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Joachim W. Deitmer, FB Biologie mit FB Mathematik und ITWM

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1 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

2 A model reduction approach to the kinetics of the monocarboxylate transporter MCT1 and carbonic anhydrase II MCT MCT 1 H+ 1 Lac- Joachim Almquist1, Henning Schmidt1, Patrick Lang2, Dieter Prätzel-Wolters2, 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

3 Methods - Electrophysiological Techniques and Mathematical Modeling
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

4 Functionally expressed proteins in Xenopus oocytes: Interactions with carboanhydrases?
Messung von Membranströmen in ‚Voltage-Clamp‘ Messung von cytosolischem pH und Na+ mit ionen selektiven Mikroelektroden Struktur-Funktion-Analyse durch gezielte Mutationen Funktionelle heterologe Expression von Membrantransportproteinen in Frosch-Oozyten (Xenopus laevis) Carboanhydrase II Modell der Wechselwirkung zwischen Carboanhydrase und Membrantransporter (NBCe1=Natrium-Bikarbonat-Kotransporter NBCe1 und Carboanhydrase ko-exprimiert

5 The Model Monocarboxylate transporter MCT 1 H+ 1 Lac-
Ordinary differential equation model of the transporter states shown in the cartoon

6 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.

7 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).

8 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

9 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 Protonen-Pufferung in Zellen

10 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

11 Thank you! Gilt das nur für den Betze?


13 Measurement of buffer capacity
CO2 +H2O 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 βt = intrinsic + CO2/HCO3—dependent ßCO2 ≈ 2.3 [HCO3-] dpHi Addition/injection of acid By measuring pHi, ß can be determined!

14 Model Reduction One possible set of constraints that can be used to reduce the ODE-system. Solving this set of equations yields a explicit rate expression for the cross-membrane transport of MCT1 substrates,T.

15 Substrate Inhibition Predictions
Model reduction with different assumptions on transporter properties leads to predictions of inhibition by single substrate presence.

16 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.

17 Voltage dependence of the total buffer capacity (ßT) of oocytes expressing NBC
Becker & Deitmer (2004) J. Biol. Chem. 279,

18 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)?

19 Data from Substrate Inhibition
The inhibition predictions are tested in experiments.

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