Membrane Permeability Suggested Additional

Membrane Permeability
Suggested Additional (research level) Reading:
Stein, W.D. (1986) Transport and Diffusion across Cell Membranes. Academic Press
Objectives
To discuss:
• the flow or “transport” of molecules across
biomembranes
• the methods we use to study this
• the broad categories of transport across
biomembranes and,
• the physical properties of membranes that
contribute to the solute permeability of lipid bilayers.
This discussion will allow us to understand:
1.
The definition of membrane transport
2.
How we measure transport
3.
When transport is protein-mediated or simple,
non-mediated, transbilayer diffusion.
4.
When transport is passive or active
5.
Why cells need mediated transport systems
6.
The differences between channels and carriers
What is Membrane Transport?
Membrane transport is defined as the movement of molecules across cell
membranes.
There are two classes of membrane transport.
Rapid, stereoselective, saturable, protein-mediated transport.
Slow, non-specific diffusion of molecules across the cell membrane.
Why are biologists interested in transport?
Non-mediated (protein-independent) transport is slow and
membranes are impermeable to small polar molecules
Mediated (protein-dependent) transport is rapid, highly
selective (one gene product typically transports one
substrate) and is often regulated by cytokines and metabolic
demand
Mediated transport is responsible for some forms of drug
resistance
Defects in transport are responsible for many diseases
Transporters are “inside-out” proteins and present significant
technical challenges to structural biologists.
How do we measure transport?
Epithelia
Cells
side 2
side 1
*
measure:
influx or efflux
(v21 or v12)
*
*
side 2
(blood side)
side 1 (blood side)
*
measure:
absorption or
secretion
(v21 or v12)
Basic Principles
Uptake, efflux & exchange
315
Erythrocyte sugar transport
Table 1
Sugar transport measurements in human erythrocytes. Adapted from Ref. [8].
from: Erythrocyte Sugar Transport
A. CARRUTHERS and R.J. ZOTTOLA
1996 Elsevier Science B.V. Handbook of Biological Physics
Volume 2, edited by W.N. Konings, H.R. Kaback and J.S. Lolkema
Adapted from Naftalin, R. J. and Holman, G. D. (1977).
In “Membrane Transport in Red Cells” (eds. J. C. Ellory and V. L. Lew), pp. 257-300. New York: Academic Press.
Methods of detecting transported
molecules
1 Chemical
• Atomic absorption, e.g. Na, Mg, K, Ca.
• Analytical, e.g. HPLC separations and quantitation of
amino acids, nucleotides etc.
• Biochemical, e.g. assays of sugars or nucleosides
using enzyme-coupled measurements.
• Mass Spectrometry of small molecules
Methods of detection
2 Radio-Chemical
Using radiotracers in transport studies
we assume isotopes are chemically equivalent
tracer
H
parent molecule
H
OH
14C
HO
H
H
12C
HO
O
H
H
H
HO
OH
O
H
OH
t1/2 = 5730 yr
H
OH
OH
HO
stable
22Na
23Na
t1/2 = 2.6 yr
stable
45Ca
40Ca
t1/2 = 162.7 days
stable
H
OH
Radioisotopes are much easier to detect and quantitate than specific
molecules which may require chromatography for separation and
quantitation.
Radioisotopes and parent compounds compete for interaction with a common
substrate binding site.
e.g. [14C]-D-glucose and [12C]-D-glucose compete for transport by the glucose
transporter GluT1.
Uptake of extracellular [14C]-Dglucose by cells is competitively
inhibited by increasing levels of
extracellular [12C]-D-glucose.
dpm of
[14C]-D-glucose
inside cells
[12C]-D-glucose
11
dpm of [14C]-D-glucose can be expressed as mol glucose
10 µL 100 µM D-glucose = 20,000 dpm
10 x 10-6 x 100 x 10 -6 mol D-glucose = 20,000 dpm
1 dpm = (1000 x 10-12/20,000) mol glucose = 50 x 10-15 mol
Thus if 106 cells take up 5000 dpm [14C]-D-glucose in 1 min,
the rate of sugar import is calculated as:
5000 50x10−15
x
60
106
mol.cell-1.s-1
= 4.2 x 10-18 mol.cell-1.s-1
Methods of detection
3 Electrochemical
•
Cation-selective microelectrodes, e.g. H+, Ca2+, Na+.
