1
CARRIER-MEDIATED TRANSPORT
MBOC Chapter 11
2
Goals for this class
1. How to recognize when transport is protein-mediated.
2. Understanding the major differences between the kinetic
behavior of channels and carriers?
3. How to distinguish active and passive transport.
3
Channels and Carriers
There are two classes of proteinmediated transport systems:
1) channels
2) carriers
4
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.
Channels have very high kcat approaching 105 per sec.
5
Carriers
Passive
Sugar Transporters Amino acid transporters
Nucleoside transporters
Purine transporter(s)
Pyrimidine transporter(s) Nucleotide transporter(s)
Amine Transporter(s)
Na/Ca exchanger(s) Na/H exchangers
Cl/HCO3 exchanger(s)
Active
Na,K,Cl co-transporter(s) Phosphate, H co-transporter(s)
Neurotransmitter/cation co-transporter(s)
Sugar/cation co-transporters
Amino acid/cation co-transporters
Cation ATPase(s) (Na/K, Ca, H)
Drug resistance transporter (P-glycoprotein(s))
What are the properties of carrier-mediated
transport systems?
DEFINITIONS:
Intracellular - SIDE 1
Extracellalar – SIDE 2
2
1
CFTR protein expression in well-differentiated human
bronchial epithelial cultures. Well-differentiated
cultures derived from human bronchial epithelial tissues
were immunostained with CFTR and tubulin antibodies
and analyzed on a Leica SP2 laser confocal microscope
with three independent laser lines (364, 488, and 568
nm). The image represents an overlay of the DIC
(grayscale), CFTR (redorange), cilia (tubulin, green),
and nuclei (DAPI, blue) confocal planes and depicts an
epithelial cell sheet that contains a group of ciliated
cells surrounding a goblet cell (bottle-shaped cell with
no cilia). CFTR is expressed only at the apical
membrane of ciliated cells, but not goblet cells.
Magnification x190.
from: Mol. Biol. Cell 2005 16: 2154-2167
DEFINITIONS:
trans - opposite side of the membrane
cis – same side of the membrane
transport is always measured in the direction cis to trans
Transport is like a reversible reaction
j2→1


S2 

 S1
j
1→2
Both unidirectional uptake and efflux proceed simultaneously. When
uptake (j2->1) is equal to exit (j1->2) the system is at equilibrium.
Unidirectional transport is measured in the direction cis to trans using
radiotracers
NET transport describes a measurement in which the difference between
unidirectional uptake and exit is measured.
Net uptake =j2→1 − j1→2
A positive value indicates net uptake, a negative value would indicate
net exit
Transport (like all enzyme catalyzed reactions) is a
saturable process
80
v=V
[S]/(K +[S])
max
m
60
6
60
70
uptake pmol/10 cells/s
70
6
uptake pmol/10 cells/s
80
50
40
30
carrier-mediated
20
50
40
20
10
10
0
0
20
30
[S]
40
50
leakage
0
80
70
60
6
10
uptake pmol/10 cells/s
0
v = k[S]
30
50
40
v = k[S] +
V [S]/(K +[S])
30
max
m
20
10
carrier-mediated + leakage
0
0
10
20
30
[S]
40
50
10
20
30
[S]
40
50
mM NaCN plus 4 pg. ml" FC
uptake measurements at 37 or a
What are the properties of carrier-mediated
poisoned cells at 37 "C follow
characterized by rate constants
transport systems?
respectively. Transport stimula
fold) are indistinguishable. Th
cose spaces of controland po
s+ 2 1 0 4 4
The e.S2 complex
a
indistinguishable and are nots
wateracontent
of the erythrocy
In order for substrate to be translocated, it must first form
complex
uptake by control and poison
with its carrier. How do we know this?
"0 loo
0
4
4
control cells at ice-temperatur
0
50
100
150
200
250
suming that thefree water cont
are identical, k for uptake on i
a. Saturable substrate uptake Time min
represents a 90-fold reduction
uptake.
These results indicate that s
cytes is passive (the equilibrium
not exceed the water content o
an activation energy of 20 kcal
range 4-37
"C), and(1993)
is stimulat
Diamond
& Carruthers
J.abolic
Biol. Chem
268, 6437-6444
poisons.
Theseresults
whether sugar uptake occurs vi
pathways.
Fig. 1B illustrates arepresent
time course of sugar uptake sti
0 10 20 30 40 50 60
FCCP was determined. Uptake
[3-O-methylglucose],mM
presence or absence of 50 FM c
FIG. 1. Effects of FCCP (4 pg.ml-') and cyanide (1 mM) protein-mediated sugar transpo
poisoning on 3-0-methylglucose uptake by erythrocytes ob- exposed either to FCCP (4 pg. m
tained from a single pigeon. A, time course of 0.8 mM 3-0- alone. Aliquots of the suspens
methylglucose uptake by control (0,A) and poisoned (0)cells. intervals and 3-0-methylglucos
Uptake was measured at 37 "C ( 0 , O )and at4 "C (A). Poisoned cells 10-min period. Based upon the
were treated with FCCP and CN for 2 h at 37 'C, and control cells
control cells during a 10-min i
[cytochalasin Blf,, =
where c p q , and cpmtOhlrefer to cpm p
and suspension samples, respectively. A
of this methodology is provided in Helg
b. Competitive-inhibition
uptake
3912 Biochemistry,of
Vol.
30,No. 16, 1991
(1987).
A
300
T
~ " " " " " " ' " " " " " " " " ~
6
3
200
t
1
-0.5
0
0.5
[D-Glucose] mM
1
c
1.5
FIGURE 4: D-Glucose dependence of zero-trans Dglucose uptake by
human erythrocytes. (A) Effects of maltose on D-ghCose uptake in
the absence of cytochalasin B. The data are presented as a Hanes-'
Woolf plot. Ordinate: [~-glucose]/rateof D-ghCOSe uptake in min
(assuming 2.8 X I O l 3 cells suspended in saline + 200 mM maltose
or sucrose contain 1 L of water). Abscissa: D-glucose concentration
in mmo1.L-I. Uptake in the absence of maltose is shown by the open
circles (0)and in the presence of 200 mM maltose by the filled circles
( 0 ) .The number of duplicate measurements per data point is 3 or
more. Each point represents the calculated mean f 1 standard error.
