Outline for Lecture #5

C2006/F2402 '14 OUTLINE FOR LECTURE #5 Last updated 02/03/14 05:49 PM

(c) 2014 Dr. Deborah Mowshowitz , Columbia University, New York, NY

Handouts: 5A-- Measurement & Classification of Transport* 5B -- Models for Active Transport

*Handout 5A is in the CW handout folder -- accessible to registered students via the left menu on the Courseworks (CW) web site. All handouts with copyrighted material are in this folder. (Extra paper copies available after lecture in Boxes on 7th floor of Mudd.)

I. Review of Classification of types of transport. See handout 4C, Sadava table 6.1, or Becker table 8-1.

Note: The terms used for the various types of transport are variable, and you will see different terms used in different sources. The features of each type (1-5 on handout 4C) are more important than the names.

II. How is Transport of Small Molecules Measured?

A. Need a suitable experimental set up. A common method: using RBC ghosts. How is it done?

Put resealed ghosts in solution with some concentration of X on the outside and none on the inside.

At appropriate time intervals, you take a sample, centrifuge out the ghosts, and measure the amount of X in the ghosts.

You repeat with different starting concentrations of [X]out

B. What do you learn from doing this?

You can tell active transport from passive.

You can tell if a carrier or pump protein is required for transport (as vs. none or a channel).

You can measure the properties of the transport protein (if any) -- equivalents of Km and Vmax..

C. Details of experimental set up -- See handout 5A.

Put ghosts in solution with some concentration of X inside and outside:

Co = concentration outside = [X]out ( [X]out = some fixed value to start)

Ci = concentration inside = [X]in ( usually [X]in = 0 to start)

You measure Ci as a function of time. This generates 'curve #1' -- the top set of three curves on 5A.

You repeat with different starting values of Co. This generates the data plotted in 'curve #2' -- the second set of three curves on 5A.

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D. How to check your understanding? Be sure you can answer the following: In terms of types of transport, which cases on Handout 4C does this procedure allow you to tell apart? Which set of curves on handout 5A will give you the information you need? How do you get the 'Km and Vmax'? (Details of each case & meaning of each curve are discussed in detail below.) III. What do Results of Measurement Look Like? What do they mean?

A. Curve # 1 -- Uptake of X vs time: Measure [X]in at increasing times at some starting, outside concentration of X; plot conc. of X inside vs. time. Curve always levels off -- but at what value? This allows you to distinguish active and passive transport.

1. For active transport of neutral molecules, at equilibrium, [X]in will exceed [X]out.

2. For passive transport of neutral molecules, at equilibrium, [X]in will equal [X]out.

Notes: (1) If X is charged, the situation is more complicated, as explained below.

(2) Concentration of X outside is essentially fixed and is the same as the starting concentration outside (Co). This is because the amount taken up is relatively small. Why? Because the volume inside the cells is much smaller than the volume outside the cells.

Questions: If you measure carrier-mediated uptake a second time, using a higher starting concentration of X, what will happen? (1) Will the slope of curve #1 (flux) be the same? (2) Will the curve level off at the same value?

B. 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 (flux) vs. initial concentration of [X]out. See handout or Becker fig. 8-5 (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.

C. Summary Comparison of Curve #1 vs Curve #2. For both curves, you are considering the reaction Xout ↔ Xin. So what's the big difference? (Most of this should be discussed as we go along, but is summarized here for reference.)

1. In Curve #1, you are looking at how the concentration of Xin varies with time (starting with a fixed concentration of Xout, and no X inside.).

a. Curve shows uptake as a function of time.

b. (Initial) Slope of the curve = rate of uptake (with time as the variable) |

c. Plateau value = yield = final value of [X]in when curve #1 levels off (when rate in = rate

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out).

d. Note that curve #1 ALWAYS levels off.

e. Same idea as plotting P formed (or S used up) vs time for an enzyme catalyzed reaction.

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 [X]out.

a. Curve shows uptake as a function of concentration of X added (outside).

b. (Initial) Slope of the curve = rate of change of uptake (with [X]out as the variable) = flux

c. This curve levels off * only if a protein must bind to X and/or change conformation significantly in order to move X.

d. Same idea as a plotting V vs S for an enzyme-catalyzed reaction. Gives you the properties of the transport protein.

