C2006/F2402 '05 Outline for Lecture #4 -- updated 01/27/05 09:14 AM

2005 Deborah Mowshowitz, Department of Biological Sciences, Columbia University, New York NY

Note: References for Purves are given first for the 7th edition and then in ( ) for the 6th edition if different. For Becker, refs. in ( ) are for the 4th ed.  For older editions see older schedules.

Handouts: 4B -- Comparison of Types of Transport   4A. Kinetics of Types of Transport

I. Cell-Cell (& Cell-ECM) Connective Structures ;

IV. Cell-Cell (& Cell-ECM) Connective Structures  -- For Pictures see Purves 5.6, & Becker Ch. 11 (exact figures listed below).  For comparisons, see table below and/or Becker table 11-3.

    A. Cell-Cell Junctions

        1. Gap junctions -- Becker fig. 11-23 (11-20); Purves  5.6 & 15.16 (15.18). 

        2. Plasmodesmata -- Purves 15.17 (15.19) or Becker 11-28 (11-25). The plant's equivalent of a gap junction. 

        3. Adhesive (anchoring) junctions -- Spot (with IF) = desmosomes vs belt (with MF) = adherens junctions. Becker fig. 11-19 (11-15) & 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).

        4. Tight Junctions -- Becker fig. 11-20 (11-17)

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.  Resemble half of an adhesive junction (adherens junction or desmosome).(Becker Fig. 11-12. I think diagrams are not quite right but pictures are good.) 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. Plaque (thickening) forms on inside of cell near junction

b. Plaque connects transmembrane protein to IF or MF on inside of cell.

2. Differences from cell-cell adhesive junctions

a. Transmembrane protein is an integrin, not a cadherin

b. Transmembrane protein connects to ECM, or to solid support cell is growing on,  instead of to 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 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.  What does a real cell look like? Where are the junctions, etc.?  -- Becker Fig. 11-18 (11-14) or handout 3A, top.

    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 Purves 41.2 (40.2))

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

b. Nervous -- individual cell is called a neuron. Specialized for conduction of messages.

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

d. Epithelial -- see handout 3A -- example of cells with many types of junctions

1. Cells tightly joined 

2. Make up linings of external and internal surfaces

3. Usually sheets. Can have one or more layers

4. Often rest on noncellular support material = basal lamina = part of ECM secreted by cells

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

6. Usual functions: selective absorption (selective transport across sheet), protection, secretion (from glands)

7. An example: epithelial layer surrounding the gut. See handout 3A and Becker fig. 11-18 (11-14) or Purves 41. 3 (40.3) .

This leads to the next topic: How does the intestinal epithelium (& various types of junctions) function in transport?

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 (& various types of junctions) function in transport? How do substances get across an epithelial cell layer?

III. Types of Transport Across Membranes (of small molecules/ions). For an overall summary, see table below and handout 4B.  For reference, types of transport are numbered 1-5 on handouts & chart below.  Also see Becker, fig. 8-2 or Purves 5.1. (Terminology differs somewhat between texts -- see note at bottom of handout 4A. However what is meant by cases 1-5 is the same for all handouts and texts.)

    A. Basic Types of transport -- classified by type of protein (or none) involved (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 -- 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. (Passage through a channel may be referred to as diffusion,  facilitated diffusion, or neither, depending on the text.) 

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   

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, through a channel, or with help of carrier protein. Cases 1- 3. 

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 act. 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 , where [X] in  exceeds  [X] out

(b). ATP + H2O --> ADP + Pi

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 , as above

(b). [Y] high ---> [Y] low

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

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)

    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 animations of cell and molecular processes.

Try problem 2-2.

IV. 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), and looking at the yield -- what is the final value of [Xin]?  What is the value  of [Xin] when curve #1 plateaus?

2. In Curve #2, you are looking at the the rate of uptake (flux) for different starting concentrations of Xout. What is the slope of curve #1 (for different  starting concentrations of Xout)?

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

    A. Simple Diffusion (Case 1)

1. Curve #1 (uptake or concentration of substance X inside plotted vs. time) plateaus at [X]in = [X]out.

2. Curve #2 (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 = 0; at equil. [X]in = [X]out).
Actual free energy change 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 & b are common to many proteins and are only listed here for comparison (details elsewhere). Method c is unique to transmembrane proteins. (This section is about regulation of activity of pre-existing levels of protein. Synthesis and therefore protein levels are also regulated, as will be explained 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. Channels (Case #2) -- whatever is not covered today will be done in the next lecture.

1. Curve #1 -- Same as above except

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

b. Channels often conduct ions. This has consequences. Curve #1 plateaus as above with  [X]in = [X]out  only if X is neutral or there is no electric potential -- see point 4 below. 

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 -- see Purves 5.10. This is a current hot topic of research.)

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. 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. 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 the best term for transport through a channel is "channel mediated diffusion."

5. Gating

a.  Some Channels are gated = % time any particular 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 Purves 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. 9-9 & 9-10.

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

b. 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? See below.

7. 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). These can both "push" ions the same way or in opposite directions.

b. Result of charge: Keq not usually 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).

See problem 2-6, A. Can you rule out transport through a channel?

Next Time: Remaining cases of transport, and an example of how the various types of transport are used. How glucose gets from lumen of intestine --> muscle and adipose cells.