• Voltage electrodes
• Voltage clamp (whole cell, patch clamp, single
channels)
voltmeter
extracellular
electrode
intracellular
glass electrode
bath
cell
d
The red curve shows what happens when the cell contains voltage gated channels. The green curve shows what
would have happened in the absence of these channels.
Voltage clamp allows ion flow across the cell membrane to be measured as current flow while
membrane potential is held constant (clamped) using a feedback amplifier.
Ion channels expressed in Xenopus
oocytes can be studied by twomicroelectrode voltage clamp. The
oocyte is penetrated by two
microelectrodes, one for voltage-sensing
and one for current injection.
Membrane potential is measured by
the voltage-sensing electrode and a
high input impedance amplifier
(amp1).
This is compared with a command
voltage, and the difference is brought to
zero by a high gain feedback amplifier
(amp 2).
The injected current is monitored using a current-to-voltage converter thereby
providing a measure of total membrane current.
modified from:
http://www.sci.utah.edu/~macleod/bioen/be6003/labnotes/W05-voltage-clamp-lab
FIG. 1. Amino acid sequence of the N-terminal (ligand binding) domain of the 5-HT3 receptor. Sequen
domain 1, with tryptophan residues highlighted in bold type. The putative signal sequence is shown underlin
transmembrane topology of the 5-HT3 receptor, illustrating extracellular N and C termini and transmembrane
FIG. 2. Electrophysiological responses of 5-HT3 receptor mutants W183Y and W195S compared with WT. Responses of single
cells (representative of at least four different cells) are shown at maximal and EC50 concentrations of 5-HT.
rate constants between the open and desensitized states of the
stability of the desensiReceptor Ligand Binding Domain*
tized state in the mutant receptors. Changes in stability of the
different states of the receptor are likely to affect the equilibrium binding data, which depends on the interplay between
these different states at equilibrium; this interplay will differ
in the presence of agonists, where desensitization is obligatory,
and antagonists, which may bind preferentially to either the
closed or desensitized state. Thus, if our hypothesis is correct,
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 8, Issue of February 25, pp.
receptor, perhaps indicating decreased
5620
–5625,
2000
The Role
of Tryptophan
Residues in the 5-Hydroxytryptamine3
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 8, Issue of February 25, pp. 5620 –5625, 2000
Printed in U.S.A.
(Received for publication, July 16, 1999, and in revised form, November 22, 1999)
Avron D. Spier‡§ and Sarah C. R. Lummis‡¶!
From the ‡Neurobiology Division, Medical Research Council Laboratory of Molecular Biology, Hills Road,
Cambridge, CB2 2QH and the ¶Department of Biochemistry, University of Cambridge, Tennis Court Road,
Cambridge, CB2 1GA, United Kingdom
Aromatic amino acids are important components of
the ligand binding site in the Cys loop family of ligandgated ion channels. To examine the role of tryptophan
residues in the ligand binding domain of the 5-hydroxytryptamine3 (5-HT3) receptor, we used site-directed mutagenesis to change each of the eight N-terminal tryptophan residues in the 5-HT3A receptor subunit
to tyrosine or serine. The mutants were expressed as
homomeric 5-HT3A receptors in HEK293 cells and analyzed with radioligand binding, electrophysiology, and
immunocytochemistry. Mutation of Trp90, Trp183, and
Trp195 to tyrosine resulted in functional receptors, although with increased EC50 values (2–92-fold) to 5-HT3
receptor agonists. Changing these residues to serine ei90
183
nACh receptors indicate that the ligand binding site is located
in discontiguous regions of the extracellular N-terminal domain, and this has been further confirmed by the construction
of a chimeric protein consisting of the N-terminal domain of the
"7 neuronal nACh receptor subunit linked to the C-terminal
portion of the 5-HT3A receptor subunit, which showed nACh
receptor pharmacological properties and 5-HT3 receptor channel properties (11). Labeling and mutagenesis studies have
identified a number of N-terminal amino acids in nACh subunits that are probably involved in ligand binding; these are
mostly aromatic amino acids and include Trp"54, Trp"86,
Tyr"93, Trp"149, Trp"187, Tyr"190, Cys"192, Cys"193, and
Tyr"198 (12–25).
Sequence alignments between the nACh and 5-HT receptors
FIG. 3. Dose-response curves for
mutant W60S (open circles), W
Methods of detection
4 Photochemical
•
Cation-sensitive dyes (e.g. H+, Ca2+, K+).
