Joachim W. Deitmer, FB Biologie
mit FB Mathematik und ITWM
Die Rolle der Neuroglia bei der
Bildung, Funktion und Plastizität
von Synapsen
Räumlich-zeitliche Interaktionen
zelluläre Signalmoleküle
A model reduction approach to the kinetics
of the monocarboxylate transporter
MCT1 and carbonic anhydrase II
Joachim Almquist1, Henning Schmidt1, Patrick Lang2, Dieter PrätzelWolters2, Joachim W. Deitmer3, Mats Jirstrand1, and Holger Becker3
1Fraunhofer-Chalmers Centre, Gothenburg, Sweden
2Institut für Techno- und Wirtschaftsmathematik (ITWM) Fraunhofer
Gesellschaft, Kaiserslautern, Germany
3Technische Universität Kaiserslautern, Kaiserslautern, Germany
Neuron-glia interactions
MCT
MCT
1 H+
1 Lac-
Aim of this Project
To determine the mechanism of the
monocarboxylate transporter (MCT1)
and to present a mechanistic
hypothesis of how MCT1 interacts
with the enzyme carbonic anhydrase
II (CAII). The modelling process
might provide ideas for this.
To derive a rate expression for the
MCT1 that also includes the effect of
CAII. This could be used in other
models.
Methods Electrophysiological
Techniques and
Mathematical Modeling
Protein expression in Xenopus oocytes
Injection of rat MCT1-cRNA
Injection of CAII (isolated from bovine erythrocytes)
Microelectrodes for intrcellular pH measurements
ODE modelling
Model reduction
Functionally expressed proteins in Xenopus oocytes:
Interactions with carboanhydrases?
- Messung von Membranströmen in ‚Voltage-Clamp‘
- Messung von cytosolischem pH und Na+ mit ionenselektiven Mikroelektroden
- Struktur-Funktion-Analyse durch gezielte Mutationen
Funktionelle heterologe Expression
von Membrantransportproteinen in
Frosch-Oozyten (Xenopus laevis)
NBCe1 und Carboanhydrase ko-exprimiert
Carboanhydrase II
Modell der Wechselwirkung zwischen Carboanhydrase und
Membrantransporter (NBCe1=Natrium-Bikarbonat-Kotransporter
Monocarboxylate
transporter
MCT
1 H+
The Model
1 Lac-
Ordinary differential equation model of the
transporter states shown in the cartoon
CAII Included in Model
The effect of CAII is included in the model as an increased rate of
proton uptake and release on the intracellular side of the transporter.
Comparing Measurements with Simulations
Efflux experiments with and without CAII
(A,B) are compared with the model (C).
Influx experiments with and without
CAII (A,C) are compared with the
model (B,D).
Kooperationen
• Prof. D. Prätzel-Wolters, FB Mathematik und ITWM
• ITWM: Dr. P. Lang
• Fraunhofer-Chalmers-Centre, Göteborg, Schweden:
Prof. M. Jirstrand, J. Almquist
Erfolge/Fortschritte: Erstes Paper über das Modell in
Revision
Bisher dem Projekt zugewiesene Mittel: 30 T€ für 2008
Weiterer Fahrplan
• Erweiterung des Modells mit Voraussagen
(Einbeziehung von verschiedenen Isoformen und
Mutanten der Carboanhydrase sowie mit NBCe1
• Experimentelle Überprüfung dieser Voraussagen und
Simulierung weiterer Parameter
• Neues Projekt (DFG-Antrag wird gerade geschrieben):
Mechanismen und Modellierung der ProtonenPufferung in Zellen
Future Projects
• Measuring, analyzing and modelling of the capacity
and dynamics of cellular H+ buffering
- Spatial dynamics of buffering within a cell and
role of carbonic anhydrases
- The role of acid/base-coupled membrane
transporters, such as the NBC, for buffering
Thank
you!
Gilt das nur für den Betze?
Measurement of buffer capacity
CO2 +H2O
CA
H+ + HCO3-
Henderson- Hasselbalch equation:
pH = pK‘ log [HCO3-]/[CO2]
[HCO3-]i = 10(pHi-pK‘) x [CO2]
(pK‘=6.1)
Buffer capacity = acid/base injection / dpHi
dpHi
βt = intrinsic + CO2/HCO3—
dependent
ßCO2 ≈ 2.3 [HCO3-]
Addition/injection of acid
By measuring pHi, ß can be determined!
Model Reduction
One possible set of constraints that can be used to reduce the ODEsystem. Solving this set of equations yields a explicit rate expression
for the cross-membrane transport of MCT1 substrates,T.
Substrate Inhibition Predictions
Model reduction with different assumptions on transporter properties leads to
predictions of inhibition by single substrate presence.
A Hypothesis for the MCT1-CAII-mechanism
A hypothesis for the MCT1-CAII-mechanism. One or several CAII molecules
close to the inner mouth of MCT1 might be working as a proton antenna. If
proton uptake and release are the rate-limiting steps of transport, MCT1
turnover could be increase by this antenna.
Voltage dependence of the total buffer
capacity (ßT) of oocytes expressing NBC
A
140
B
%
100
NBC (n = 11)
ßT (mM)
100
80
0.7 mM/mV
NBC - H2O
60
40
H2O (n = 6)
Relative changes (%)
120
90
80
70
60
ßT (%) n = 11
 Im (%) n = 11
50
 Na+ (%) n = 10
40
20
-100
-80
-60
Em (mV)
-40
-20
-100
-80
-60
-40
-20
Em (mV)
Becker & Deitmer (2004) J. Biol. Chem. 279, 28057-28062
Aim of these studies
• Measuring and modelling (simulating) H+ buffer
capacity
• Predicting parameters of cellular buffering
• Testing predicted parameters in experiment
• What consequences do our findings might have
for pH-dependent processes in systems (cells,
tissue, organs)?
Data from Substrate Inhibition
The inhibition predictions are tested in experiments.

FB Biologie: Allgemeine Zoologie