Biochemistry of Metabolism

Membrane Transport

Copyright 1999-2007 by Joyce J. Diwan.

All rights reserved.

This discussion will focus on selected examples of

transport catalysts for which structure/function
relationships are relatively well understood.
Transporters are of two general classes:
carriers and channels.
These are exemplified by two ionophores (ion carriers
produced by microorganisms):
valinomycin (a carrier)
gramicidin (a channel).

Valinomycin
H3C

CH3
CH O

CH

O
O

CH
H3C

L-valine

H
C

O
N

H
C

CH

CH3 H3C

D-hydroxy-

isovaleric acid

CH3 O
O

CH

CH3

D-valine

L-lactic

acid

Valinomycin is a carrier for K+.

It is a circular molecule, made up of 3 repeats of
the sequence shown above.

Puckering of the ring,

stabilized by H-bonds, allows
valinomycin to closely
surround a single unhydrated
K+ ion.

Valinomycin
O
O

K+

O
O
Six oxygen atoms of the
O
ionophore interact with the
Hydrophobic
bound K+, replacing O atoms
of waters of hydration.
Valinomycin is highly selective for K+ relative to Na+.
The smaller Na+ ion cannot simultaneously interact with all 6
oxygen atoms within valinomycin.
Thus it is energetically less favorable for Na + to shed its
waters of hydration to form a complex with valinomycin.

Valinomycin
O
O
O

K+
O

O
O

Hydrophobic

Whereas the interior of the valinomycin-K+ complex is

polar, the surface of the complex is hydrophobic.
This allows valinomycin to enter the lipid core of the
bilayer, to solubilize K+ within this hydrophobic milieu.
Crystal structure (at Virtual Museum of Minerals & Molecules).

Val-K+

Val-K+
K+

K+
Val

Val
membrane

Valinomycin is a passive carrier for K+. It can bind or

release K+ when it encounters the membrane surface.
Valinomycin can catalyze net K+ transport because it can
translocate either in the complexed or uncomplexed state.
The direction of net flux depends on the electrochemical
K+ gradient.

Proteins that act as carriers are too large to move

across the membrane.
They are transmembrane proteins, with fixed
topology.
An example is the GLUT1 glucose carrier, in plasma
membranes of various cells, including erythrocytes.
GLUT1 is a large integral protein, predicted via
hydropathy plots to include 12 transmembrane
-helices.

conformation
change

conformation
change

Carrier-mediated solute transport

Carrier proteins cycle between conformations in

which a solute binding site is accessible on one side of
the membrane or the other.
There may be an intermediate conformation in which a
bound substrate is inaccessible to either aqueous phase.
With carrier proteins, there is never an open channel
all the way through the membrane.

conformation
change

conformation
change

Carrier-mediated solute transport

The transport rate mediated by carriers is faster than in

the absence of a catalyst, but slower than with channels.
A carrier transports one or few solute molecules per
conformational cycle, whereas a single channel opening
event may allow flux of many thousands of ions.
Carriers exhibit Michaelis-Menten kinetics.

Classes of
carrier
proteins

Uniport
A

Symport
A

Antiport
A

Uniport (facilitated diffusion) carriers mediate

transport of a single solute.
An example is the GLUT1 glucose carrier.
The ionophore valinomycin is also a uniport carrier.

Symport (cotransport) carriers

bind two dissimilar solutes
(substrates) & transport them
together across a membrane.
Transport of the two solutes is
obligatorily coupled.
A gradient of one substrate, usually an ion, may drive uphill
(against the gradient) transport of a co-substrate.
It is sometimes referred to as secondary active transport.
E.g: glucose-Na+ symport, in plasma membranes
of some epithelial cells
bacterial lactose permease, a H+ symport carrier.

Lactose permease catalyzes uptake

of the disaccharide lactose into
E. coli bacteria, along with H+,
driven by a proton electrochemical
gradient.
It is the first carrier protein for which an atomic
resolution structure has been determined.
Lactose permease has been crystallized with
thiodigalactoside (TDG), an analog of lactose.

The substrate binding

site is at the apex of an
aqueous cavity between
two domains, each
consisting of six transmembrane -helices.

TDG
substrate
analog

Lactose Permease

PDB 1PV7

In the conformation observed in this crystal structure,

the substrate analog is accessible only to what would be
the cytosolic side of the intact membrane.
Residues essential for H+ binding are are also near the
middle of the membrane.

conformation
change

conformation
change

Carrier-mediated solute transport

As in simple models of
carrier transport based on
functional assays, the tilt of
transmembrane -helices
is assumed to change,
shifting access of lactose &
H+ binding sites to the other
side of the membrane during
the transport cycle.

