C2006/F2402 '08 Outline for Lecture #4 -- updated 01/30/08 02:05 PM

2008 Deborah Mowshowitz, Department of Biological Sciences, Columbia University, New York NY

Handouts: 4B -- Comparison of Types of Transport   4A. Kinetics of Types of Transport 4C. Epithelial Cells (see Becker fig. 17-17)

If you haven't seen it yet, you should try out the cell animation video. It's at several places on the web. A shorter version of this video (set to music) is available on many web sites including http://www.youtube.com/watch?v=CVUnzk40npw and http://multimedia.mcb.harvard.edu/media.html. The Harvard web site also has a longer version with narration instead of the music, and several other nice videos.

I. Cell-Cell (& Cell-ECM) Connective Structures, cont.  -- For Pictures see Sadava 5.7 (5.6), & Becker Ch. 17 (11) -- exact figures listed below.  For comparisons, see table below and/or Becker table 17-3 (11-3). For diagrams, see handout 4C or 3A. (The same diagrams are on both handouts. They are repeated on 4C for note taking convenience).

    A. Cell-Cell Junctions.  For the molecular details (FYI only) see fig. 17-12 (11-13).

        1. Gap junctions -- Becker fig. 17-21 (11-23); Sadava  5.7 (5.6) & 15.19 (15.16). 

        2. FYI: Plasmodesmata -- Sadava 15.20 (15.17) or Becker 17-25 (11-28). The plant's equivalent of a gap junction. 

        3. Tight Junctions -- Becker fig. 17-19 (11-20)    

      4. Adhesive (anchoring) junctions -- Spot (with IF) = desmosomes vs belt (with MF) = adherens junctions. Becker fig. 17-18 (11-19) & 17-13 (11-14) for molecular details (for your interest only). For a nice EM picture see http://trc.ucdavis.edu/mjguinan/apc100/modules/Integument/_index.html (click on generic desmosome). For diagram of adherens junction see picture from Alberts. Also has nice pictures of desmosomes, gap junctions, etc.

Summary Table of Animal Cell-Cell Junctions:

Name of Junction

Important Structural Features

Function

Gap Junction

Connexons;  small gap between cells (2-4 nm)

Passage of small molecules and ions (signaling & nutrition)

Tight Junction

Fusion of ridges of membrane -- no gap at ridge.

Water Tight Seal between cells; divide membrane into regions

Adhesive or Anchoring
Junctions

Intracellular Plaques with filaments. Classified by:
    (a) Shape of plaque: spot vs belt
    (b) Type of filaments:
        IF (desmosomes) vs MF (adherens junctions)
Cell/cell protein connectors = cadherins
Larger gap between cells (25-35 nm)

Strength

  B. Cell - ECM junctions.  Connect cytoskeleton to ECM or solid support. 

These junctions resemble half of an adhesive junction (adherens junction or desmosome). See handout 4C (or 3A). Also Becker Fig. 17-11 (11-12). Diagrams in 5th ed. are not quite right but pictures are good. Diagrams look ok in 6th. For a better diagram see http://celljunctions.med.nyu.edu/images/figure1.gif. For a nice EM picture see http://trc.ucdavis.edu/mjguinan/apc100/modules/Integument/skin/hemidesmosome/hemidesmosome2.html

1. Similarities to cell -cell adhesive junctions

a. Transmembrane protein connected by linker proteins to IF or MF on inside of cell.

b. Plaque (thickening) often forms on inside of cell near junction (especially in hemidesmosomes) -- contains some of linker proteins

2. Differences from cell-cell adhesive junctions

a. Transmembrane protein is an integrin, not a cadherin (See Becker fig. 17-10 (11-11) for a diagram of structure of integrin.)

b. Transmembrane protein connects to ECM. (Not to extracellular domain of protein from another cell. )

3. Two Types.

a. Hemidesmosomes  = "half desmosome";  connect to IF on inside of cell. Connect to basal lamina on outside.

b. Focal Adhesions  = "half adherens junction" --  connect to MF on inside of cell and ECM (can be on a solid support) on outside.

At this point, it is a good idea to make a chart for yourself that classifies (or compares and contrasts) all the types of junctions, whether cell-cell or cell-ECM. Also try problems 1-12 & 1-14.


II. Types of Specialized Cells & an Example
 What does a real cell look like? Where are the junctions, etc.? 

    A. Cells & Tissues

a. Specialization. All cells in multicellular organism are specialized; there is no "typical cell."

b. Types. About 200 different cell types per human.

c. Tissue = Group of cells with similar structure & function that work as a unit.

d. 4 Major cell/tissue types -- muscle, nerve, connective, epithelial

e. Terminology Note: "tissue" is also used in a nonspecific way to mean a group of cells derived from an organ or system as in "kidney tissue." A kidney is an organ made up of many different tissue types.

    B. The Four major Tissue Types  (See Sadava 40.7(41.2 ))

a. Muscle -- three kinds (skeletal, smooth & cardiac muscle); all specialized for contraction. (Sadava 40.4)

b. Nervous -- individual cell is called a neuron. Specialized for conduction of messages. (Sadava 40.6)

c. Connective -- cells dispersed in a matrix. (Sadava 40.5.) Extracellular matrix can be solid (as in bone), liquid (as in blood) or semi-solid (gel like) as in cartilage, adipose tissue. (Note fat in adipose tissue is stored inside the adipose cells in vesicles, not between the cells in the matrix.) See Sadava 40.4 in 6th ed. Connective tissue surrounds other tissues and provides support, protection and transport of materials to and from tissues (in blood).

d. Epithelial -- for an example see handout 4C. For a similar diagram, see Becker fig. 17-17 (11-18). For a picture, see Sadava 40.3 (41. 3).