The straight lines drawn through the points were calculated by the
method of least squares. The following constants Characterize Dglucose uptake. Control:
= 0.069 mM, V, = 89.3 pmol.(2.8
X IO" cells)-Imin-'. Maltose: K, e
= 1.496 mM, V,,, = 80.1
pmob(2.8 X IO1) cells)-lmin-l. (B) P8ects of maltose on D-glucose
uptake in the
1 presence of cytochalasin B (998 i 24 nM). Ordinate
t
and abscissa as in (A). Uptake in the absence of maltose is shown
by the open
circles (0)and
in the presence
of 20Q1mM maltose1.5
by
-0.5
0
0.5
10 pL) ofofthe
suspension
Calculation
Michaelis
and were
Velo
rates" of trans
Sugarsedimented
Uptake. "Initial
by centrifugation,
from supernatant
at least two incubation
intervals
were sampled.
pendent
of time) (see
at each
[~-glucose]
extraction
above),
the sae
concentrations of [~-glucose]which, ba
tochalasin
calculated
experiments,
spanB]
thewas
range
[D-glucos
IlO
,,(,,,,were used. The [D[
glucose]
Carruthers
&K,Helgerson(1991)
were
analyzed
by3907-3915
linearization
[cytochalasin
Blf,, (Hane
=
Biochemistry
30,
glucose]/uptake versus [D-glucose])to o
and q\.Linear regression and nonline
whereand
c p unweighted)
q , and cpmtOhl
(weighted
wererefer
per
and
suspension
samples,
respe
software package KaleidaGraph 2.1
of this
methodology is provide
Reading,
PA).
(1987).
RESULTS
Calculation of Michaelis
Dependence
of D-Glucose
Uptake
o
"Initial
rates
Sugar Uptake.
tration.
Figure
4 shows
thatincubation
Pglucose u
from
at least
two
at ice-water temperature is well app
pendent of time) atand
each
[~saturation kinetics. Km(app)
Vmm
for
concentrations
of
[~-glucose
69 pM and 89.3 pmol.(L of cell water)
experiments,
span
the range
Nature
of Transport
Inhibitions
Prod
Cytochalasin
Maltose
lOK,,,(increases
,,,,were used
glucose]B.I
(V,, were
is unchanged)
analyzedwhile
by cytochalasin
linearizati
for sugar
uptake [Km(app)
is unchange
glucose]/uptake
versus
[D-glu
inhibitions
suggests
that
maltose
acts
and q\.Linear regression asan
of D-glucose uptake while cytochalasin
(weighted
unweighted)
petitive
inhibitor ofand
transport.
Ki(app)v
software
package
KaleidaG
cytochalasin B inhibitions of D-glucos
Reading,
PA).
periments
are 9.7
mM and 126 nM, r
Inhibitions of Transport Produce
Presence
of Maltose and Cytochalasin
RESULTS
rizes an experiment in which 0.1 mM
The first step in transport is thus formation of
the e.S2 complex
k−2
k1




e
+
S
e.S
e.M 2 
M
+





2
2
2
k
k
−1
2
dead-end
inhibited
complex
unoccupied
carrier
catalytic
complex
2000 single straight line with slope = l/Vma, and x intercept = -Km(app).
Interpretation of analysis for a one-site carrier mechanism.
e Interpretation of analysis for a two-site carrier mechanism.
The e.S1 complex
if a and X are unity. 'Interpretation if X is unity.
dInterpretation
1500 - L@)form
versus [SI, where dC- tP is
fData are plotted in the form [S]/(U'~
Intracellular
substrate
can
also
a complex
with over
its carrier. How do
the increment in unidirectional transport produced
by trans sugar
we know
in this?
the absence of trans sugar (u"). ZNot applicable here.
that observed
a. Saturable substrate efflux
Dependence of the initial rate of 30MG exit on
intracellular 3-O-methylglucose concentration. Ordinate:
sugar exit rate in pmol (L of cell water)-l min-l. Abscissa:
[30MGi] in mM. Exit is shown in the absence (0) and
presence (●) of 50 µM phloretin. In the presence of
phloretin, exit is described by the relationship, v (exit in
pmol (L of cell water) -l min-l) = KL[30MG], where KL was
calculated by linear regression (shown by the straight line
drawn through the points) as (4.4 ± 0.3) X l0-4 min-1. In the
absence of phloretin, exit is described by the linear
component described in the presence of phloretin plus a
saturable component with Km(app) of 0.9 ± 0.1 mM and
Vmax of 65 ± 8 pmol L-l min-l. Each point consists of
triplicate determinations shown as mean ± SD
Pod
mM
-
500
- 2 -
Helgerson & Carruthers (1989)
Biochemistry 30, 4580-4594
4: (A) Inh
(inverted triangles) a
D (open circles), an
rates of entry and ex
Abscissa: log [inhib
triplicate at 1 mM s
inhibition of exit by
inhibition = 87 i 6
= 0.1 1 0.02 pM,
of uptake by cytoch
FIGURE
[30MGJ
mM
3: Sugar uptake and efflux from rat erythrocytes. (A)
Dependence of the initial rate of DG uptake on DG concentration.
Ordinate: sugar uptake rate in pmol (L of cell water)-' min-I.
FIGURE
lo00
*
Biochemistry, Vol. 28, No. 11, 1989 4581
A
FIGURE 4: (A) Inhibition of 3-0-methylglucose exit by phloretin
(▽) and DG uptake by cytochalasins B (●), D (O), and E (∆).
Q-
Ordinate: inhibition of initial rates of entry and exit (%) and rate
of 30MG exit in pmol L-1 min-1. Abscissa: log [inhibitor] (M).
Uptake and exit were measured in triplicate at 1 mM sugar. The
curves drawn through the points are inhibition of exit by
phloretin, Ki(app) = 0.31 ± 0.02 µM, maximum inhibition = 87 ±
6%; inhibition ot entry by cytochalasin B, Ki(app) = 0.11 ± 0.02
µM, maximum inhibition = 96.4 ± 2.8%; inhibitions of uptake by
cytochalasins D and E (assuming 96% maximum inhibition), Ki
(app) = 125 ± 11 and 87 ± 18 µM, respectively. Points are shown
as mean & SD (for inhibition) of at least three determinations.