•
Membrane potential- sensitive, environment-sensitive,
volume-sensitive dyes.
• Cation- or nucleotide-sensitive bioluminescent proteins
(e.g. aequorin, luciferin/luciferase)
• Engineered sensors (e.g. glucose binding proteins
coupled to green fluorescent protein)
GlcSNFR
Fluorescence (%)
10
12.5 mM
5 mM
8
4 mM
6
3 mM
4
2 mM
1 mM
2
0
0.001
0 mM
0.01
0.1
1
Time (s)
10
100
3 Substrate interacts with
2 Substrate transported
fluorescent sensor
across the bilayer
4 Interaction
changes
fluorescence
intensity
1 Irradiation
Methods of detection
5 Others
• Capacitance changes (volume flow across epithelia)
• Scintillator glass (reacts to ß particles)
• Light scattering (volume-dependent changes in light
scattering by cells)
SCIENCE Volume 290, 2000
pp 481-486
Structure of a GlycerolConducting Channel and
the Basis for Its
Selectivity
Fu, Daxiong; Libson, Andrew;
Miercke, Larry J. W.;
Weitzman, Cindy; Nollert,
Peter; Krucinski, Jolanta;
Stroud, Robert M.
Department of Biochemistry
and Biophysics, School of
Medicine, University of
California, San Francisco, CA
94143-0448, USA.
Fig. 4. Relative rates (µ) for conductance of a selection of carbohydrates into protein-free liposomes (black bars) and into GlpFcontaining proteoliposomes (hatched bars). Structures are indicated in the Fisher diagrams. Error bars represent the standard deviation
from 10 stopped-flow accumulations. ( ) An example of the stopped-flow assay that measures rates of transport of different
carbohydrates into reconstituted vesicles, applied in this example to ribitol, a conducted alditol. Vesicles were reconstituted with GlpF
(red) or without GlpF (green) and then treated with 100 mM carbohydrate at time = 0, or with buffer at time = 0 (blue), and the change
in vesicle size monitored by light scattering at 440 nm. Vesicle size initially decreases rapidly as water diffuses through the lipids in
response to the osmotic challenge. The vesicles reswell with a time constant that depends on conductivity. Changes in light scattering
were therefore fitted by two exponentials Y = [ AW (1 − e−[lambda]t ) − a0 ] + (e− µt ) + a[inf inity] . The first time-constant corresponds to the rapid
water efflux ([lambda] > 5 s ). The second corresponds to the slower rate of reswelling with time constant µ. The black lines represent
the computed fits based on these two exponentials. The time course for a over the entire range of molar ratios of lipid to tetrameric
complex tested (950 - [infinity]). Liposomes with and without GlpF were formed by dilution into reconstitution buffer (20 mM Hepes,
pH 7.2) containing 2 mM DTT as described for aquaporins . A molar ratio of 14,000 lipids (total acetone/ether-extracted polar lipids;
Avanti) to 1 GlpF tetramer (90 mg of lipid/1 mg GlpF) was routinely used unless otherwise specified. After formation and
centrifugation, liposomes were extensively dialyzed against reconstitution buffer for the first day with 2 mM DTT and for 3 days
without DTT. Light scattering was measured with a Kin Tek stopped-flow model SF-2001 at 25°C. Vesicle diameterswere 130 ± 20
nm as measured by electron microscopy and 138 nm ± 36 nm as measured by dynamic light scattering with a DynaPro 801 from
Protein Solutions.
How do we know when transport is protein-mediated?
Specificity is one key piece of evidence. e.g. human erythrocytes are
100,000 times more permeable to D-glucose than they are to L-glucose.
D-Glucose
L-Glucose
CH2 OH
CH2 OH
O
O
H
OH
H
OH
H
H
OH
OH
OH
H
H
H
H
OH
OH
H
H
OH
Metabolically depleted human erythrocytes are 1,000 fold more permeable to
potassium (at. wt. = 39.09) than they are to sodium (at. wt. = 22.99) ions.
Insulin-stimulates D-glucose (but not L-glucose) uptake by adipose and
skeletal muscle by 10 - 50-fold!
This tells us that specific, stereoselective systems mediate transport of Dglucose and K and insulin-stimulation of glucose transport!!
