4 Transport
-
Upload
choi-eun-sang -
Category
Documents
-
view
220 -
download
0
Transcript of 4 Transport
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 1/39
Membrane Transport
Copyright © 1999-2007 by Joyce J. Diwan.
All rights reserved.
Biochemistry of Metabolism
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 2/39
This discussion will focus on selected examples oftransport 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).
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 3/39
Valinomycin is a carrier for K +.
It is a circular molecule, made up of 3 repeats of
the sequence shown above.
N CH C OHC
CH
CH3H3C
O
C N
CH
CH3H3C
O
HC
CH
CH3H3C
C O CH
CH3
C
O
H
O
H
3
Valinomycin
L-valine D-hydroxy- D-valine L-lacticisovaleric acid acid
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 4/39
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
O O
Hydrophobic
O
K+
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.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 5/39
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).
Valinomycin
O
O O
O O
Hydrophobic
O
K+
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 6/39
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.
Val Val
Val-K + Val-K +
K +
membrane
K +
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 7/39
Proteins that act as carriers are too large to moveacross 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 viahydropathy plots to include 12 transmembrane
a-helices.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 8/39
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.
conformationchange
conformationchange
Carrier-mediated solute transport
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 9/39
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 perconformational cycle, whereas a single channel opening
event may allow flux of many thousands of ions.
Carriers exhibit Michaelis-Menten kinetics.
conformationchange
conformationchange
Carrier-mediated solute transport
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 10/39
Classes ofcarrier
proteins
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 Antiport
A A B A
B
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 11/39
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.
Symport
A BSymport (cotransport) carriers
bind two dissimilar solutes
(substrates) & transport themtogether across a membrane.
Transport of the two solutes is
obligatorily coupled.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 12/39
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.
Symport
A B
Lactose permease catalyzes uptake
of the disaccharide lactose into
E. coli bacteria, along with H+,
driven by a proton electrochemical
gradient.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 13/39
In the conformation observed in this crystal structure,
the substrate analog is accessible only to what would bethe cytosolic side of the intact membrane.
Residues essential for H+ binding are are also near the
middle of the membrane.
TDGsubstrateanalog
Lactose Permease PDB 1PV7
The substrate bindingsite is at the apex of an
aqueous cavity between
two domains, each
consisting of six trans-membrane a-helices.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 14/39
As in simple models ofcarrier 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
substrateanalog
Lactose Permease PDB 1PV7
conformationchange
conformationchange
Carrier-mediated solute transport
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 15/39
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.
Antiport (exchange diffusion) carriers
exchange one solute for another across a
membrane.
Usually antiporters exhibit "ping pong"
kinetics.
Antiport
A
B
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 16/39
Example of an antiport carrier:
Adenine nucleotide translocase (ADP/ATP exchanger)
catalyzes 1:1 exchange of ADP for ATP across the inner
mitochondrial membrane.
ATP 4
ADP 3
mitochondrialmatrix
adenine nucleotide translocase
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 17/39
Active transport enzymes couple net solute movement
across a membrane to ATP hydrolysis.An active transport pump may be a uniporter or
antiporter.
S1 S2
ATP
ADP + Pi
Side 1 Side 2
Active
Transport
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 18/39
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, whichcatalyze transmembrane movements of various organic
compounds including amphipathic lipids and drugs,
will not be discussed here.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 19/39
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.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 20/39
P-class pumps (cont):
Ca++-ATPases, in endoplasmic reticulum (ER) and
plasma membranes, catalyze ATP-dependent
transport of Ca++
away from the cytosol, into theER 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.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 21/39
The reaction mechanism
for a P-class ion pump
involves transientcovalent modification
of the enzyme.
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.
P-Class Pumps
ATP
C
O
O P O-
O-
O
C
O
OH
ADP
Enzyme-
Enzyme-
Pi
H2O
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 22/39
In this diagram of SERCA reaction cycle,
conformational changes altering accessibility of
Ca++-binding sites to the cytosol or ER lumen aredepicted as positional changes.
