Download Outline for Lecture #5

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 K m
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 'K m 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 X out ↔
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.
b. K eq = 1; Standard free energy change (ΔGo ) = 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 + H2 O → 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 (antidiuretic 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).
b. K eq not = 1 and standard free energy (ΔGo ) 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),
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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
Active
Transport
Type of
"Port"
Pictures in
Becker Figures in
Sadava
a. Na+ /K+ pump Primary
Antiport
figs. 8-11 & 812
6.14 (6.16)
+
b. Na /Glucose cotransport *
Secondary
Symport
fig. 8-13
6.15 (6.17) Example
*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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