C2006/F2402 '07 OUTLINE FOR LECTURE #5  Last updated 02/01/07 02:35 PM

(c) 2007 Dr. Deborah Mowshowitz , Columbia University, New York, NY

Handouts:   5ATransport of glucose through body -- , 5B -- Models for Active Transport , 5C--Receptor Mediated Endocytosis 

I. Transport of Small Molecules, cont.

    A. Channels

1. Gating

a.  Most 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. 13-8 & 13-9 (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? Why isn't the concentration of K⁺ on both sides of the membrane the same? See below.

2. 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 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).

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

    B. Active Transport -- How does it work? See previous lecture for links to animations & references to pictures in texts.

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

f. 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: 3 Na⁺ out per 2 K⁺ in. Some of charge differential balanced by anion transport. Cells are negative on inside relative to outside, but most of charge imbalance is NOT due to 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; loss of both glucose & Na⁺ does the same -- flips protein so it faces the "other" way.

d. Direction of glucose transport & reversibility: 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, glucose always goes into the cell with Na⁺. Why?

3. 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 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.

    (2). Protein that controls type of ear wax, and presumably some other (important function), such as the amount of sweat. Recently discovered; see article from NYTimes based on article in Nature Genetics.

d. Important Related Protein: Protein defective in cystic fibrosis belongs to same super family 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.

4. 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 irreversible. 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 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.

    A. How glucose gets from lumen of intestine → muscle and adipose cells. An example of how the various types of transport are used. (Handout 5A and Becker fig. 11-22 in 5th ed; not in 6th.) Steps in the process:

1. Role of Active transport -- Needed to get glucose from lumen to inside of epithelial cell.

        a. Primary active transport -- Na⁺/K⁺ pump keeps intracellular [Na⁺] low.

        b. Secondary active transport -- Glucose enters epithelial cells by Na⁺/Glucose co-transport

2. Role of Passive Transport & Phosphorylation

a. Passive Transport -- Used  to move glucose the rest of the way -- out of epithelial cells, in & out of capillaries, and into body cells. 

b. Phosphorylation of glucose -- Used in the body cells to keep the free glucose level at the "end of the road" low, and ensure that the glucose gradient is "downhill" from epithelial cells to capillaries to body cells.

3. Role of Diffusion: Glucose and other small molecules (but not macromolecules) diffuse in and out of capillaries through the liquid filled spaces between the cells, not by diffusing across the cell membrane. Note that proteins are too big to enter or leave capillaries this way.

4. Role of GLUT transporters (another protein/gene family)

a. GLUT proteins are responsible for carrier mediated transport of glucose. All passive glucose transport across membranes depends on a family of proteins called GLUT 1, GLUT 2, etc. This family of genes and transport proteins is responsible for all carrier mediated transport of glucose. 

b. Different family members (genes and proteins) are expressed in different cell types. GLUT 1 protein is found in plasma membrane of RBC & most other cells, GLUT 2 protein on BL surface of intestinal epithelial cells, GLUT 4 protein in muscle and adipose, etc. (Note all genes for all proteins are present in all these cell types -- DNA is the same!)

c.  All the genes and corresponding proteins are similar, but have significant structural and functional differences. This is another example of a gene/protein family. All the proteins have a similar overall  structure -- 12 transmembrane segments, COOH and amino ends on intracellular side of membrane, etc. For a picture click here;  For diagram and table click here.

d. Position & Action of GLUT 4 is insulin dependent. GLUT 4 is the only insulin dependent member of the family. Insulin triggers insertion of GLUT 4 protein into the plasma membrane, by triggering vesicle fusion, as explained above. All the other proteins are located constitutively in their respective membranes. 

e. Direction of transport. Note that one member of this family (GLUT 2) is responsible for ferrying glucose OUT of epithelial cells; different members are responsible for helping glucose ENTER most other cells. All family members bind glucose on one side of the membrane, change conformation and release glucose on the other side of the membrane. Which way the glucose goes (overall) depends on the relative concentrations of glucose on the two sides of the respective membrane, not on which GLUT protein is used.

Try problem 2-9.

III. Ways that Big Molecules Enter Cells -- types of Endocytosis. In all cases net effect is that cell membrane folds in and pinches off, forming a vesicle in the cytoplasm that contains material from the outside.

    A. Pinocytosis = bulk phase endocytosis; no receptor. Cells take in random samples of surrounding fluid containing a random selection of extracellular substances. See Becker fig. 12-13. 

    B. Phagocytosis -- in specialized cells only -- extensions of cells (pseudopods) reach out and engulf solids. See Becker fig. 12-14. Vesicle that is formed is called a phagocytic vesicle (or vacuole) or phagosome. 

