C2006/F2402 '06 Outline for Lecture #4-- updated 01/25/06 11:27 AM
2006 Deborah Mowshowitz, Department of Biological Sciences,
Columbia University, New York NY
Handouts: 4B -- Comparison of Types of Transport 4A. Kinetics of Types of Transport
I. Epithelial cell structure, cont. A few features not explained last time:
A. Role of tight junctions in maintaining separate (apical and basolateral) domains of plasma membrane
B. Location of MF in adherens junction -- MF go around cell in a band -- see Becker, fig. 17-17 (11-18).
This leads to the next topic: How does the intestinal epithelium function in transport? How do substances get across an epithelial cell layer?
II. Types of Transport Across Membranes (of small molecules/ions). For an overall summary, see table on handout 4B. For reference, types of transport are numbered 1-5 on handouts & below. Also see Becker, fig. 8-2 or Purves 5.1.
A. Role of Protein -- Basic Types of transport classified by role of transport protein (if any) (See handout 4B)
1. No protein involved -- Simple Diffusion (case 1). Effective only for hydrophobic molecules (such as steroid hormones), gases, and very small molecules that can diffuse across lipid bilayers. See Becker table 8-1 & figure 8-5.
2. Protein involved -- transport protein is a channel, permease (carrier or exchanger) or pump. Cases 2-5.
a. channel (case 2) -- protein forms a pore allowing passage of hydrophilic materials across the lipid bilayer.
b. transporter -- permease, carrier or pump -- protein binds to substance(s) on one side of bilayer, protein changes conformation and releases substance on other side of bilayer. Cases 3-5.
B. Other ways of classifying transport -- by (1) how energy is supplied and (2) by direction.
1. Active vs. passive -- whether substances flow down their gradients (passive transport -- cases 1-3) or are pushed up their gradients by using energy (active transport -- cases 4 & 5). See Becker table 8-2 .
a. Passive transport -- substance moves down its concentration gradient. This can be by simple diffusion, passage through a channel, or with the help of carrier protein. Cases 1- 3.
X out → X in ΔG (high concentration) (low concentration) <0
b. Active transport -- substance moves up its concentration gradient (as in reaction (a) below) with the help of "pump" protein and expenditure of energy (one of the reactions labeled (b) below). See Becker fig. 8-9 or Purves 5.14 (5.13) for comparison of the 2 kinds of active transport.
(1). Primary (or direct) active transport (Case 4) -- energy for transport is supplied by hydrolysis of ATP. In other words, the following two reactions are coupled:
(a). X out → X in ΔG (low concentration) (high concentration) >0 (b). ATP + H2O → ADP + Pi. <<0 Net: X out + ATP + H2O → X in + ADP + Pi <0
Examples: pumps that move Ca++ into the ER or H+ into vesicles such as endosomes or lysosomes.
(2). Secondary (or indirect) active transport (Case 5) -- energy is supplied by some 2nd substance running down ITS gradient (reaction (b) below). The following two reactions are coupled:
(a). X out → X in ΔG (low concentration) (high concentration) >0 (b). [Y] high → [Y] low <<0 Net: X out + [Y] high → X in + [Y] low <0
Where is the high concentration of Y? Which way does Y move? In some cases, Y moves in the same direction as X (symport); in other case it moves in the opposite direction (antiport). See table below.
An example: Glucose/Na+ co-transport -- glucose is pushed up its gradient by energy derived from Na+ going down its gradient. X = glucose; Y = Na+.
Important Note: ATP may have been used to establish the gradient of [Y], but ATP is not directly involved here. For example: The Na+/K+ pump can be used to establish a Na+ gradient. (This is primary active transport, and uses ATP.) Once a Na+ gradient exists, the Na+ running down its gradient provides the energy to move glucose. (This is secondary active transport, and does not require ATP.)
2. Direction things move -- See Becker fig. 8-7 or Purves 5.12 (5.11.)
|Type of Transport||
|One substance moves.||
Carrier mediated transport of Glucose; Ca++ transport into ER
|Two or more substances move in same direction.||
|Two or more substances move in opposite directions.||
Na+/K+ pump (active
-- driven by hydrolysis of ATP);
* An exchanger can be considered passive transport (facilitated diffusion), since the concentrations of the substances themselves drive the reaction . Alternatively, it can be considered secondary active transport, because movement of one of the substances down its gradient can drive transport of the other substance up its gradient.
C. Summary Table -- See Handout 4B. For animations of transport done by Steve Berg at Winona State University go to Facilitated Diffusion Primary Active Transport. Berg's web site has many nice pictures & animations of cell and molecular processes.