Keep in mind that SERCA is a large protein that
maintains its transmembrane orientation.
The ER Ca++ pumpis called SERCA:
Sarco(Endo)plasmic
R eticulum
Ca++-ATPase.
E
E-Ca++
2
2Ca++
ERcytosol membrane lumen
2Ca++
E~P-Ca++2 E~P-Ca++
2
ADP
Pi
ATP
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 23/39
Reaction cycle:
1. 2 Ca++ bind tightly
from the cytosolic
side, stabilizing the
conformation that
allows ATP to react
with an active site
aspartate residue.
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
E-Ca++2
2Ca++
ERcytosol membrane lumen
2Ca++
E~P-Ca++2 E~P-Ca++
2
ADP
Pi
ATP
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 24/39
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.
E
E-Ca++
2
2Ca++
ERcytosol membrane lumen
2Ca++
E~P-Ca++
2 E~P-Ca++
2
ADP
Pi
ATP
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 25/39
This X-ray structure
of muscle SERCA
(Ca++-ATPase) shows
2 Ca++
ions (coloredmagenta) bound between
transmembrane a-helicesin the membrane domain.
2 Ca++
Asp351
Muscle SERCAPDB 1EUL
membrane
domain
cytosolicdomain
Active site Asp351, which is transiently phosphorylated
during catalysis, is located in a cytosolic domain, far
from the Ca++ binding sites.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 26/39
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.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 27/39
This simplified cartoon represents the proposed
variation in accessibility & affinity of Ca++-binding sitesduring the reaction cycle.
Only 2 transmembrane a-helices are represented, and thecytosolic domain of SERCA is omitted.
Ca++
enzyme phosphorylation phosphatehydrolysis
SERCA Conformational Cycle
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 28/39
More complex diagrams & animations have beencreated 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).
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 29/39
Channels cycle between open & closed conformations.
When open, a channel provides a continuous pathwaythrough the bilayer, allowing flux of many ions.
Gramicidin is an example of a channel.
closed
conformation
change
open
Ion
Channels
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 30/39
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-
D-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).
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 31/39
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 ina lipid bilayer separating 2
compartments containing salt
solutions.
Gramicidin dimer
(PDB file 1MAG)
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 32/39
With voltage clamped at some value, current (ion flow
through the membrane) fluctuates.
Each fluctuation, attributed to opening or closing of onechannel, is the same magnitude.
The current increment corresponds to current flow
through a single channel (drawing - not actual data).
c u r r e n t
time
ion flow
through one
channel
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 33/39
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.
open closed
Proposed mechanism of
gramicidin gating
Gating (opening &closing) of a
gramicidin channel
is thought to
involve reversibledimerization.
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 34/39
Channels that are proteins
Cellular channels usually consist of large protein
complexes with multiple transmembrane a-helices.
Their gating mechanisms must differ from that ofgramicidin.
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)
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 35/39
Patch
Clamping
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.
electrode
electrode electrode
A
B
glass pipet
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 36/39
Patch
Clamping
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.
reference
electrode
membrane patch
electrode in
patch pipet
amplifier with
voltage control
salt solution
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 37/39
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 & closedstates reflects the rate of ion flux through one channel.
View a video of an oscilloscope image during a patch
clamp recording.
open
closed c u r r e n t
ime
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 38/39
Patch clamp recording at 60 mV. Consecutive traces
are shown. Note that at a negative voltage, increased
current is a downward deflection.
20
pA
102 msec per trace
8/12/2019 4 Transport
http://slidepdf.com/reader/full/4-transport 39/39
Current Amplitude
Histogram
Occupancy of differentcurrent 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 thechannel is closed, is due to
leakage of the patch seal and
membrane permeability. -60 -55 -50 -45
A lit d i A
O c c u p a n c y o f c u r
r e n t l e v e l s