Protein-mediated vs non-mediated transport
Uptake in the
presence of an
inhibitor
Total uptake
v = k[S] +
V [S]/(K +[S])
100
50
0
0
10
vInhibited
= k[S]
Uptake
150
20
30
40
[S] mM
protein-mediated
+ leakage
50
100
50
0
0
10
20
30
v =Uninhibited
V [S]/(K
+[S])
- Inhibited Uptake
150
m
uptake pmol/106 cells/s
uptake pmol/106 cells/s
max
uptake pmol/106 cells/s
150
Difference
40
50
[S] mM
leakage or non-mediated
max(difference) m
100
50
0
0
10
20
30
40
[S] mM
protein-mediated
50
Passive versus Active Transport
For some cells exposed to certain solutes, the equilibrium, intracellular
concentration of solute is identical to that outside the cell.
e.g. erythrocytes & D–Glucose
Because equilibrium equilibrium [D-glucose]i = [D-glucose]o, the red cell glucose
transport system is described as “passive” – the distribution of sugar across the cell
membrane is the same as that produced by simple passive diffusion (although
simple diffusion would take much longer to equilibrate the sugar)
Baker, P. F. and Carruthers, A. (1981). Sugar transport in giant axons of Loligo. J. Physiol. (Lond.) 316, 481-502.
For different cells or solutes, the equilibrium, intracellular concentration of
solute is not identical to that outside the cell.
e.g. D–Glucose content of epithelial cells of small intestine.
Because
[D-glucose]i = 20 [D-glucose]o
the epithelial cell glucose transport system is described as “ACTIVE” – the
distribution of sugar across the cell membrane is NOT that produced by
simple passive diffusion.
When charged species are examined (e.g. Na+) we must consider the
effect of the membrane potential (V) on transmembrane solute
distributions
Most cells are characterized by a membrane potential
difference (V) of -70 mV (inside negative with respect to
the outside).
If we examine the levels of cations and anions in serum and cytosol
Species [Extracellular]
mM
[Intracellular] Equilibrium potential
mM
mV
VDF
mV
Na+
140
15
+57.3
-127.3
K+
5
121
-81.2
+11.2
Ca2+
1.5
0.0002
+119.2
-189.2
Cl-
125
9
-70.3
-0.3
Species
[Extracellular]
mM
[Intracellular]
mM
Equilibrium potential
mV
Cl-
125
9
-70.3
Consider Cl-. The Cl- concentration gradient is directed into the cell. Thus Cl- tends to diffuse along
the concentration gradient into the cell. The interior, however, is negative with respect to the
outside and Cl- ions are pushed out along the electrical gradient. An equilibrium is achieved
when Cl- influx = Cl- efflux. The membrane potential at which this equilibrium exists is the
equilibrium potential. Its magnitude is calculated from the Nernst equation as follows:
RT [Cl−o ]
ECl =
ln
= −70.3mV
FZ Cl [Cli− ]
where R is the gas constant (1.987 cal/
deg/mol)
T is absolute temperature (37˚C = 310˚K)
F is the faraday (23060 cal/volt/mol)
ZCl is the valence of Cl (-1)
VDF = Vm - Veq
negative for cation means uptake
0 means no driving force
positive for cation means exit
negative for anion means exit
0 means no driving force
positive for anion means uptake
Species
[Extracellular]
mM
[Intracellular]
mM
Equilibrium potential
(Veq) mV
Na+
150
15
+57.3
K+
5.5
150
-81.2
Ca2+
1.5
0.0002
+119.2
Cl-
125
9
-70.3
Because ECl = V (membrane potential), no forces other than those
represented by the chemical and electrical gradients (the electrochemical
gradient) need be invoked to explain the distribution of Cl- across the cell
membrane.
Because ENa, EK and ECa ≠ V, this suggests that other processes intervene
to exclude Na and Ca and to accumulate K. These are transport
processes and must be ACTIVE.
Species
[Extracellular]
mM
[Intracellular]
mM
Equilibrium potential
(Veq) mV
VDF
mV
Net flow
Na+
150
15
+57.3
-127.3
in
K+
5.5
150
-81.2
+11.2
out
Ca2+
1.5
0.0002
+119.2
-189.2
in
Cl-
125
9
-70.3
+0.3
~in
The direction of the electrochemical gradient for net flow (VDF) is obtained as
VDF = VM - Veq
Species
VDF
Direction of gradient
Cation
+
out
Cation
0
none
Cation
-
in
Anion
+
in
Anion
0
none
Anion
-
out
Selective transport is protein-mediated
Transporters are classic enzymes – they accelerate the rate at which a
molecule achieves its equilibrium distribution across the cell membrane by
providing (literally) an alternative reaction pathway. These are the PASSIVE
transporters.