C
.-.e
c
.#
c)
-
1008
7
-log [ I ]
6
5
4
M
2500
(B) Effects of intracellular and extracellular [30MG] on Ki(app)
for CCB and phloretin inhibition of sugar transport. The initial
rate of either 30MG exit (●) or entry (O) at varying [30MG]
(0.1-5 mM) was measured over a range of [CCB](0-50 µM).
Ki(app) for inhibition of transport was obtained as -x intercept
from linear regression analysis of plots of [I] /inhibition of
transport versus [I], where I represents the inhibitor CCB. The
results are plotted in the form Ki versus [30MG]. The lines
drawn through the points were calculatd by the method of least
squares and in theory extrapolate to -Ki(app) for 30MG exit and
entry, respectively, on the x axis. Leakage-mediated sugar
fluxes (estimated as influx in the presence of 50 µM CCB and
exit in the presence of 50 µM phloretin) were subtracted from
i total fluxes to obtain "saturable" transport. The results are
Q shown as mean ±1 SD of three duplicate estimates.
800
2000
K
-
exit
entry
600
1500
i(app)
nM
).
-
-
400
lo00
500
-
200
- 2 - 1
-1
0
1
0
1
2
3
4
5
6
2
3
4
5
6
[30MG] mM
[3MG] mM
FIGURE 4: (A) Inhibition of 3-0-methylglucose exit by phloretin
(inverted triangles) and DG uptake by cytochalasins B (filled circles),
Helgerson & Carruthers (1989)
Biochemistry 30, 4580-4594
The first step in exit is thus formation of the
e.S1 complex
k−4
k3
4
k−3




e.CCB1 
CCB
+
e
+
S




 e.S1
1
1
k
dead-end
inhibited
complex
unoccupied
carrier
catalytic
complex
The transport reaction is thus seen as:
1
k1


e + S2 
e.S


2
k
external substrate binding
−1
2
3
k5


e.S2 
e.S


1
k
−5
k−3


e.S1 
e
+
S


1
k
translocation of bound substrate
release of translocated substrate
3
Questions
Can e.S1 and e.S2 co-exist?
Is there only one form of unoccupied carrier?
Can e.S1 and e.S2 co-exist?
Ligand binding studies help here
exofacial ligand
cell
endofacial ligand
permeant?
yes (ncma)
[3H]-CCB
D-glucose
yes (cmb)
D-glucose
maltose
no
ethylidene glucose yes (ncm, slow))
ATB-BMPA
ancm
(low affinity)
no
- non-carrier-mediated or diffusion; bcm - carrier-mediated
Exofacial photolabelling of human erythrocyte glucose transporter
centrifugation, and then the supernatant was removed and retreated to obtain a second precipitate. The pellets were washed
by resuspension in 2 xS50 1 of 0.5 % Mega 10 solution (as
described above) and the washings were combined with the first
supernatant. To the combined supernatant sample was added
15O Ial of solubilizing buffer containing 10% (w/v) SDS and
36% (v/v) glycerol, with 10% mercaptoethanol. Protein was
released from the antibody pellets (usually the combined first and
second pellets) with 200,ul of the same solubilizing buffer. Both
the supernatant and the pellet samples were then subjected to
electrophoresis. The positions of the photolabelled peaks were
compared with those of the molecular-mass-marker proteins
(BSA, 66 kDa; ovalbumin, 45 kDa; soya-bean trypsin inhibitor,
20 kDa; lysozyme, 14 kDa), as indicated in the Figures.
Electrophoresis
Samples in solubilizing buffer (see above) were run on 16 cm
100% (or 12 %) polyacrylamide gels using the Laemmli [28]
discontinuous buffer system. The gels were stained briefly with
Coomassie Blue, then destained and sliced (three slices/cm). The
slices (in scintillation vials) were dried at 80 ° C for 2-3 h, then
dissolved in 300 ,1 of H202 [containing 2 % (v/v) NH3 solution]
for a further 2-3 h at 80 'C. Liquid-scintillation fluid was then
added and the radioactivity was counted.
RESULTS
Structure and photodecomposition of ATB-BMPA
The structure of ATB-BMPA is shown in Fig. 1. It contains
two D-mannose groups linked through their C-4 hydroxy groups
to 2-propylamine. The photolabile 4-(1-azi-2,2,2-trifluoroethyl)benzoic acid group [29] is coupled to the amino group on the
bridge and between the two D-mannose groups. In this position
the bulky group does not interfere with the interaction of the
transporter with the hexose hydroxy groups, which are required
for binding [30].
By using our apparatus, the half-time of photolysis of ATBBMPA in water is approx. 30 s (Fig. 2). This is considerably
longer than the half-times observed with phenylazides [17] but, as
shown below, the resulting carbene is more selective than the
nitrenes derived from the activation of phenylazides.
Determination of the affinity constant for ATB-BMPA
We have used two methods to determine the affinity (or halfmaximal inhibition constant, K1) of the erythrocyte glucosetransport system for ATB-BMPA. Fig. 3 shows the inhibition of
100 /LM-D-galactose uptake at 20 'C. The calculated K1 was
297 + 53 ,/m. The new method for determining the same parameter is based on inhibition of the equilibrium binding of ASABMPA. The displacement of radiolabelled ASA-BMPA by non-
617
0.08
0.06
A 0.04
0.02
0
390
Wavelength (nm)
Fig. 2. Photochemical decomposition of ATB-BMPA
ATB-BMPA (150 /M in water) was irradiated in a Rayonet
photochemical reactor with 300 nm lamps for 0, 15, 30 and 45 s as
indicated. The absorption maximum at 350 nm gave an e value of
380 M-l cm-l
labelled ATB-BMPA gave a K1 of 368 + 59/tM at 20 'C. The
equilibrium binding was also studied at 0-4 'C, but no change in
the affinity constant was observed (K. = 338 + 37/M at 0-4 C)
(Fig. 4).
