C2006/F2402 '04 Outline for Lecture #4 -- updated 01/28/04 11:13 AM

2004 Deborah Mowshowitz, Department of Biological Sciences, Columbia University, New York NY

Note: References for Purves are given first for the 6th edition and then in ( ) for the 5th edition if different. 

Handouts: 4A -- Transport from Lumen of Intestine  4B -- Models for Active Transport   4C. Types of Transport

I.  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 Purves 40.2)

a. Muscle -- specialized for contraction. 

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

c. Connective -- cells dispersed in an extracellular 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 or Becker 11-1.

d. Epithelial  -- 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. Usual functions: selective absorption (transport), protection, secretion.

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

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?

Now try problems 1-12 to 1-14. By now you should be able to do all the problems in problem set #1.

II. Types of Transport Across Membranes (of small molecules/ions). For an overall summary, see bottom of handout 3B.  For reference, types of transport are numbered 1-5 on handout 3B & chart below. Also see Becker, fig. 8-2.

    A. Basic Types of transport -- classified by type of protein (or none) involved (See handout 3B)

 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 for comparison of the 2 kinds of act. transport.

(1). Primary 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

(2). Secondary 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 5.9.)

Type of Transport

What Moves

Example(s)

 uniport 

one substance moves

Carrier mediated transport of Glucose

 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; anion exchanger

    C. Summary Table:

Type of Transport

Type of Protein

Direction X Moves

Source of Energy to transport X

Example(s)

Name

Function

1

Simple Diffusion

None

Down its gradient

Gradient of X

Restricted to very small molecules and hydrophobics

How glucose enters capillaries

2

Channel

Transmembrane   channel

down its
gradient

gradient of X

water channels in kidney & RBC; many types of ion channels

control vol. of urine & RBC; flux of ions 

3

Facilitated ** Diffusion or carrier mediated transport

Carrier or Permease

down its
gradient

gradient of X

Glucose transporter in many plasma  membranes

How Glucose exits epithelial cells to body; enters adipose tissue

RBC anion Exchanger*

Maximizes CO2 transport by blood

4

Primary Active Transport**

Pump

up its gradient

ATP

Na+/K+ pump

Maintain high [K+], low [Na+] in cells

5

Secondary Active Transport

Pump or co-transporter

up its gradient

Not ATP (directly): usually a gradient of some substance other than X

Glucose/Na+ Co-transport

How Glucose enters epithelial cells from lumen

 

* In some older books band 3 protein is called an anion channel. It is now clear it transports ions but is not a channel.

**Note: These links are to animations of transport done by Steve Berg at Winona State University. His web site has many nice animations of cell and molecular processes.

III. How transport is measured (Example = in RBC ghosts) -- Two types of curves. Handout 4C. 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 5.10 in 5th ed. 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)?

IV. 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 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)

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: 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.

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

(2). dephosphorylation -- removal of phosphate groups --  catalyzed by phosphatases.

c. removal/insertion of carrier into membranes. Newly made transporter or pore protein is inserted into the membrane of a vesicle, by a mechanism to be discussed later. Fusion of the vesicle with the plasma membrane inserts transport protein into plasma membrane where it can promote transport. Budding of a vesicle back into the cytoplasm removes the transport protein and stops transport. Insertion/removal of transport protein by fusion/budding of vesicle can be reversible. An example of this is given below in topic IV.

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

    C. Channels (Case #2)

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 44.6 (41.6)  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 is not entirely understood, but is a current hot topic of research.)

4. Terminology. Diffusion through a channel is sometimes 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 44.15 (41.14).

(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?.

    D. 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. Enzyme-like protein involved -- acts as transporter or pump.

3. Energy: Not readily reversible; Keq not = 1 and standard delta G not = zero. Overall reaction usually has large, negative standard delta G because in overall reaction transport of X (uphill, against the gradient) is coupled to a very downhill reaction, as shown in II. B. above. 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).

4.  Secondary 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 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 (see handout 4B,  texts & animations for models). Click on links for animations.

Example

Type of Active Transport

Type of "Port"

Pictures in Becker 

Pictures in Purves

a.

Na+/K+ pump  

Primary

Antiport

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

5.12

b.

Na+/Glucose co-transport 

Secondary

Symport

fig. 8-13 (8-12)

5.13 

7. 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 delta G 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 irreversible. Most (but not all) individual "pump" proteins work only one way in cells, because the standard delta G 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. 

(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.

When we finish the mechanisms of active transport, try problems 2-3 & 2-5.

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