Some active transporters exploit high energy intermediates (ATP-hydrolysis) to
catalyze rapid net solute movement against a concentration gradient (uphill) these are Primary Active Transporters.
Yet other active transporters exploit Na+, K+ or H+ gradients to drive a molecule
against an electrochemical gradient - these are Secondary Active Transporters.
Active transporters make an endergonic reaction (Keq < 1) more exergonic (Keq > 1) by
coupling the first reaction (e.g. Na export from low to high concentration) to a second
exergonic reaction (e.g. ATP-hydrolysis) through common intermediates
As with other enzymes, membrane transporters display saturation kinetics and
competitive or non-competitive inhibition by relatively low concentrations of
specific inhibitors.
Standard Free Energy Changes are Additive
Consider the following reactions:
A
B
∆G˚1
≡
B
C
A
C
∆G˚total
∆G˚2
The ∆G˚ of sequential reactions are additive, thus
∆G˚total = ∆G˚1 + ∆G˚2
This principle of bioenergetics explains how an endergonic reaction (Keq <
1) can be improved (more product formed) by coupling it to a highly
exergonic reaction (Keq >>1) through a common intermediate.
Reaction 1 - Na export
∆G = RT ln Nao + zFV
Nai
where z is +1; F (the Faraday) = 23,062 cal V-1mol-1; Nao/Nai ≈ 10; V = 70
mV (outside).
The cost to do this (∆G) is approximately 2.98 kcal per mol at 25 ˚C. This is
equivalent to an equilibrium constant of:
- ∆G
Keq1 = 10 2.303 RT = 0.0065
Reaction 2 - ATP hydrolysis
[ADP][Pi ]
Keq 2 =
= 2x10 5 M
[ATP]
Combined reaction - ATP hydrolysis driven Na export
∆Gs are additive ∴ Keqs are multiplied, hence
Keq(combined) = Keq1 * Keq2 = 1,300
∆G for ATP hydrolysis in
cells ≈ -13 kcal per mol
transported molecule
channel
protein
carrier
protein
concentration
gradient
lipid
bilayer
EN
ER
channeldiffusion mediated
PASSIVE TRANSPORT
G
Y
carriermediated
ACTIVE
TRANSPORT
Why Do Cells Need Membrane Transporters?
The lipid bilayer is an effective barrier to the movement of small
hydrophilic molecules. Two factors govern the rate at which molecules
can diffuse across the lipid bilayer. These are:
(1) the membrane solubility of the specific molecules in question and
(2) the size of the molecule that diffuses across the cell membrane.
Dissecting the characteristics of Transbilayer diffusion
Transbilayer diffusion is a first order process
transbilayer solute flux = J 12 = k (C aq1 - C 2aq)
inject substrate
100
80
1-fractional equilibration
Relative signal
•
60
40
20
1
0.1
0.01
0.001
0
100
200
time sec
0
0
50
100
150
Time in seconds
200
250
mol.L-1.s-1
•
Transbilayer diffusion is dependent on the nature of the diffusing species
(the diffusant)
37
•
The Cell Membrane is thus a Barrier to Solute Movement
Let’s examine why this is so by considering 3 concepts
1 Diffusion = Random Walk (Fig 1)
Figure 1 Simulation of the diffusion process. Three successive stages are shown of
molecules moving by random walks from: A. The first position where all molecules are at
one side of the barrier. B. An intermediate stage. C. An equilibrium distribution
Diffusion is stochastic - the probability of a molecule moving from side 1 to side 2 is related
directly to the difference in its relative concentrations at each side.