ATB-BMPA photolabelling
When ATB-BMPA was irradiated in the presence of intact
erythrocytes, good incorporation of radiolabel into the glucosetransport system occurred. No other membrane proteins were
labelled, and the photolabel was completely displaced from the
glucose transporter by high concentrations of cytochalasin B and
by D-glucose (Fig. Sa). Fig. 5(b) shows that the C-terminal
antibody that we have prepared precipitated approx. 64 % of the
glucose transporter after a single treatment. A second treatment
(pellet 2 in the Figure), gave a further 20 % (560% of the first
supernatant). Thus the combined treatments gave over 800%
precipitation. Photolabelled transporter was not precipitated by
preimmune serum (results not shown).
Fig. 5(c) shows that the new photolabel, ATB-BMPA, is
60
40
A
n
co
20
N
-N
Fig. 2.
Some substrates (D-glucose and D-galactose) and inhibitors (maltose, ethylidene
glucose,
0
200 400 600 800
phloretin and cytochalasin B) of the human red cell sugar transport system.
[ATB-BMPA] (pM)
OH
HO
0
1000
Fig. 3. Inhibition of D-galactose uptake by ATB-BMPA
The erythrocyte uptake of 100,UM-D-galactose was measured at a
range of ATB-BMPA concentrations. The reciprocal of the uptake
rate constant (slv) was plotted against the ATB-BMPA concenATB-BMPA
non-linear
tration. The K1 was determined
fitting to
by
Fig. 1. Structure of ATB-BMPA
regression
the Michaelis-Menten equation. The Ki was 297 + 53 /SM (from two
experiments with triplicate observations at each inhibitor concentration); results are means and the bars represent the S.E.M. of the
six observations at each concentration.
[CCB]free/[CCB]bound
Non-permeant sugars inhibit CCB binding to the sugar transporter
export site.
add external sugar
18
ethylidene glucose 24ºC
D-glucose 24ºC
maltose 24ºC
D-glucose at 4ºC
Helgerson, A. L., and
Carruthers, A. (1987)
Equilibrium ligand binding
to the human erythrocyte
sugar transporter. Evidence
for two sugar-binding sites
per carrier J. Biol. Chem.
262, 5464-5475.
by D-glucose (Fig. Sa). Fig. 5(b) shows that the C-terminal
interfere with the interaction of the
antibody that we have prepared precipitated approx. 64 % of the
19
hydroxy groups,
which
are required
glucosephotoreactive
transporter after a single
A cell–impermeant
bis–mannose
derivative
treatment. A second
treatment competitively
(pellet 2 in the Figure), gave a further 20 % (560% of the first
inhibitsofgalactose
uptakeThus
by the
erythrocytes
(below)
but800%
its labeling of
the half-time of photolysis
ATBsupernatant).
combined treatments
gave over
. 30 s (Fig. 2). This
is considerably glucose
precipitation.
Photolabelledistransporter
was not
by
erythrocyte
transporter
abolished
byprecipitated
cytochalasin
B (next page).
bserved with phenylazides [17] but, as
preimmune serum (results not shown).
carbene is more selective than the
Fig. 5(c) shows that the new photolabel, ATB-BMPA, is
ctivation of phenylazides.
y constant for ATB-BMPA
ods to determine the affinity (or halfnt, K1) of the erythrocyte glucose-
BMPA. Fig. 3 shows the inhibition of
at 20 'C. The calculated K1 was
thod for determining the same paron of the equilibrium binding of ASAof radiolabelled ASA-BMPA by non-
PA
60
40
A
n
co
20
N
-N
0
0
200 400 600 800 1000
[ATB-BMPA] (pM)
Fig. 3. Inhibition of D-galactose uptake by ATB-BMPA
The erythrocyte uptake of 100,UM-D-galactose was measured at a
range of ATB-BMPA concentrations. The reciprocal of the uptake
rate constant (slv) was plotted against the ATB-BMPA concentration. The K1 was determined by non-linear regression fitting to
the Michaelis-Menten equation. The Ki was 297 + 53 /SM (from two
experiments with triplicate observations at each inhibitor concentration); results are means and the bars represent the S.E.M. of the
six observations at each concentration.
Holman, G. D., Parkar, B. A., and Midgley, P. J. (1986) Exofacial photoaffinity labelling of
the human erythrocyte sugar transporter Biochim Biophys Acta 855, 115-26.
20
Red cell glucose transporter was photolabelled
with 3H-ATB-BMPA in the presence of increasing
D-[glucose] or in the presence of 50 µM CCB (☐).
Membranes were harvested, proteins separated
by SDS-PAGE and each lane of the gel was
sliced into 2 mm sections and counted.
24
20
0
_
I
200
600
^|~~~p
400
[ATB-BMPA] (pM)
66 kDa
V
45 kDa
*
V
a*
*
0
64-
-6E 48.
E
2._
'5 320
16
0
l~
0
4
Fig. 4. Inhibition of ASA-BMPA binding by ATB-BMPA
The binding of 0.1 ,uCi (1O pmol) of ASA-BMPA to 200 #1 of a
80 %-cytocrit erythrocyte suspension was measured over a range of
ATB-BMPA concentrations. The free/bound ratio was calculated
by comparison with [14C]sucrose as described in the Materials and
methods section. The free/bound ratio in the presence of ATBBMPA was multiplied by the bound/free ratio in the absence of
inhibitor (n) and was plotted against the ATB-BMPA concentration.
The K1 was calculated by non-linear regression fitting to the
Michaelis-Menten equation. At 20 ° C (0) the Ki = 368+ 59/tM
(from observations at 16 ATB-BMPA concentrations). At 0 ° C (M)
the K1 = 338 + 37 /uM (from observations at 14 ATB-BMPA
concentrations).
Q
E 16
-6
, 12
0
V
I
._2
I
10 12 14 16 18 20
Slice no.
x
I
8
cr
x
_
4
I
6
0
4
V
66 kDa
8
then all the sites would covale
BMPA.
V
45 kDa
12
16
.5
0
'D
0
^
E
,D
Enzyme treatments
Fig. 6(a) shows trypsin treatm
ATB-BMPA-labelled cells. The
converted into an 18 kDa frag
labelled 50 kDa transporter rem
we treated isolated membranes
them with ATB-BMPA subseque
less conversion into the 18 kDa p
was labelled first.
Fig. 6(a) also shows that, aft
BMPA-labelled glucose transpo
region of the gel, and the 18 kD
4 kDa C-terminal peptide, as
tated by the anti-(C-terminal pe
We also studied the proteolys
Slice no.
