Fighting Fatigue

1

• Fighting Fatigue
• The Structure function and regulation of
  monocarboxylate (lactate) transporters

Andrew Halestrap Department of Biochemistry
University of Bristol
2
(No Transcript)
3
a-Cyano-4-hydroxycinnamate inhibits lactate and
pyruvate transport into mitochondria and red
blood cells
Mitochondria
4

• Use of BCECF to measure monocarboxylate transport

Cells loaded with the pH sensitive fluorescent
dye, BCECF
BCECF-AM
BCECF-AM
BCECF
Lactate
Lactate
Monocarboxylate transport measured using BCECF to
detect the decrease in pHi
pHi
5
Lactate transport into different cell
lines measured using BCECF fluorescence
ELT
COS
5mM L-lactate
NBL-1
440/490 ratio
NBL-1
440/490 ratio
COS
ELT
20 s
10
20
5
30
2
L-lactate
mM
1

• 4 min

6
Topology of MCT1
Confirmed by selective proteolysis and labelling
of red blood cells
K
K
K
K
413
413
290
290
D
D
302
302
R
R
R
P
R
P
F
F
R
R
360
360
313
313
N
N
C
C
7
Relatedness of human MCT isoforms
MCT8 is a thyroid hormone transporter and MCT10
(TAT1) i a sodium independent aromatic amino
acid transporter
The natural substrates for MCT5, MCT6 , MCT7,
MCT9 and MCT11-MCT14 are unknown but probably do
not include lactate and pyruvate
MCT8
MCT10
MCT9
MCT14
MCT5
MCT12
MCT11
MCT13

• MCT1

Known to transport lactate, pyruvate and ketone
bodies
MCT2
MCT3
MCT4
MCT6
MCT7
8

• Cladogram showing possible evolution of MCT
  family

Known to transport lactate, pyruvate and ketone
bodies
Human
C elegans
Bacteria
Not responsible for lactate transport
Yeast
9
Tissue distribution of MCT1 and MCT4

• Rat tissues

67kDa
43kDa
MCT1
30kDa
67kDa
43kDa
MCT4
30kDa
Msc
Liv
Hrt
10
MCT4 is expressed in white muscle and MCT1 in red
muscle
MCT1
MCT4
300
ST
200
MCT1
MCT expression as a percentage of
that in white tibealis anterior
100
SOL

• WG

WTA
EDL
PL
RTA
RG
SOL
100
MCT4
75
20mm
50
25
Succinate dehydrogenase
20
40
60
80
100
Percentage of slow oxidative
and fast oxidative glycolytic fibres
11
MCT1 in heart is concentrated at the intercalated
disks and t-tubules
12
MCTs are absent in b-cells of pancreatic islet of
Langerhans
Insulin
Glucagon
MCT1
MCT2
MCT4
13
Distribution of MCTs in rat testis and lung
MCT2

• MCT1

Testis X16
Testis X63
Lung X16
14
MCT2 distribution in coronal sections through rat
cerebellum
Higher staining intensity
d2-glutamate receptor
MCT2 peptide
MCT2 peptide

• MCT2

MCT2
Molecular layer
Purkinje cell bodies
Granule cell layer
100 mm
White matter
Data from Ole Ottersen's laboratory (Linda
Bergesen et al, 2001, Exp. Brain Res 136 523-534
15
MCT2 and the d glutamate receptor are colocalized
at synapses between parallel fibres and Purkinje
cell spines
MCT2 antibody peptide
GluR2/3 antibody
MCT2 antibody
200 nm
200 nm
200 nm
MCT2 d2-antibody
Mitochondrion
Parallel fibres
Purjinke cell spines
200 nm

• Data from Ole Ottersen's laboratory (Bergesen et
  al, 2001, Exp. Brain Res 136 523-534)