TDG
substrate
analog

Lactose Permease

PDB 1PV7

Antiport (exchange diffusion) carriers

exchange one solute for another across a
membrane.
Usually antiporters exhibit "ping
pong" kinetics.
A substrate binds & is transported.
Then another substrate binds & is transported in the other
direction.
Only exchange is catalyzed, not net transport.
The carrier protein cannot undergo the conformational
transition in the absence of bound substrate.

mitochondrial
matrix
ATP 4
ADP 3
adenine nucleotide translocase

Example of an antiport carrier:

Adenine nucleotide translocase (ADP/ATP exchanger)
catalyzes 1:1 exchange of ADP for ATP across the inner
mitochondrial membrane.

Active
Transport

ADP + Pi

S2

S1
ATP
Side 1

Side 2

Active transport enzymes couple net solute movement

across a membrane to ATP hydrolysis.
An active transport pump may be a uniporter or
antiporter.

ATP-dependent ion pumps are grouped into classes

based on transport mechanism, as well as genetic &
structural homology.
Examples include:
P-class pumps
F-class (e.g., F1Fo-ATPase to be discussed later)
& related V-class pumps.
ABC (ATP binding cassette) transporters, which
catalyze transmembrane movements of various organic
compounds including amphipathic lipids and drugs,
will not be discussed here.

P-class ion pumps are a gene family exhibiting

sequence homology. They include:
Na+,K+-ATPase, in plasma membranes of most
animal cells is an antiport pump.
It catalyzes ATP-dependent transport of Na+ out of a
cell in exchange for K+ entering.
(H+, K+)-ATPase, involved in acid secretion in the
stomach is an antiport pump.
It catalyzes transport of H+ out of the gastric parietal
cell (toward the stomach lumen) in exchange for K+
entering the cell.

P-class pumps (cont):

Ca++-ATPases, in endoplasmic reticulum (ER) and
plasma membranes, catalyze ATP-dependent
transport of Ca++ away from the cytosol, into the ER
lumen or out of the cell.
Some evidence indicates that these pumps are
antiporters, transporting protons in the opposite
direction.
Ca++-ATPase pumps function to keep cytosolic Ca++
low, allowing Ca++ to serve as a signal.

O
Enzyme-C

The reaction mechanism

for a P-class ion pump
involves transient
covalent modification
of the enzyme.

OH

ATP

Pi

ADP

H2O

O
Enzyme- C

O
O

P-Class Pumps

O-

O-

At one stage of the reaction cycle, phosphate is transferred

from ATP to the carboxyl of a Glu or Asp side-chain,
forming a high energy anhydride linkage (~P).
At a later stage in the reaction cycle, the Pi is released by
hydrolysis.

E~P-Ca++2

E~P-Ca++2

ADP

The ER Ca++ pump

is called SERCA:
Sarco(Endo)plasmic
Reticulum
Ca++-ATPase.

ATP
2Ca++

2Ca++
E-Ca++2
E

Pi
cytosol

membrane

In this diagram of SERCA reaction cycle,

conformational changes altering accessibility of
Ca++-binding sites to the cytosol or ER lumen are
depicted as positional changes.
Keep in mind that SERCA is a large protein that
maintains its transmembrane orientation.

ER
lumen

Reaction cycle:

ADP

E~P-Ca++2

E~P-Ca++2

1. 2 Ca++ bind tightly

ATP E-Ca++
from the cytosolic
2
side, stabilizing the
2Ca++
conformation that
E
allows ATP to react
Pi
with an active site
cytosol
membrane
aspartate residue.

2Ca++

ER
lumen

2. Phosphorylation of the active site aspartate induces a

conformational change that
shifts accessibility of the 2 Ca++ binding sites from one
side of the membrane to the other, &
lowers the affinity of the binding sites for Ca ++.

ADP
ATP
2Ca++

E~P-Ca++2

E~P-Ca++2
2Ca++

E-Ca++2
E

Pi
cytosol

membrane

ER
lumen

3. Ca++ dissociates into the ER lumen.

4. Ca++ dissociation promotes
hydrolysis of Pi from the enzyme Asp
conformational change (recovery) that causes Ca++
binding sites to be accessible again from the cytosol.

Asp351

This X-ray structure

of muscle SERCA (Ca+
+-ATPase) shows
2
Ca++ ions (colored
magenta) bound between
transmembrane -helices
in the membrane domain.

cytosolic
domain

2 Ca++

PDB 1EUL

membrane
domain

Muscle SERCA

Active site Asp351, which is transiently phosphorylated

during catalysis, is located in a cytosolic domain, far
from the Ca++ binding sites.