16
12-
0
4-
0-
(c)
(a) ATB-BMPA photolabelling of the glucose transporter in intact erythrocytes. The labelling by ATB-[2-3H]B
of D-glucose (-) and at 1 mM- (0), 5 mM- (A), 25 mM- (-) and 100 mM- (A) D-glucose. Binding and labelling w
cytochalasin B (Ol). Membranes were prepared and the labelled proteins were analysed by electrophoresis. On
of the electrophoresis gel is shown for clarity. There were no other labelled peaks in other regions of the gel.
BMPA-labelled glucose transporter. Glucose transporter was labelled in intact erythrocytes by ATB-[2
suspensions were prepared. Glucose transporter was then immunoprecipitated with anti-(C-terminal pepti
Materials and methods section. The radioactivity was mainly associated with the first pellet (@). The super
treatment with antibody gave a second pellet (0), but some transporter remained in the supernatant (A).
electrophoresis. (c) ATB-BMPA photolabelling of the glucose transporter in isolated erythrocyte membranes. Me
buffer were labelled with ATB-[2-3H]BMPA in the presence of 300 mM-D-mannitol (-) or 300 mM-D-glucos
washed in phosphate buffer and analysed by electrophoresis. In this and succeeding Figures the arrowheads (V
proteins (see the text).
Fig. 5. Photolabelling of the glucose transporter
Holman, G. D., Parkar, B. A., and Midgley, P. J. (1986) Exofacial photoaffinity labelling of
the human erythrocyte sugar transporter Biochim Biophys Acta 855, 115-26.
21
These data suggest that the cytochalasin B (export) and bis-mannose
(import) sites are mutually exclusive.
The reaction thus far
ATB-BMPA.e
ATB-BMPA+ e +S2
e.S2
CCB.e
CCB+ e +S1
e.S1
is there only one form of e
or does e undergo
conformational changes in
the absence of S?
A quick review of Channels
S1
E
ES
E
k 2
àààà
Ü
á
ààà
à
k S
k1 S 1
ààà
Ü
á
àà
à
k
2 2
1
The simple channel contains only a single form of E (unoccupied channel).
Lieb and Stein demonstrate the following
(Lieb, W. R., and Stein, W. D. (1974) Testing and characterizing the simpler pore, Biochim. Biophys. Acta 373, 165-177.)
Experiment
Vmax
Parameter
Value
substitute numbers
Zero-trans
exit
V12zt
[Et ]k−2 [Et ] * 4 = 4[Et ]
Zero-trans
entry
V21zt
[Et ]k−1 [Et ] * 2 = 2[Et ]
equilibriumexchange
V
ee
[Et ]
k−1k−2
k−1 + k−2
[Et ]
2*4 8
= [E ]
2+4 6 t
thus Vexchange < Ventry or Vexit
S2
23
Why is Vexchange < Ventry or Vexit?
direction of transport measurement
S1
e.g. Na22
γ-emitter
S2
ES
e.g. Na23
When S2 > S1 the unidirectional flow of S in the direction 1 to 2 is
impeded because the channel becomes filled at side 2 and, since S1
and S2 cannot pass one-another in the single-file channel, S2 blocks the
flow of S1 from side 1 to 2.
Thus Na23 at side 2 will inhibit unidrectional efflux of Na22 from cells
when mediated by a channel.
Thus extracellular Cs inhibits unidirectional K efflux through voltage-gated
K channels in squid giant axons.
Adelman, W. J., Jr., and French, R. J. (1978) Blocking of the squid axon potassium channel by external caesium ions J
Physiol 276, 13-25.
24
Returning to Carriers
For carrier systems, the presence of transported substrate at
the interior of the cell can be without effect on, inhibit or even
stimulate substrate influx! Let’s examine this with a carriermediated transport mechanism – the sugar transporter.
25
26
27
Internal dialysis of the giant axon of L. forbesi
porous
dialysis tubing
nonporous
dialysis tubing
0.6-1.2 mm
≥ 10 cm
Unidirectional 14C-3MG uptake by an internally dialyzed giant axon and
effect of intracellular unlabelled sugar
C-3MG outside, varying [12C-3MG] inside, collect isotope taken up by the cell
14
Baker, P. F., and Carruthers, A. (1981) 3-O-methylglucose transport in internally dialysed giant axons of Loligo
J. Physiol. (Lond.) 316, 503-525.
28
unidirectional 14C-3MG exit by an injected giant axon and effect of
extracellular sugar
Key:
2DODG = 2-deoxy-D-glucose
3OMG = 3-O-methylglucose
DG = D-glucose
C-3MG inside, varying [12C-sugars] outside, collect
isotope exported by the cell
14
Baker, P. F., and Carruthers, A. (1981) Sugar transport in giant axons of Loligo J. Physiol. (Lond.) 316, 481-502.
29
30
31
Red cell reversible hemolysis
Artificial cytosol
+
transport
Substrate
4 ˚C
hypotonic
lysis
red cell
in
r
e
t
a
b
fo
40
at
37
ºC
m
u
c
n
i
substrate
resealed “ghost”
measure
transport
(substrate efflux)
Figure 4 unidirectional 3MG exit from rat red cells and effects of
extracellular sugar
C-3MG inside, varying
[12C-sugar] outside,
collect isotope
exported by the cell
14
Key to external sugars:
⃝
▲
Glucose
3OMG
Helgerson, A. L., and Carruthers, A. (1989) Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the
alternating conformation carrier model for sugar transport Biochemistry 28, 4580-4594.
32
33
The Carrier
One simple mechanism that can account for this result is called
the simple carrier which is shown in schematic and in KingAltman forms below.
OUT
e2.M
e2.S
e2
ko
k-o
k1
k-1
e1
e1.S
e1.CCB
IN
The key to this mechanism is that
in the absence of substrate, the
carrier, e, can exist in one of two
forms - e1 or e2 - that present a
substrate binding site to either
side 1 or to side 2 of the
membrane (where the subscripts 1
and 2 refer to intracellular and
extracellular sides respectively).
Cytochalasin B (CCB) binds only
to e1 and bismannose (M) binds
only to e2.
34
35
How can this model account for transacceleration of fluxes?