16
MCT2 has a C-terminal PDZ-binding motif

• MCT2 NTHNPPSDRGKESSI
• d2 LGLNLGNDPDRGTSI
• GluR2 YKEGINVYGIESVKI
• GluR3 YREGYNVYGTESVKI
• Kir.2.1 SPVLEPRPLRRESEI
• -----------------------
• MCT1 QQNSSGDPAEEESPV
• MCT4 PEKNGEVVHTPETSV

Does MCT2 bind to PSD 93 like the d2 glutamate
receptor?
17
Characterising the properties of individual MCT
isoforms
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Characterisation of MCTs by expression in Xenopus
laevis oocytes

• MCT cRNA

Microinject Xenopus oocytes with mRNA
MCT cRNA
Translation to MCT protein
MCT protein
Translocation to membrane
Measure transport with radiotracer or BCECF
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(No Transcript)
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• Kinetics of MCT4 expressed in oocytes

Km 28 mM
Km 153mM
Km 519 mM
22
Kinetics of L-lactate transport into Xenopus
oocytes transfected with MCT1, MCT2 and MCT4
5000
250
MCT2
4000
200
MCT1
3000
150
Lactate uptake via MCT1/4 (pmol/10 min per oocyte)
Lactate uptake via MCT2 (pmol/10 min per oocyte)
2000
100
MCT4
1000
50
Uptake measured with 14C-lactate
0
0

• 0

10
20
30
40
50
Lactate concentration (mM)
Stefan Bröer, Angelika Bröer, Hans-Peter
Schneider, Roger Dallwig and Joachim W. Deitmer,
Tübingen
23
Substrate affinities of MCT isoforms
200
150
Km mM
MCT1
MCT2
100
Ki mM
50
0

• L-Lactate

Pyruvate
Acetoacetate
D-Lactate
D-b-OHB
KIC
24
Anti-FLAG
Secondary only
MCT 8 injected
Water injected
25
Edith Friesma Theo Visser
26
Compartmentation of lactate metabolism in muscle
and brain
Neurons
Red muscle fibres
Interstitial fluid
Glucose
Glucose
Glucose
Lactic
Lactic
Lactic
Pyruvate
acid
acid
acid
MCT1
Oxidation
Blood
Endothelium
ATP
27
The lactate carrier is closely associated with
another membrane protein of the immunoglobulin
superfamily
28
CD147
CD147 (OX47) is a T-cell surface glycoprotein
that belongs to small subset of the
immunoglobulin superfamily.
Highly conserved transmembrane region
Widely expressed
May play a general role in cell surface
recognition but physiological ligand is not known
29
Colocalisation of MCT1 and CD147 in rat heart
cells and pancreatic islets
MCT1
Overlay
CD147
b-cells have little MCT1 or CD147
30
CD147 antibodies immunoprecipitate MCT1 and MCT4
but not MCT2
Y3
RBL
H4IIE
2
2
4
1
1
1
2
1

• Anti-MCT

kDa
45
31
HELA
CD147 is required to enable translocation of MCT1
and MCT4 to the plasma membrane
HELA cells (Tet off)
Single transfection
Dual transfection
CD147
CD147
MCT1
Overlay
MCT1
Overlay

• MCT4

CD147
MCT4
20 mm
20 mm
32
CD147 does not enable MCT2 translocation to the
plasma membrane
COS cells (CMV)
Single transfection
Dual transfection
MCT1
CD147
MCT1
Overlay
MCT2
CD147
Overlay

• MCT2

CD147
20 mm
20 mm
33
Cross-linking antibodies against CD147 cause
capping of BOTH CD147 AND MCT1 and MCT4. This
shows that the two proteins remain associated in
the plasma membrane
Capped
Controls
CD147
CD147
CD147
CD147
MCT1
GLUT-1
MCT1
MCT4
MCT4
Overlay

• Overlay

Overlay
GLUT-1
20 mm
34
The transmembrane and intracellular domain of
CD147 is required for MCT1 expression
Active
Active
Conserved glutamate
Inactive
35
The principle of FRET
36
MCT1cCFP is properly targeted to the plasma
membrane and catalytically active when
co-expressed with CD147cYFP
MCT1cCFP single transfected
MCT1cCFP single transfected
Non-transfected
MCT1cCFP/CD147cYFP co-transfected
MCT1cCFP / CD147cYFP cotransfected