1. Cells tightly joined -- use junctions described above to connect cells to each other and/or to ECM.

2. Make up linings of external and internal surfaces

3. Usually sheets. Can have one or more layers

4. Cells are Polarized -- two sides of cell layer are different. Contain different proteins and/or lipids in different domains (areas) of membrane.

(a). Terminology: Side of cell membrane facing lumen = apical surface; side facing body = basolateral (BL) surface.

(b). What keeps the two domains separate? Tight junctions.

5. Often rest on noncellular support material = basal lamina = part of ECM secreted by cells (on BL side)

6. Can form glands = epithelial tissues modified for secretion. (More details on formation, structure & function of glands later in course.)

7. Usual functions: selective absorption (selective transport across sheet), protection, secretion (from glands). See below for more on transport.

8. An example: epithelial layer surrounding the gut. See handout 4C or Becker Fig. 17-17 (11-18)

Now try problem 1-13. By now you should be able to do all the problems in problem set #1.


This leads to the next topic: How does the intestinal epithelium function in transport? How are substances are transported across membranes?


III. 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 Sadava  table 5.1.

    A. Classification of Transport -- three basic criteria for classifying transport

        1. Role of transport protein (if any)

        2. How energy is supplied

        3. Direction things move

    B. 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. If Protein involved -- transport protein can be 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.

    C. Other 2 ways of classifying transport  -- by how energy is supplied and 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 a carrier protein. Cases 1- 3. 

  [X] high, out [X] low, in ΔG <0
  (high concentration outside)   (low concentration inside)  

Note: We are arbitrarily calling the two sides of the membrane 'in' and 'out'. Transport can be from inside to outside or vice versa. However it always goes from high concentration to low concentration. In most of the examples discussed below, we are looking at transport into a cell (or vesicle), but transport out of cells is just as important.

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 Sadava  5.15 (5.14)  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] low, out [X] high, in ΔG >0
  (low concentration outside)   (high concentration inside)  
         
(b). ATP + H2O ADP + Pi ΔG <<0
         
Net: X out + ATP + H2O X in + ADP + Pi  ΔG <0
  (X moves up its gradient at expense of ATP)  

Examples: pumps that move Ca++ into the ER or H+ into vesicles such as lysosomes. (The ER stores Ca++. Lysosomal enzymes work best at acid pH, unlike most other enzymes that work best at neutral pH.)

        (2). Secondary (or indirect) active transport (Case 5) -- energy for transport is supplied by some 2nd substance running down ITS gradient (reaction (b) below). The following two reactions are coupled:

(a). [X] low, out [X] high, in ΔG >0
  (low concentration outside   (high concentration inside)  
         
(b). [Y] high
on one side of membrane
[Y] low
on other side of membrane
ΔG  <<0
         
Net: X out + [Y] high X in + [Y] low ΔG  <0
  (X moves up its gradient while Y flows down its gradient)  

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 (details below or next time) 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 Sadava 5.12 (5.11.)

Type of Transport

What Moves

Example(s)

 Uniport 

One substance moves.

Carrier mediated transport of Glucose; Ca++ transport into ER

 Symport

Two or more substances move in same direction.

Glucose/Na+ co-transport

Antiport

Two or more substances move in opposite directions.

Na+/K+ pump (active -- driven by hydrolysis of ATP);
Anion exchanger  (passive -- driven by concentrations of Cl- & bicarb)*

* An exchanger can be considered passive transport (carrier mediated), 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.

IV. How transport is measured  

A. Need a suitable experimental set up. A common method: using RBC ghosts. 

B. Measurements of Uptake Generate Two Kinds of Curves --  See handout 4A

C. 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?

D. 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 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  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. These saturating levels are not usually reached in practice.

E.  Curve #1 vs Curve #2.  For both curves, you are considering the reaction Xout → Xin. So what's the big 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 [X]in 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.  (Same idea as a V vs S curve for an enzyme.)

V. 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). 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 5.12 (5.11)

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

        Carrier can be considered an enzyme that catalyzes:

Xout     Xin

        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 transport proteins 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 (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. Examples --

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

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

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 Xout X in).

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).

Q: How do you tell the two types of active transport apart?

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 will be discussed next time). Click on links for animations.

Example

Type of Active Transport

Type of "Port"

Pictures in Becker 

Pictures in Sadava

a.

Na+/K+ pump  

Primary

Antiport

figs. 8-11 & 8-12 (8-10 & 8-11)

5.14 (5.13)

b.

Na+/Glucose co-transport 

Secondary

Symport

fig. 8-13 (8-12)

5.15 (5.14) 

  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. Important Features:

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.

3. Mechanism.   The problem is how to reconcile the two important features listed above -- the combination of high speed (& capacity) and high specificity.

See Sadava 44.6 for comparison of ion pumps and ion channels;  Becker p. 203 (209)  for comparison of carrier and channel proteins. 

Mechanism of specificity has been recently figured out for one channel. For pictures see Sadava 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.

4. Terminology. 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.) 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?