

c. Conformational  Change: Binding of both glucose & Na⁺ probably flips protein so it faces the "other" way. One possibility:  loss of both glucose & Na⁺ does the same -- flips protein so it faces the "other" way. Another possibility: Protein is like a swinging door or 'rocking banana' -- Na⁺ can get in from either side, and glucose only binds after Na⁺.

d. Direction of glucose transport & bidirectionality: Either form (with or w/o Na⁺) can face in or out. Therefore this pump is reversible in the sense that Na⁺ can theoretically be used to pump glucose into or out of a cell. However, in normal cells, when this protein is used, glucose always goes into the cell with Na⁺. Why? (Think mechanism, not rationale.)

    B. Other Features of Active Transport (for reference)

1. ABC Transporters

a. Large super family of transporters.  Wide range of substances, not just cations, can be transported. Different proteins transport different classes of substances. All ABC transporters are similar in structure.

b. Type of primary active transport. ATP binds to a cytoplasmic protein domain called an ATP binding cassette. ATP hydrolysis drives movement of substance being transported.

c. Examples:

    (1). MDR protein = multiple drug resistance protein. Pumps many different hydrophobic drugs out of cells; causes lack of response to many cancer treatments. First eukaryotic ABC transporter to be discovered.

    (2). CFTR: Protein defective in cystic fibrosis (CFTR) belongs to same super family as ABC transporters and is similar in structure, but acts as a channel (for Cl^-), not a transporter. ATP probably opens and closes channel.  See Becker Box 8B.

    (3). Some flippases -- transporters that move certain lipids from  the P (or cytoplasmic) side to the E (or lumen) side of cell membranes.

FYI: Becker calls all transporter proteins that help move lipids across the membrane 'flippases'; other books use different terminology to distinguish various types of lipid transporters.

2. Are pumps reversible?

a. Theoretically, all pumps (like Na⁺/K⁺ pump) are reversible -- a pump can break down ATP and use the energy to drive ions up their gradient, or (if ion gradient is large enough), ions running down their gradient can provide enough free energy to drive phosphorylation of ADP to ATP. Therefore, proteins that catalyze active transport are sometimes called "ATPases" or pumps, whether their normal function is to hydrolyze ATP or to synthesize ATP.

b. Practically speaking, inside cells, most pumps are unidirectional. Most (but not all) individual "pump" proteins work only one way in cells, because the standard free energy for the "usual" direction is very negative. Therefore it takes very high concentrations of products (very high ATP or very high ion concentrations, depending on the reaction) to push the reaction in the "reverse" direction. The concentrations needed to reverse the reaction are not reached in cells, but can be achieved in test tubes (by adding ATP, setting up ion gradients, etc.). So in vitro (in test tubes), but not in vivo (in living cells), you can make the pumps run in either direction. Two examples of important pumps that are reversible (in vitro), but usually run in one direction (in vivo):

(1). In the inner membranes of mitochondria and chloroplasts, chemical or light energy is used via electron transport to set up a proton gradient, which then runs down; driving phosphorylation of ATP. So these systems almost always act to make ATP while ions run down their gradient. (Diff. proteins are used in the two organelles.)

(2). The Na⁺/K⁺ pump in the plasma membrane almost always uses up ATP --  this system drives ions up their gradients at the expense of ATP.

For more examples, see Becker table 8-3.

Now try problems 2-3 & 2-5.

Next Time: Wrap up of Small Molecule Transport. Then: Putting all the Methods of Transport of Small Molecules Together or What Good is All This? Then details of how large molecules cross membranes.  (Some specific examples -- fates of LDL, EGF, and Fe-transferrin.)