38
2 Chemical Potential
The chemical potential of a molecule is comprised of those components
of a molecule (j) that enable it to perform work.
a. Concentration, Cj (osmotic work)
b. Charge, Zj e ψ where
Z = valence (electrical work)
e = electron charge
ψ = electric potential
c. Volume, Vj (work against applied pressure)
d. Mass, mj (gravitational work)
e. Chemical structure (chemical work)
39
Nobel, 1974 shows that chemical potential (µ) of molecule j (µj)
µj = µjo + R T lnCj + Zj e F ψ
+ V j P + mj g h
µjo = chemical potential of substance j in standard state when ψ = 0,
h = 0, P and T are standard and Cj = 1M in a particular solvent. As
gravity and ∆P unimportant here,
µj = µjo + R T lnCj + Zj F e ψ
3
40
Equilibrium Distributions
3.1 The Partition coefficient, K
Imagine glycerol is added to a mixture of oil and water. The mixture is shaken until the
concentrations of glycerol in oil and water no longer change (equilibrium is achieved).
The mixture is allowed to stand (phase separation occurs) and the oil and water
phases are assayed for glycerol content.
At equilibrium, glyceroloil is in equilibrium with glycerolwater
i.e. µjoil = µjwater
As glycerol is uncharged, an electrical term is not needed and
µ oj oil + RT ln C j oil = µ oj water + RT ln C j water
µ oj oil − µ oj water = RT (lnC j water − lnC j oil)
or K oil/water = exp[( µ oj water − µ oj oil) / RT ]
i.e. K is determined by differences in standard state chemical potential of j in oil and water
41
Koil/water = exp [(µjowater - µjooil)/RT]
each µjo determined by energetics of interaction
between j and solvent
glycerol has three - OH groups resulting in strong Hbonding to H2O and is thus in a more energetically
favorable state in H2O
∴ µjowater < µjooil
∴ Koil/water < 1.
Now let’s use these principles to examine trans-membrane diffusion
S1
Membrane
S2
aq
C
1
m
C1
b
a
c
λ
m
C
2
aq
C
2
Permeability depends upon:
• partitioning into the membrane Kj (processes a and c)
• mobility within the membrane µj (process b)
• Thickness of the membrane (λ)
43
mols of substrate crossing the membrane per sec
molar flux
flux across a unit surface area
hence,
J 12 = k (C aq1 - C 2aq)
mol.mL-1.s-1
J 12 = P $ A (C aq1 - C 2aq)
mol.mL-1.s-1
k = P$A
{A = surface area (in cm2) of that number of cells containing 1 mL water; P =
permeability coefficient in cm.s-1; C=mol.cm-3}
It can be shown that
KDm
P=
λ
Permeability is positively related to K
and Dm (where Dm - diffusion coefficient - is
related to mobility within the membrane)
44
We will now use measurements of the permeability of
human red blood cells to a variety of small compounds to
determine whether this hypothetical relationship is true.
45
Fig 2 shows a plot of log P vs. log K where P is the
permeability of red cells to substances and K is Partition
coefficient for species in hexadecane/water.
The data are listed and
numbered in Table A.I
There is reasonable
agreement!
However, low MW species lie
above line e.g. H2O
high MW species lie below
line (see Table 1 for molecular
species)
46
Why? Is Dm greater for small species?
P = KDm/λ, it thus follows that Dm = Pλ/K.
Assuming K is identical to that for hexadecane and H2O and assuming λ = 40 Å, Dm
is calculated and shown in Fig 3
Dm < Dwater and is inversely proportional to MW!
47
If we make plot of log Dm vs diffusant volume (van der Waal’s vol), the
relationship is clear - the larger the molecule, the lower the Dm
logDm = logDmv=0 − mv ⋅ V
The red cell lipid bilayer, like all solvents and polymers,
contains “void space” or free volume (the volume of the
constituent molecules < total volume).
In order for a molecule to diffuse within the bilayer, it must
move from one free volume to another. These free volumes
are transient in nature and for any given polymer (bilayer) have
a characteristic average size.
The average free volume in the red cell lipid bilayer is 8.4 cm3/
mol. This is close to the van der Waal’s volume of a
methylene group of a hydrocarbon which is less than the van
der Waal’s volume of water (10.6 cm3/mol)!!
∴ explains steep size dependence of Dm in red cells!
49
To illustrate this, let us examine the
water permeability of a lipid bilayer
as it undergoes the ordered to
disordered phase transition.
endothermic
water
permeability
So Why Do Cells Need Mediated Transport systems?
The lipid bilayer is an effective barrier to the movement of small hydrophilic
molecules. For an average hydrophilic metabolite such as a sugar or an amino
acid, low membrane solubility (K ≤ 1 x 10-7) and great molecular size (30 to 70
cm3/mol) offer significant resistance to movement either into the cell or out of
the cell. This allows the cell to retain important metabolic intermediates.
