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

H
OH

t1/2 = 5730 yr

OH

OH

HO

stable

22Na

23Na

t1/2 = 2.6 yr

stable

45Ca

40Ca

t1/2 = 162.7 days

stable

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 L 100 M 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 50x1015
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

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 dierence 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 (292-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 (1225).
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

4 mM
6

3 mM

2 mM
1 mM

2
0
0.001

0 mM
0.01

0.1

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 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 () 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 t ) + 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 . 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 25C. 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

OH

OH

OH

OH

OH

OH

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

10

vInhibited
= k[S]
Uptake

150

20

30

40

[S] mM

protein-mediated
+ leakage

50

100

50

10

20

30

v =Uninhibited
V [S]/(K
+[S])
- Inhibited Uptake

150

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

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 & DGlucose
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. DGlucose 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+

121

-81.2

+11.2

Ca2+

1.5

0.0002

+119.2

-189.2

Cl-

125

-70.3

-0.3

Species

[Extracellular]
mM

[Intracellular]
mM

Equilibrium potential
mV

Cl-

125

-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 [Clo ]
ECl =
ln
= 70.3mV
FZ Cl [Cli ]
where R is the gas constant (1.987 cal/
deg/mol)
T is absolute temperature (37C = 310K)
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

-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

-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

none

Cation

in

Anion

in

Anion

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:

G1

Gtotal

G2

The G of sequential reactions are additive, thus

Gtotal = G1 + G2
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

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

Lets 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 where
Z = valence (electrical work)
e = electron charge
= 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 () of molecule j (j)

j = jo + R T lnCj + Zj e F

+ V j P + mj g h

jo = chemical potential of substance j in standard state when = 0,

h = 0, P and T are standard and Cj = 1M in a particular solvent. As
gravity and P unimportant here,
j = jo + R T lnCj + Zj F e

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. joil = jwater
As glycerol is uncharged, an electrical term is not needed and

oj oil + RT ln C j oil = oj water + RT ln C j water

oj oil oj water = RT (lnC j water lnC j oil)

or K oil/water = exp[( oj water 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 [(jowater - jooil)/RT]

each jo 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
jowater < jooil
Koil/water < 1.

Now lets use these principles to examine trans-membrane diusion

S1

Membrane

S2

aq
C
1
m
C1

m
C
2

aq
C
2

Permeability depends upon:

partitioning into the membrane Kj (processes a and c)

mobility within the membrane j (process b)

Thickness of the membrane ()

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=

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/, it thus follows that Dm = P/K.
Assuming K is identical to that for hexadecane and H2O and assuming = 40 , 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 Waals 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 Waals volume of a
methylene group of a hydrocarbon which is less than the van
der Waals 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

cm.sec

Khex
-1

Dmem
2

cm .sec

Size.corrected P
-1

cm2.sec-1

Ethanediol

36.5

62

2.90E-05

1.70E-05

6.82E-07

2.24E-03

Ethanol

31.9

46.07

2.10E-03

5.70E-03

1.47E-07

9.36E-02

Glycerol

51.4

95.12

1.60E-07

2.00E-06

3.20E-08

7.27E-05

n-Hexanol

72.9

102.18

8.70E-03

1.3

2.68E-09

51.10334

Methanol

21.7

33.05

3.70E-03

3.80E-03

3.89E-07

4.90E-02

n-Propanol

42.2

60.1

6.50E-03

3.30E-02

7.88E-08

9.88E-01

Urea

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

10.60

4.22E-05

methane

15.77

2.37E-05

ethane

27.04

1.78E-05

n-propane

38.31

1.54E-05

n-hexane

68.73

7.5E-06

n-heptane

78.87

6.46E-06

n-octane

90.14

5.62E-06

methyl acetate

42.82

1E-07

ethyl acetate

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