*at physiological values of [X]out

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

All the cases below refer to the reaction [X]in ↔ [X]out. All the important features are summarized in the table on handout 5A.

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:

a. Reversibility: Rxn ( X in ↔ X out) is strictly reversible.

o b. Keq = 1; Standard free energy change (ΔG ) = 0; at equil. [X]in = [X]out

c. ΔG. 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.

Diffusion across a membrane: Used by steroid hormones, some small molecules, gases. Only things that are very small or nonpolar can use this mechanism to cross membranes.

Diffusion through liquid (no membrane involved): Materials -- usually small molecules -- can diffuse in or out of 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.) More on this next time.

B. Carrier mediated Transport = Facilitated Diffusion using a carrier protein (Case 3). Note we are deferring case 2.

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1. Curve #1 same as above (case 1)

2. Curve #2 saturates. See Becker fig. 8-5 (8-6), or Sadava fig. 6.12 (6.14)

3. Mechanism: Carrier acts like enzyme or permease, with Vmax, Km etc.

Carrier can be considered an enzyme (permease) 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. Example -- GLUT family of proteins. Transport of glucose across a membrane (down its gradient) requires a GLUT (glucose transporter/carrier) protein. For mech. of action, see Becker fig. 8-7 (8-8).

a. Role of GLUTs: Glucose enters and/or exits most cell using a GLUT protein.

b. Different cell types make different GLUT proteins. Proteins are called GLUT1, GLUT2, GLUT4 etc.

(1). RBC contain GLUT1 (See Becker fig. 8-2)

(2). Liver cells contain GLUT2

(3). Muscle and adipose tissue contain GLUT4

c. Direction: Which way the glucose goes, in or out of the cell, depends on the relative concentrations of glucose on the two sides of the membrane, not on the GLUT protein present. An analogy: a revolving door. Some molecular examples:

(1). GLUT1 & GLUT4 transport glucose into their respective cells (in vivo).

(2). GLUT2 in liver cells can transport glucose in or out (in vivo), depending on the level of glucose in the blood.

d. Regulation: Most GLUT proteins are permanently inserted into their target membrane, but GLUT4 is not. See below.

e. Origin/Evolution: More details on the GLUT family of proteins next time.

6. Regulation: Activity of a transport protein (or any transmembrane protein) 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 (more 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.

Kinases catalyze: X + ATP → X-P + ADP

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

Phosphatases catalyze: X-P + H2O → X + Pi

P (bold) = phosphate group attached to molecule; Pi = inorganic phosphate group (in solution)

(3). Reversibility. Reactions carried out by phosphatases and kinases are generally irreversible. However the covalent modifications of the target proteins are reversible -- by using both types of enzymes.

(a) Each reaction, (1) or (2) above has a large negative ΔGo. Therefore each individual reaction is irreversible (practically speaking).

(b) Each reaction 'undoes' the effects of the other -- therefore the modifications and their effects (activation or inactivation of target) ARE reversible -- by using the 'other' enzyme.

c. Removal/insertion of carrier into membranes.

(1). Insertion during synthesis. Newly made membrane proteins are inserted into the membrane of a vesicle, by a mechanism to be discussed later.

(2). Insertion/removal Process. Vesicle can fuse with plasma membrane; process is reversible.

(a). Fusion of the vesicle with the plasma membrane (exocytosis) 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). Regulation by insertion/removal. 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 is the signal for insertion of the transporter into the plasma membrane of some cells, allowing increased glucose uptake. Details next time.

(b). Water channels (aquaporins) in kidney cells. The hormone ADH (anti- diuretic hormone) is the signal to promote insertion of the channels into the plasma membrane of the cells lining the kidney tubule. The location of the channels determines whether water is retained in the body fluids or excreted in the urine. (Most membranes have some permeability to water, but transport of large amounts of water requires aquaporins.) More details when we get to kidney. See Becker, p. 207-208 (206).

Notes: (1). 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.

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(2). Regulation by method c (removal/insertion) is known to occur in some examples of transport in cases 2 & 3. Could apply to cases 4 & 5, but I don't know of any examples.

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.

Note: This assumes we are following the reaction Xout → X in. The reaction could be written more generally, as Xside 1 → X side 2 . In that case, when the curve plateaus, what will be greater -- [X]in or [X]out?