In order for a cell to selectively regulate its metabolite content it must use
transporter molecules which accelerate the rate of entry or export of these
species into or out of the cell. Selective expression of specific transporters
allows the cell to retain or import molecules that are important for survival and
to export molecules that are incompatible with cellular survival.
Channels and Carriers
There are two classes of protein-mediated transport
systems:
1)
channels
2)
carriers
The channels form membrane-spanning pores that allow
molecules to diffuse down the electrochemical gradient into or
out of the cell.
Some channels are gated. They are opened or closed by
binding of a ligand or by altered membrane potential.
Mapping Shaker channel mutations onto the KcsA structure. Mutations in the voltage-gated Shaker K+channel that affect
function are mapped to the equivalent positions in KcsA based on the sequence alignment. Two (of 4) subunits of KcsA are
shown. Mutation of any of the white side chains significantly alters the affinity of agitoxin2 or charybdotoxin for the Shaker K+
channel. Changing the yellow side chain affects both agitoxin2 and TEA binding from the extracellular solution. This residue is
the external TEA site. The mustard-colored side chain at the base of the selectivity filter affects TEA binding from the
intracellular solution [the internal TEA site]. The side chains colored green, when mutated to cysteine, are modified by cysteinereactive agents whether or not the channel gate is open, whereas those colored pink react only when the channel is open. Finally,
the residues colored red (GYG, main chain only) are absolutely required for K+ selectivity.
D A Doyle et al. Science 1998;280:69-77
Published by AAAS
The carriers are an altogether different class of transport mechanism. The
carriers appear to present either an import or an export site to the
transported molecule but not both sites simultaneously.
GLUT1 conformational changes
e2
Kenneth Lloyd & Tony Carruthers, 2011 - e2 modeled after the FucP crystal structure and e1 modeled after the GlpT crystal structure
e1
Summary - Permeability
1.
What is membrane transport? - the movement of molecules across the
cell membrane
2.
How do you measure transport? - a variety of technologies permit
transport measurement
3.
When is transport mediated or non-mediated? - mediated transport is
protein catalyzed, rapid, stereospecific/saturable and is often inhibited by
specific toxins.
4.
When is transport passive or active? - when a cell accumulates or exports
a substrate beyond its predicted equilibrium distribution.
5.
Why do cells need mediated transport systems? - because the cell
membrane is an effective barrier to the movement of small polar
molecules.
6.
What are channels and carriers? - integral, amphipathic membrane
proteins that catalyze substrate transport through a pore or through a
substrate-promoted conformational change.
57
Table A.1
Molecule
Number
vdWvol
3
cm .mol
Mr
-1
P
cm.sec
Khex
-1
Dmem
2
cm .sec
Size.corrected P
-1
cm2.sec-1
Ethanediol
2
36.5
62
2.90E-05
1.70E-05
6.82E-07
2.24E-03
Ethanol
3
31.9
46.07
2.10E-03
5.70E-03
1.47E-07
9.36E-02
Glycerol
4
51.4
95.12
1.60E-07
2.00E-06
3.20E-08
7.27E-05
n-Hexanol
5
72.9
102.18
8.70E-03
1.3
2.68E-09
51.10334
Methanol
6
21.7
33.05
3.70E-03
3.80E-03
3.89E-07
4.90E-02
n-Propanol
7
42.2
60.1
6.50E-03
3.30E-02
7.88E-08
9.88E-01
Urea
9
32.6
60.6
7.70E-07
3.50E-06
8.80E-08
3.73E-05
Water
10
10.6
18.02
1.20E-03
4.20E-05
1.14E-05
4.24E-03
Water
1
10.60
4.22E-05
methane
2
15.77
2.37E-05
ethane
3
27.04
1.78E-05
n-propane
4
38.31
1.54E-05
n-hexane
5
68.73
7.5E-06
n-heptane
6
78.87
6.46E-06
n-octane
7
90.14
5.62E-06
methyl acetate
8
42.82
1E-07
ethyl acetate
9
52.96
5.62E-08
propyl acetate
10
63.10
1.78E-08
butyl-acetate
11
74.37
1E-08
methanol
12
22.54
5.62E-07
benzene
13
47.32
2.09E-08
Data for Fig. 6