Consider an influx cycle in the absence of intracellular substrate (S1).
The cycle is the following:
e2
e.S2
Substrate
Binding
FAST translocation
2 msec
Substrate
Translocation
e.S1
e1
SLOW Relaxation
200 msec
e2
Substrate
Dissociation
NOTE: The substrate binding association and dissociation steps are orders of
magnitude faster than translocation steps.
36
Trans-acceleration measure
uptake into cells
e.g. rat eythrocytes containing saturating 12C-D-glucose
14C-D-glucose
FAST forward
translocation of 14C-sugar
e2
Step
1
e.S2
Substrate
Binding
2 msec
Substrate
Translocation
e.S1
e1
Substrate
Dissociation
e1
Step
2
e2
or
Step
2’
e.S2
Substrate
Dissociation
FAST reverse
translocation of 12C-sugar
e.S1
2 msec
Substrate
Translocation
SLOW Relaxation
200 msec
e2
e1
Substrate
Binding
The reverse translocation occurs more rapidly than does forward relaxation. Uptake of radiolabeled
extracellular sugar (S2) is stimulated by the presence of saturating unlabeled intracellular sugar (S1).
No Trans-effect
e.g. uptake by squid
axons
e2
e.S2
37
measure 14C-D-glucose uptake into cells
containing saturating 12C-D-glucose
MEDIUM forward
translocation of 14C-sugar
e.S1
e1
100 msec
step 1
Substrate
Binding
Substrate
Translocation
Substrate
Dissociation
e1
MEDIUM Relaxation
100 msec
e2
step 2
e2
e.S2
MEDIUM reverse
translocation of 12C-sugar
e.S1
e1
100 msec
or
step 2’
Substrate
Dissociation
Substrate
Translocation
Substrate
Binding
Translocation and relaxation reactions occur at identical rates.
Unlabeled S1 has no effect on labeled S2 uptake.
38
Trans-inhibition
e2
e.S2
SLOW forward
translocation of 14C-sugar
e.S1
180 msec
e1
step 1
Substrate
Binding
Substrate
Translocation
Substrate
Dissociation
e1
Fast Relaxation
20 msec
e2
step 2
SLOW reverse
translocation of 12C-sugar
e2
e.S2
e.S1
e1
180 msec
or
step 2’
Substrate
Dissociation
Substrate
Translocation
Substrate
Binding
Translocation is slower than relaxation. Unlabeled S1 will inhibit labeled
S2 uptake. Not seen (AFAIK) for sugar uptake in any cell. Seen with sugar
exit in squid giant axons.
39
Examples of passive transport systems that are consistent with this
model include:
Trans-acceleration:
Sugar transport in human red cells, glycine transport in neurons,
nucleoside transport in red cells.
No Trans-effect:
Sugar transport in muscle and adipose.
Trans-inhibition:
Sugar transport in giant axons of Loligo forbesi
40
A variation on the simple carrier mechanism ANTIPORT
While the simple carrier model can account for the properties of a large
number of passive transport systems, a number of passive transporters
cannot transport substrate in the absence of substrate at the opposite (trans)
side of the membrane. With a slight modification, the simple carrier model
can account for these transport systems.
Consider the following model:
e2.S
OUT
e2
k1
k-1
e1
IN
e1.S
41
The difference between this model and the simple carrier is that unoccupied carrier isoforms e1 and e2
cannot isomerize in the absence of substrate. This is known as the exchange-only simple carrier. In order
for multiple rounds of substrate influx to occur, e1 must be converted back to e2 by transporting S1 out of
the cell.
e2.S
OUT
e2
k
3
-3
k
k1
k-1
-4
k
e1
IN
k
4
e1.S
You might suspect that this transport system would lead to futile cycling of substrate. However, what
normally occurs is that one substrate is transported into the cell in exchange for a different substrate
being transport out.
42
An example is Na:Ca exchange.
Here 1 Ca2+ is extruded from the
cell in exchange for 3 Na+.
The inwardly directed Na
electrochemical gradient is used to
do useful work - pump Ca out of the
cell!
What is the equilibrium distribution of Na and Ca produced by such a carrier?
If the membrane potential were 0, then at
equilibrium, passive transport requires
Na3iCao = Na3oCai or
3
Na i
Cai
3 =
Na o
Cao
In other words, Ca2+ is accumulated at one side of the membrane to an extent proportional to Na+
accumulation at the same side. If Nao/Nai = 10 then Cao/Cai = 103 = 1000.
The generic solution (where m molecules of S are
exchanged for n molecules of P) is:
m
n
S
P
m n
m n
i
i
Si Po = S o Pi or m = n
So
Po
43
.AI+O
EXCHANGE
44
Let us examine this Ca:Na exchanger in a little more detail. It seems obvious
that if 3 Na+ are exchanged for 1 Ca2+, the transport system must carry one
net positive charge during the transport cycle. This must mean that the rate
of transport is affected by membrane potential ( ∆Ψ). How can we analyze
such a system?
Let us make the following assumptions:
1.
The net charge on the Ca-occupied carrier, ZECa = 0
2.
The net charge on the Na-occupied carrier, ZE3Na = +1.
When ∆Ψ = 0 mV, the net charge carried by E3Nai or E3Nao will have no
effect on the rates of carrier isomerizations (k2 and k-2).
45
When ∆Ψ =-60 mV (inside), k-2 will be decreased to some extent (the positive
charge will be attracted by the negative potential inside the cell) and k2 will be
increased to the same extent (the positive charge will be attracted by the negative
potential inside the cell). Because ZECa= 0, k1 and k-1 are unaffected.
When ∆Ψ = +60 mV (inside), k-2 will be increased to some extent and k2 will be
decreased to the same extent. Because ZECa= 0, k1 and k-1 are unaffected.
Thus we would predict:
1. When ∆Ψ is negative (inside), Nao:Cai exchange (external Na dependent Ca
efflux) is stimulated. Thus hyperpolarization stimulates Nao:Cai exchange.
2. When ∆ Ψ is negative (inside), Nai:Cao exchange (external Ca dependent Na
efflux) is inhibited. Thus hyperpolarization inhibits Nai:Cao exchange.
3. When ∆ Ψ is positive (inside), Nao:Cai exchange (external Na dependent Ca
efflux) is inhibited. Thus depolarization inhibits Nao:Cai exchange.