3. Mechanism -- An enzyme-like transport protein is involved as in previous case.

a. X moves up its gradient. 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.

b. Most primary active transport involves movement of cations. (But see ABC transporters below.) Gradient of cations can then be used to do work, such as secondary active transport, or to propagate a signal (as in nerve).

4. Energy relationships for pumps:

a. Pumps: Reaction is not readily reversible. Although all reactions are micro-reversible, active transporters called pumps virtually always transport X in the same direction in living cells (which is either in or out for any particular pump).

o b. Keq not = 1 and standard free energy (ΔG ) not = zero. At equil. [X]in is not equal to [X]out

c. Coupling: Overall reaction usually has large, negative ΔGo (& ΔG) because in overall reaction, transport of X (uphill, against the gradient) is coupled to a very downhill reaction. The downhill reaction is either

(1). Splitting of ATP (in primary active transport), or

(2). Running of some ion (say Y) down its gradient (in secondary active transport).

5. Energy Relationships for Exchangers:

Exchangers (antiporters that exchange X and Y) are somewhat different from pumps. There are two coupled recactions as above, but exchangers may transport X in different directions in living cells, depending on the ΔG determined by the gradients of X and Y.

6. 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+). file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect5.14.html[2/3/2014 6:56:06 PM] Outline for Lecture #5

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.

7. Some Examples & Possible mechanisms (models are discussed below). Click on links for animations.

Type of Type of Pictures in Figures in Example Active "Port" Becker Sadava Transport

figs. 8-11 & 8- a. + + Primary Antiport 6.14 (6.16) Na /K pump 12

+ b. Na /Glucose co- Secondary Symport fig. 8-13 6.15 (6.17) transport *

*This is how glucose enters the epithelial cells lining the intestine or kidney tubule. Transporter is a SGLT (Sodium Glucose Transport) protein. The glucose exits those cells by facilitated diffusion using a different transporter -- GLUT2 protein.

D. Channels (Case #2) -- if this is not covered in #5, it will be covered in #6.

1. Curve #1

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

b. Transport is passive

(1). If X is neutral, the only force that drives X through the channel is the concentration of X.

(2). If X is charged (an ion) you have to take the electrical forces into account as well as the concentration. (Details below on how to do this.)

(3). In summary, Curve #1 plateaus with [X]in = [X]out only if X is neutral or there is no electric potential.

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. Gating/regulation/specificity

a. Specificity: Channels are very specific -- each channel transports only one or a very small # of related substances.

b. Most Channels are gated = % time any particular channel/gate is open is controlled (but each individual gate is either open all the way or shut).

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(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 Sadava fig. 6.10 (6.11).

(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 (13-6 & 13-7).

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

c. Other means of regulation -- some channels are regulated by insertion into the membrane or removal from the membrane as explained above for aquaporins in kidney.

d. 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 on the inside relative to the outside.

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.

4. Curve #1 for ions. 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 or electrical potential). Concentration & voltage can both "push" ions the same way or push in opposite directions.

b. Result of charge: Keq 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 pulls/pushes K+ in) balances out the concentration difference across the membrane (which pushes K+ out).

5. Mechanism.

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. One model (FYI). How to explain the combination of high speed (& capacity) and high specificity? Mechanism of specificity has been figured out for at least one channel. For pictures see Sadava text (fig. 6.12) & Becker fig. 13-8 & 13-9. For more, see Nobel Prize in

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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. (It is discussed at length in neurobiology courses.)

See Sadava fig. 45.6 for comparison of ion pumps and ion channels; Becker p. 202 for comparison of carrier and channel proteins.

6. Terminology. (Reminder from last time)

a. Facilitated diffusion? 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.)

b. Simple diffusion? 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, for physiological concentrations of X. (See above and handout 5A, 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.

c. Better Terminology: Therefore, for clarity, transport through a channel is often called "channel mediated diffusion," or "facilitated diffusion through a channel."

See problem 2-6, A. Can you rule out transport through a channel? (In this problem 'facilitated diffusion' = 'carrier mediated transport.')

Next Time: Anything we don't get to above, and models of Small Molecule Transport. Then: Putting all the Methods of Transport of Small Molecules Together or What Good is All This? Then details of how large molecules cross membranes. (Some specific examples -- fates of LDL, EGF, and Fe-transferrin.)

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