SERCA structure has been determined in the presence &

absence of Ca++, with or without substrate or product
analogs and inhibitors.
Substantial differences in conformation have been
interpreted as corresponding to different stages of the
reaction cycle.
Large conformational changes in the cytosolic domain
of SERCA are accompanied by deformation & changes
in position & tilt of transmembrane -helices.
The data indicate that when Ca++ dissociates:
water molecules enter Ca++ binding sites
charge compensation is provided by protonation
of Ca++-binding residues.

++

Ca

SERCA Conformational Cycle

enzyme
phosphorylation

phosphate
hydrolysis

This simplified cartoon represents the proposed

variation in accessibility & affinity of Ca++-binding sites
during the reaction cycle.
Only 2 transmembrane -helices are represented, and the
cytosolic domain of SERCA is omitted.

More complex diagrams & animations have been

created by several laboratories, based on available
structural evidence. E.g.:
animation (lab of D. H. MacLennan)
diagram (by C. Toyoshima, in a website of the Society of General
Physiologists - select Poster).

website of the Toyoshima Lab (select Resources for movies).

conformation
change

Ion
Channels

closed

open

Channels cycle between open & closed conformations.

When open, a channel provides a continuous pathway
through the bilayer, allowing flux of many ions.
Gramicidin is an example of a channel.

Gramicidin is an unusual peptide,

with alternating D & L amino acids.
In lipid bilayer membranes, gramicidin
dimerizes & folds as a right-handed helix.
The dimer just spans the bilayer.
Primary structure of gramicidin (A):

Gramicidin dimer
(PDB file 1MAG)

HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-DLeu-L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-NHCH2CH2OH
Note: The amino acids are all hydrophobic; both peptide ends are
modified (blocked).

The outer surface of the

gramicidin dimer, which interacts
with the core of the lipid bilayer,
is hydrophobic.
Ions pass through the more polar
lumen of the helix.
Ion flow through individual
gramicidin channels can be
observed if a small number of
gramicidin molecules is present in
a lipid bilayer separating 2
compartments containing salt
solutions.

Gramicidin dimer
(PDB file 1MAG)

c u rre n t

ion flow
through one
channel
time

With voltage clamped at some value, current (ion flow

through the membrane) fluctuates.
Each fluctuation, attributed to opening or closing of one
channel, is the same magnitude.
The current increment corresponds to current flow
through a single channel (drawing - not actual data).

Gating (opening &

closing) of a
gramicidin channel
is thought to
involve reversible
dimerization.
An open channel forms when two gramicidin molecules
join end to end to span the membrane.
This model is consistent with the finding that at high
[gramicidin] overall transport rate depends on
[gramicidin]2.

Channels that are proteins

Cellular channels usually consist of large protein
complexes with multiple transmembrane -helices.
Their gating mechanisms must differ from that of
gramicidin.
Control of channel gating is a form of allosteric
regulation. Conformational changes associated with
channel opening may be regulated by:
Voltage
Binding of a ligand (a regulatory molecule)
Membrane stretch (e.g., via link to cytoskeleton)

A
electrode

Patch
Clamping

glass pipet

B
electrode

electrode

The technique of patch clamping is used to study

ion channel activity.
A narrow bore micropipet may be pushed up against
a cell or vesicle, and then pulled back, capturing a
fragment of membrane across the pipet tip.

amplifier with
voltage control

Patch
Clamping

reference
electrode

electrode in
patch pipet

salt solution

membrane
patch

A voltage is imposed between an electrode inside the

patch pipet and a reference electrode in contact with
surrounding solution. Current is carried by ions
flowing through the membrane.

c u rre n t

open
closed
time

If a membrane patch contains a single channel with 2

conformational states, the current will fluctuate between
2 levels as the channel opens and closes.
The increment in current between open & closed
states reflects the rate of ion flux through one channel.
View a video of an oscilloscope image during a patch
clamp recording.

20
pA

102 msec per trace

Patch clamp recording at 60 mV. Consecutive traces

are shown. Note that at a negative voltage, increased
current is a downward deflection.

Current Amplitude
Histogram
O
c
c
u
p
a
n
c
yo
fc
u
rre
n
t le
v
e
ls

Occupancy of different
current levels during the
time period of a recording is
plotted against current in
picoAmperes (1012 Amp).
Peaks represent open &
closed states (note scale).
Baseline current, when the
channel is closed, is due to
leakage of the patch seal and
membrane permeability.

-60
-55
-50
Amplitude in pAmp

-45