4. When ∆ Ψ is positive (inside), Nai:Cao exchange (external Ca dependent Na
efflux) is stimulated. Thus depolarization stimulates Nai:Cao exchange.
These predictions are observed experimentally (See next pages).
46
Experimental arrangement
Voltage-clamped giant axon injected with 22Na or 45Ca and exit of
cation measured into media of varying [Ca] or [Na] at different Em.
SEAWATER
FLOWCOLLECTION
VOLTMETER
AXON
.A
or
45
Ca
VOLTAGEELECTRODES
CURRENTELECTRODES
CURRENT
47
Allen, T. J., and Baker, P. F. (1986) Comparison of the effects of potassium and membrane potential on the calcium-dependent sodium
efflux in squid axons J Physiol 378, 53-76.
48
Allen, T. J., and Baker, P. F. (1986) Influence of membrane potential on calcium efflux from giant axons of Loligo J Physiol
378, 77-96.
49
Examples of exchange only systems:
a.
ATP/ADP exchanger of mitochondria
b.
Anion exchanger of red cells
c.
Na/H exchanger of most cells
d.
Na/Ca exchanger of neurons.
50
51
A PRIMARY ACTIVE CARRIER MECHANISM
ATP Hydrolysis Drives the Transport of Sodium and Potassium Ions Across the
Plasma Membrane
Most cells have a high concentration of K and a low concentration of Na relative to
the external medium. These transmembrane ionic gradients are generated by a
specific transport system that is called the Na,K pump because the movement of
these ions is linked.
The active transport of Na and K is of great physiologic importance. Indeed, more
than a third of the ATP consumed by a resting animal is used to pump these ions.
The Na K gradient in animal cells controls cell volume, renders nerve and muscle
cells electrically excitable and drives the active transport of sugars and amino acids.
52
In 1957 Jens Skou discovered an enzyme that hydrolyzes ATP only if Na and
K are present in addition to Mg, which is required by all ATPases. This
enzyme was named the Na+K+ATPase.
53
Thus the reaction scheme was viewed as
Na+, K+, Mg2+
ATP + H2 O
ADP + Pi + H+
It was proposed that the Na+K+ATPase is an integral part of the Na K pump and
that the splitting of ATP provides the energy needed for the active transport of
these cations. This is important because in order to move Na against an
electrochemical gradient, the cell requires an input of energy. Similarly, for the
transporter to move K from the outside of the cell to the inside of the cell against
an electrochemical gradient, an input of energy is needed.
This hypothesis has been supported by the finding that the level of ATPase’s
activity is correlated with the level of pump activity. Also, variations in Na and K
levels have parallel effects on the ATPase activity in transport. Finally both the Na
+ +
K ATPase and the NaK pump activities are specifically inhibited by cardiotonic
steroids.
54
The isolation of the Na+K+ATPase and its reconstitution into artificial lipid bilayers
confirms that the ATPase and the pumping activity are mediated by the same
molecular complex, the NaK pump. The results of these various studies have
confirmed the identity of the Na+K+ATPase and the NaK pump and have revealed new
insight into the class of transport mechanisms called carriers.
The NaK pump consists of two types of subunits: α and β subunits that are
associated in the membrane as an α2β2 tetramer. Hydrophobicity analyses and
membrane localization studies suggest that the α chain contains at least eight
transmembrane helices.
Much of the α chain plus its ATPase
activity is located on the cytosolic side of
the membrane. The small portion of the
α chain on the extracellular side
contains the binding site for cardiotonic
steroid inhibitors. The β chain which
contains a single membrane helix does
not appear to be essential for ATPase or
transport function.
55
56
What is the catalytic mechanism of Na and K pumping? An important clue is the
discovery that the ATPase is phosphorylated by ATP in the presence of Na and Mg.
The site of phosphorylation is the side chain of a specific aspartate residue. This βaspartylphosphoryl intermediate or EP is then hydrolyzed if K is present.
Phosphorylation does not require K whereas dephosphorylation does not require Na
or Mg.
The reaction scheme can thus now be viewed as:
57
Na-dependent phosphorylation and K-dependent dephosphorylation are not
the only critical reactions. Because the pump serves to extrude Na and to
import K, it is reasonable to conclude that Na binds at the cytosolic surface
of the transporter and dissociates at the exoplasmic surface of the ATPase
while K binds at the exofacial surface and is released at the endofacial
surface of the transporter.
Kinetic evidence also suggests that the pump interconverts between two
states: e1 and e2. Na binds to the e1 or intracellular state while potassium
binds to the e2 or extracellular state of the carrier. The catalytic cycle may
now be represented by:
58
The binding cavity alternately faces the inside and outside of the cell in each
transport cycle
Insufficient data exist to form a detailed structural model for NaK pumping.
However, the following model was proposed by Jardetzky and by Glynn.
1) The pump must contain a cavity large enough to permit entry of a small
molecule.
2) The pump must be able to assume 2 conformations such that the cavity is open
to the inside in one form and to the outside in the other form.
3) The affinity for the transported species must be different in the 2 conformations.
59
60
Modified from: Structural changes in the calcium pump accompanying the dissociation of calcium
Chikashi Toyoshima & Hiromi Nomura; NATURE 418 605 (2002)
61
Pump Stoichiometry and thermodynamics
Measurements of electrogenic Na and K movements (net current movement is
generated during transport) support the view that 3 Na and 2K are transported with
each full cycle of the pump. In the red cell, the free energy change for Na transport
from 15 mM inside the cell to 140 mM outside the cell at a membrane potential (V) of
–30 mV inside is given by:
ΔG = RT ln
140
+ zFV
15
{where R is the gas constant (1.986 cal/deg/mol), T is absolute temperature (25˚C = 298˚K), F is the
Faraday (23060 cal/volt/mol), z is the valence of Na (+1), V = 0.03 (Na moved to the outside which
is +ve relative to inside)}
For Na, ∆G = 2.0 kcal/mol (at 25 ˚C) x 3 (3 mol Na are transported per cycle)
∆G = 6 kcal per cycle.
Similarly for K (120 mM inside, 5 mM outside)
∆G = 1.2 kcal/mol x 2 = 2.4 kcal per cycle.