    C. RME = receptor mediated endocytosis. Cells take in specific substances from surrounding fluid using a receptor. See Becker fig. 12-15 (diagram) & 12-16 (micrograph). Different cell types have different combinations of receptors.

IV.  RME -- Receptor Mediated Endocytosis

        A. General and/or important Features. 

1. Receptors -- Need specific receptor for each substance (or class of closely related substances) to be transported.

2. Concentrates substances transported -- moves them up their gradient.

3. Requires energy -- multiple stages in process use ATP or GTP. Energy must be required because substances move against their gradients. Energy is required to form the vesicles and to process and/or transport the vesicles inside the cell.

4. Role of clathrin -- A peripheral membrane protein is needed to deform membrane and allow vesicles to form -- provides a coat. (See Becker figs. 12-15 to 2-18 and/or Purves 5.16.)  Other proteins are required as well, but will not be discussed.

a. Clathrin is coat protein for vesicles forming from plasma membrane and trans-Golgi. (trans side of Golgi = "far end" = side away from nucleus and ER = last part that proteins travel through as they are processed in Golgi. Also called "TGN" for "trans Golgi Network." See Purves 4.12 (5.18) or Becker fig. 12-8 for labeled pictures.)

b. Budding of other membranes involve different "coat" proteins. Best known are COPI & COPII which are involved in ER-Golgi transport. (See Becker for details if interested. Types of coats are summarized in table 12-2.) 

5. It's a cycle --  Exocytosis balances endocytosis so cell surface area stays the same. See Purves 5.15 (5.14 & 5-18) or Becker fig. 12-15. For LDL receptor, it takes about 10-20 minutes for one "round trip." 

6. Topology -- material can enter and/or exit cell without being in contact with cytoplasm. Material can remain inside a vesicle or outside cell at all times.

7. Possible fates of endocytosed material -- Where does vesicle go? Where do receptor &/or ligand end up?

a. Transcytosis --  vesicle crosses cell and fuses with opposite cell surface.

b. Degradation -- vesicle fuses with lysosomes and contents are degraded.

c. Return to surface --  material recycles -- vesicle fuses with plasma membrane.

d. Sorting -- vesicle fuses with endosomes (sorting vesicles), and different parts of the endocytosed material are directed to different destinations (a, b, or c).

    B. Stages of Cycle (Numbers match steps on handout 5C.)  Click here for animation.

(1). Receptors bind material (ligand) to be internalized

(2). Receptors are in (or migrate to) coated pits (clathrin-coated parts of membrane)

(3). Membrane starts to invaginate to form coated vesicle. A single vesicle can contain more than one type of receptor plus ligand.

(4). Coated vesicle forms (pinching off of vesicle is an energy requiring step)

(5). Uncoating occurs relatively quickly (uncoating requires energy)

(6). Vesicle is acidified to become endosome (or fuses with pre-existing endosome), and sorting of receptor(s) and ligand(s) begins.  

• A single endosome may contain many different receptors and ligands, and different ones are sorted differently. (Some examples are given in detail below.) 
• The uncoated, acidified vesicle can be called an endosome, early endosome, or a sorting vesicle.
•  Acidification requires energy to run proton pump -- to move H⁺ into vesicle at expense of ATP. Pump is in membrane of vesicle.

Note: Details of sorting and recycling -- the remaining steps -- vary with material endocytosed. More details below for individual cases.

(7). Endosome splits.  The substance we are following, and/or its receptor, can end up in either half.

In example shown on handout, one half gets the receptor and one half gets the ligand, as is the case for LDL. Other examples will be discussed in class and are outlined in detail below. 

Note: endosome may not simply split in one step; process of sorting may be gradual. Pieces of different composition may gradually bud off as internal composition of remainder changes. 

(8). What Happens to the Different Parts of the Endosome?

8A. Fate of vesicle with materials to be recycled (receptors and/or carriers) -- this vesicle fuses with plasma membrane in step 9. (In case of LDL, this vesicle would contain the receptor for LDL.)

8B. Fate of vesicle with material that remains inside the cell -- Vesicle delivers contents to appropriate cell compartment. (For LDL, vesicle delivers LDL to  lysosomes, so material is degraded.)

(9). Exocytosis occurs -- returns receptors and/or other components to the plasma membrane or outside of cell.

Next time: Wrap up of how large molecules cross membranes.  (Some specific examples -- fates of LDL, EGF, and Fe-transferrin.) How do large molecules get in and out of cells? How do newly made proteins get to the right place?