Download Membrane Transport

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
N
CH C
O
O
H
C
CH
H3C
L-valine
H
C
O
N
H
C
H
CH
CH3 H3C
D-hydroxy-
C
O
CH
C
3
CH3
D-valine
isovaleric acid
CH3 O
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.
Six oxygen atoms of the
ionophore interact with the
bound K+, replacing O atoms
of waters of hydration.
Valinomycin
O
O
+
O
K
O
O
O
Hydrophobic
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
a-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.
Uniport
Classes of
carrier
proteins
A
Symport
A
B
Antiport
A
B
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.
Uniport
Symport (cotransport) carriers
bind two dissimilar solutes
(substrates) & transport them
together across a membrane.
A
Symport
A
A
B
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.
Uniport
A
Symport
A
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.
B
A
The substrate binding
site is at the apex of an
aqueous cavity between
two domains, each
consisting of six transmembrane a-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 a-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
Uniport
Symport
Antiport (exchange diffusion)
carriers
A
A
B
exchange one solute for another across a
membrane.
Antiport
A
 Usually antiporters exhibit "ping pong"
kinetics.
A substrate binds & is transported.
B
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
O-
O-
P-Class Pumps
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++
E-Ca++2
2Ca++
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:
E~P-Ca++2
ADP
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++.
E~P-Ca++2
E~P-Ca++2
ADP
ATP
2Ca++
E-Ca++2
2Ca++
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 a-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 a-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 a-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 b-helix.
The dimer just spans the bilayer.
Gramicidin dimer
(PDB file 1MAG)
Primary structure of gramicidin (A):
HCO-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-TrpD-Leu-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)
current
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).
Proposed mechanism of
gramicidin gating
Gating (opening &
closing) of a
gramicidin channel
is thought to
involve reversible
dimerization.
open
closed
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 a-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.
current
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
Occupancy of current levels
Occupancy of different
current levels during the
time period of a recording is
plotted against current in
picoAmperes (1012 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