Total ∆G = 2.4 + 6 kcal per cycle = 8.4 kcal
62
Cost of Transport = 8.4 kcal
In the red cell at 25 ˚C,
[ATP] = 5 x 10-3 M
[ADP] = 0.125 x 10-3 M
[Pi] = 1.25 x 10-3 M
∆G˚ for ATP hydrolysis = -7300 cal per mol
∆G for ATP hydrolysis =
[ADP][Pi]
ΔG = ΔG˚+RT ln
= −13443 cal per mol
[ATP]
Thus sufficient free energy is released upon hydrolysis of 1 molecule of ATP
to move 3 Na and 2 K against the prevailing electrochemical gradients.
63
Natural Inhibitors and their applications
The cardiotonic steroids obtained (originally) from the foxglove
Digitalis purpurea have been used as cardiac stimulants for
centuries.
Their application at low doses results in increased force of
contraction of heart muscle. It is, therefore, a drug of choice in
treating congestive heart failure. It is thought that inhibition of the
Na,KATPase results in diminished transmembrane Na gradients.
This in turn produces less Ca efflux via the Na/Ca exchange
transporter (an antiporter), raising sarcoplasmic Ca levels and
thereby enhancing contraction of the contractile apparatus.
Inhibition of Na reabsorption by the renal tubule results in diuresis
(water loss). This can be achieved by application of NaKATPase
inhibitors which block active Na transport out of the epithelial cell
into the interstitium or by application of substances such as
furosemide which inhibit Na and Cl symport from the renal tubule
lumen into the epithelial cell tubule .
64
Several other findings have been obtained:
1) N-ethylmaleimide inhibits the pump and traps Na22 in an occluded
(nonreleasable) state.
2) Intracellular vanadate inhibits the pump and traps Rb86 (a substitute for K) in an
occluded (nonreleasable) state.
3) In the absence of Ko and Ki and in the presence of high Nao and low Nai, the
pump synthesizes ATP from ADP and Pi. When the Na gradient is reversed in the
absence of K, ATP is hydrolyzed. Under both conditions, the pump catalyzes Na/Na
exchange.
4) In the absence of Na but in the presence of Pi, the pump catalyzes K/K
exchange.
5) E2.P dephosphorylation is inhibited by cardiotonic steroids
65
These allow further refinement of the model:
66
Co-transport Systems: Two substrates are carried on a single
transporter
These transport systems are exemplified by active sugar transport by
epithelial cells. Sugar transport in these cells is complicated by the
additional presence of a facilitated diffusion mechanism for sugars e.g.
In mammalian cells, active transport of 3-O-methylglucose and α
−methyl glucoside can occur and this is inhibited by phloridzin. Passive
transport is selective, only 3-O-methylglucose is transported and this is
inhibited by cytochalasin B. The passive transporter cannot transport α
−methyl glucoside and is relatively insensitive to phloridzin, while the
active transporter is relatively insensitive to cytochalasin B .
67
basal or
apical, mucosal or
68
A typical uptake experiment with cultured epithelial cells would
show the following:
Fig 11
3OMG + cytochalasin B
α MG -control
70
S1/S2
35
Phloridzin blocks accumulation
of both 3-O-methylglucose and
α-methyl glucoside.
3OMG - control
+ Phloridzin
0
0
minutes
In the absence of cytochalasin
B, 3-O-methylglucose is
transported out of the cells by
the passive transporter and is
thus not accumulated to the
same extent as α-methyl
glucoside.
60
Active transport of sugar (S2) is critically dependent upon the presence of
Na ions at side 2 of the membrane.
69
Uptake of S2 is more precisely
dependent upon the transmembrane
Na electrochemical gradient.
Accumulation of S is driven by the
downhill flow of Na.
Fig 12
[Νa] mM
2000
125
90
cpm of
3OMG
in cells
What is the free energy available in the
transmembrane Na gradient? This is
computed in the usual way:
55
1000
20
125 + phloridzin
0
0
minutes
10
Nain
ΔG = RT log e
+ ZFΔψ
Naout
where Z is +1; F (the Faraday) = 23,062 cal V-1mol-1; Nao/Nai ≈ 10; ∆Ψ = -70
mV.
∆G is approximately –2.98 kcal per mol at 25 ˚C. This is equivalent to an
equilibrium constant of:
Keq = 10
-∆G
2.303RT
= 153
Thus the inwardly directed Na electrochemical gradient would permit D-glucose levels
inside the cell 153-fold greater than those present outside.
70
Examples of cotransport systems
a.
Sugar/Na in mammalian epithelial cells.
b.
Amino acid/Na in mammalian cells.
c.
Neurotransmitter precursor/H symport into synaptic vesicles
d.
Neurotransmitter/Na symport brain
e.
Sugar/H+ and amino acid/H+ in bacteria.
71
Typical kcat values for carriers
Passive uniporters - 10 to 1000 per sec
Passive antiporters - 100 - 2000 per sec
Active transporters - 10 to 500 per sec
72
Points you should understand from this class and
the membrane dynamics class
1. Equilibrium distributions of solutes across cell membranes
and impact of membrane potential on the equilibrium
distributions of charged species
2. Experiments leading to the fluid mosaic model for membrane
structure
3. Properties of lipid bilayers that determine transbilayer
diffusion of molecules
4. How can you tell that transport is protein-mediated?
5. What are the major differences between the kinetic behavior
of channels and carriers?
6. How do you distinguish active and passive transport?
73
Landmark Papers
Abramson, J., Smirnova, I., Kasho, V., Verner, G., Kaback, H. R., and Iwata, S. (2003) Structure and
mechanism of the lactose permease of Escherichia coli Science 301, 610-5.
Huang, Y., Lemieux, M. J., Song, J., Auer, M., and Wang, D. N. (2003) Structure and mechanism of the
glycerol-3-phosphate transporter from Escherichia coli Science 301, 616-20.
Both bacterial transporters are members of the Major Facilitator
Superfamily of carriers. They show remarkably similar topologies
Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., and Gouaux, E. (2005) Crystal structure of a bacterial
homologue of Na+/Cl--dependent neurotransmitter transporters, Nature 437, 215-223.
This bacterial homolog of the Na-dependent neurotransmitter transporters
is completely unlike the two MFS transporters both in terms of topography
and quaternary structure.