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



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).



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



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.



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+

http://www.soils.wisc.edu/virtual_museum/valinomycin/index.html






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 +



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.



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



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




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



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.



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.



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.



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




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



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



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



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.



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 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.



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



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



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++


2Ca++

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

2

ADP

Pi

ATP



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++


2Ca++

E~P-Ca++

2 E~P-Ca++

2

ADP

Pi

ATP



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.



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.



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



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).

http://www.utoronto.ca/maclennan/rint1.htm

http://www.sgpweb.org/symposium2005.html

http://www.iam.u-tokyo.ac.jp/StrBiol/

http://www.iam.u-tokyo.ac.jp/StrBiol/

http://www.sgpweb.org/symposium2005.html

http://www.utoronto.ca/maclennan/rint1.htm



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



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).



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)



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



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.



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)



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



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



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

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/carriers.htm





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



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

4 transport

Documents