• Rate of transport DF.s-1
• The use of fluorescence resonance energy transfer
  (FRET) to study the interaction between CD147 and
  MCT1

37
MCT1
CFP laser excitation
Single transfection MCT1nCFP
No FRET
YFP laser excitation
CD147
s
s
s
s
No FRET
Single transfection CD147cYFP
CFP laser excitation
CD147
MCT1
s
s
s
s
FRET
Co-transfection MCT1nCFP CD147cYFP
38
CD147
MCT1
s
s
s
s
Emission scan from MCT1CFP-N CD147YFP-C Co
transfection, CFP laser excitation.
Emission scan from MCT1CFP-N CD147YFP-C Co
transfection, YFP laser excitation.
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(No Transcript)
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Summary of FRET data
No FRET
FRET
41
MCT1
K
K
K
K
413
413
290
290
R
R
R
P
R
P
R
F
F
D
302
313
360
360
N
N
Highly conserved
Highly conserved
motifs characteristic
motifs characteristic
C
of the MCT family
C
of the MCT family
However, charge swops with site directed
mutagenesis do not support the interaction of
CD147 with helix 8
42
pCMBS disrupts the interaction between CD147 and
MCT1
pCMBS inhibits MCT1 and MCT4 but not MCT2.
However even if all extracellular cysteine groups
mutated to alanine, pCMBS inhibits.
Does pCMBS interact with CD147 which is not
involved with MCT2 expression?
43
pCMBS disrupts the interaction between CD147 and
MCT1
Co-immunoprecipitation experiments
CD147
MCT1
Inhibitors
44
pCMBS reduces FRET
1.2
1.1
18
1
23
p lt 0.001
0.9
50
480 / 530nm Fluorescence emission ratio
0.8
41
0.7
0.6
0.5
0.4
pCMBS
pCMBS
Control
Control
MCT1cCFP CD147nYFP
MCT1cCFP CD147cYFP
45
pCMBS prevents capping
pCMBS Treatment
DIDS
Control
CD147
CD147
CD147
MCT1
MCT1
MCT4
46
MCT2 interacts with GP70 rather than CD147
Co-expression in COS cells
GP70cCFP
MCT2cYFP
47
Regulation of MCT activity
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(No Transcript)
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Effect of 7 day chronic stimulation of rat hind
limb muscle on MCT1 expression and rate of
lactate uptake
1.0
0.8
MCT1 Content
0.6
MCT1 expression
0.4
0.2
0.0
RTA
WTA
EDL
1.0
0.8
Rate of lactate uptake
0.6
0.4
0.2
0.0
RTA
WTA
EDL
50
(No Transcript)
51
Changes in Connexin-43 and MCT1 mRNA in
congestive heart failure
SHAM
NORTHERN
WESTERN
MI
140
140
100
100
STAINING DENSITY
May be post-trancriptional regulation of MCT1
expression?
60
60
20
20
CX43
MCT-1
CX43
MCT-1
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(No Transcript)
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Hypoxia up-regulates MCT4 but not MCT1 by
transcriptional regulation
Western Blots
Northern Blots
MCT-1
MCT-4
COS cells
MCT-4
Actin
CD147
Cobalt
Hypoxia
Cobalt
Normoxia
Normoxia
Probably transcriptional control through
Hypoxia-inducible factor 1 (HIF-1)
54
Hypoxia increases MCT4 mRNA in HeLa Cells
Quantification of MCT4 mRNA by Real time PCR
Western blot
hMCT4
Hypoxia
Normoxia
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Conditions which regulate MCT expression
MCT1
Increased expression
Skeletal muscle upon chronic exercise (rats) or
training (humans)
Heart on recovery from myocardial infarction
Cell cycle in PM and G1 phases
Decreased expression
Skeletal muscle upon denervation (rats) or spinal
cord injury (humans)