Joachim W. Deitmer, FB Biologiemit FB Mathematik und ITWM

Räumlich-zeitliche Interaktionenzelluläre Signalmoleküle

Die Rolle der Neuroglia bei derBildung, Funktion und

Plastizitätvon Synapsen

A model reduction approach to the kineticsof the monocarboxylate transporter

MCT1 and carbonic anhydrase II

Neuron-glia interactions

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

1 H+ 1 Lac-

MCT

MCT

Protein expression in Xenopus oocytesInjection of rat MCT1-cRNA Injection of CAII (isolated from bovine erythrocytes)Microelectrodes for intrcellular pH measurementsODE modellingModel reduction

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.

Aim of this Project

Methods - Electrophysiological

Techniques and Mathematical

Modeling

Functionally expressed proteins in Xenopus oocytes: Interactions with carboanhydrases?

Carboanhydrase II

Funktionelle heterologe Expressionvon Membrantransportproteinen inFrosch-Oozyten (Xenopus laevis)

- Messung von Membranströmen in ‚Voltage-Clamp‘- Messung von cytosolischem pH und Na+ mit ionen- selektiven Mikroelektroden- Struktur-Funktion-Analyse durch gezielte Mutationen

NBCe1 und Carboanhydrase ko-exprimiertModell der Wechselwirkung zwischen Carboanhydrase und

Membrantransporter (NBCe1=Natrium-Bikarbonat-Kotransporter

The Model

Ordinary differential equation model of the transporter states shown in the cartoon

Monocarboxylatetransporter

1 H+ 1 Lac-

MCT

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 Protonen-Pufferung 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?

dpHi

Measurement of buffer capacity

Henderson- Hasselbalch equation:pH = pK‘ log [HCO3

-]/[CO2]

[HCO3-]i = 10(pHi-pK‘) x [CO2] (pK‘=6.1)

Addition/injection of acid

Buffer capacity = acid/base injection / dpHi

CO2 +H2O H+ + HCO3-

CA

βt = intrinsic + CO2/HCO3—

dependent

ßCO2 ≈ 2.3 [HCO3-]

By measuring pHi, ß can be determined!

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.

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

-1 0 0 -8 0 -6 0 -4 0 -2 0 2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

N B C (n = 11 )

H O (n = 6 )2

E (m V )m

ß T (m

M)

N B C - H O2

A

0 .7 m M /m V

ß (% ) n = 11T

N a (% ) n = 1 0+

I (% ) n = 11m

E (m V )m

%

Rel

ati v

e ch

ange

s (%

)

B

-1 0 0 -8 0 -6 0 -4 0 -2 0 4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

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.