Try problem 2-2.
III. How transport is measured (Example = in RBC ghosts) -- Two types of curves. Handout 4A. How do you characterize transport?
A. Curve # 1: Measure uptake of X with time 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, [Xin] at equilibrium will exceed [Xout].
2. For passive transport of neutral molecules, [Xin] at equilibrium will equal [Xout].
(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?
B. Curve #2: Measure initial rate of uptake of X (from curve #1) at varying concentrations of added (outside) X; plot rate of uptake vs. concentration. See handout or Purves 5.11 (5.10 in 5th ed.; not in 6th) 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 Vmax 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.)
C. For both curves, you are considering the reaction Xout ---> Xin. So what's the difference?
1. In Curve #1, you are looking at how the concentration of Xin varies with time (starting with a fixed concentration of Xout).
a. (Initial) Slope of the curve = rate of uptake (with time as the variable).
b. Plateau value = yield = final value of [Xin] when curve #1 levels off.
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 Xout.
IV. Kinetics and Properties of each type of Transport -- How you tell the cases apart.
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: Rxn ( X in <--> X out) is strictly reversible. (Keq = 1; Standard free energy change (ΔGo) = 0; at equil. [X]in = [X]out).
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)
1. Curve #1 same as above.
2. Curve #2 saturates. See Becker fig. 8-6, or Purves 5.11 (5.10 in 5th ed.; not in 6th)
3. Mechanism: Carrier acts like enzyme or permease, with Vmax, Km etc. See Becker fig. 8-8.
4. Energy as above -- substance flows down its gradient, so transport is reversible, depending on relative concentrations in and out.
5. Regulation: Activity of transport proteins can be regulated at least 3 ways -- methods a-c below. Methods a & b are common to many proteins and are only listed here for comparison (details elsewhere). Method c is unique to transmembrane proteins.
Note: 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.
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 + Pi
P (bold) = phosphate group; Pi = inorganic phosphate (in solution)
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 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. (An example 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. 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 Xout --> X in).
2. Curve #2 saturates as in previous case.
3. Mechanism -- An enzyme-like transport protein is involved as in previous case. However 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.
4. Energy: Not readily reversible; Keq not = 1 and standard free energy (ΔGo) not = zero. Overall reaction usually has large, negative ΔGo because in overall reaction, transport of X (uphill, against the gradient) is coupled to a very downhill reaction. The downhill reaction is either
a. Splitting of ATP (in primary active transport), or
b. 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.
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).
5. How do you tell the two types of active transport apart? Primary is directly dependent on splitting of ATP; secondary will continue even in the absence of ATP until the gradient of Y runs down.
Try problem 2-2.
6. Some Examples & Possible mechanisms (models will be discussed next time). Click on links for animations.
Type of Active Transport
Type of "Port"
Pictures in Becker
Pictures in Purves
figs. 8-11 & 8-12 (8-10 & 8-11)
fig. 8-13 (8-12)
D. Channels (Case #2)
1. Curve #1 -- Same as cases #1 & #3 above -- with the following important exceptions:
a. Very high rate of transport -- Initial slope of Curve #1 very steep.
b. Channels often conduct ions. This has consequences. Curve #1 plateaus with [X]in = [X]out only if X is neutral or there is no electric potential -- this will be discussed next time.
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. Mechanism. Lack of saturation and high rate of transport indicate that max. capacity of channel is very large and is not easily reached. This is explained by one or both of the following:
a. Binding of ion to channel protein is weak (Km >> 1), and/or
b. No major conformational change of channel protein is required for ion to pass through.
See Purves 44.5 (44.6) for comparison of ion pumps and ion channels; Becker p. 203 (209) for comparison of carrier and channel proteins. Note that channels are very specific in spite of features a & b -- each channel transports only one or a very small # of related substances.
Mechanism of specificity has been recently figured out for one channel. For pictures see Purves 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.
4. Terminology. Diffusion through a channel is usually called "facilitated diffusion" because a protein is needed (to form the channel) for transport across the membrane. (As in your texts, and on handout 4B.) However, 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, as explained in point #2 above. (See handout 4A, 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. Perhaps transport through a channel should be called "channel mediated diffusion," or "facilitated diffusion through a channel."
See problem 2-6, A. Can you rule out transport through a channel?
Next Time: Wrap up of transport -- we'll do whatever features described above we don't get to, some more aspects of channels & active transport, and an example of how the various types of transport are used. How glucose gets from lumen of intestine --> muscle and adipose cells. Then, how do big molecules get into cells?