Assays

The potassium influx after the addition of a certain amount of KCl to a potassium free medium, followed by the injection of glucose will be measured by using the MIFE and FLISE technique. This reveals a time course of potassium. Also the external potassium concentrations will be measured.

In parallel with the potassium influx the efflux of protons is monitored by measuring the external proton concentration changes with MIFE or FLISE.

Further fluxes (ammonium, chloride, calcium) will be measured dependent on the capacities of the MIFE/FLISE technique.

The external potassium changes will be monitored by the MIFE and FLISE technique. This allows an estimation of internal potassium changes by determining an initial concentration.

The external pH changes will be monitored by the MIFE and FLISE technique. This allows an estimation of internal pH changes by determining an initial pH. pH changes will be also determined by using green fluorescent protein dyes. Relating the proton efflux and the change of internal pH allows an estimate of the proton buffering capacity.

The current-voltage relation for Trk1,2 will be determined for various external potassium concentrations.

Current- voltage relations for will be determined for various internal potassium concentrations.

The main input is the ENA review paper (Function and Regulation of the Saccharomyces cerevisiae ENA Sodium ATPase System, Ruiz&Ariño 2007) and the papers referenced. Another source are the papers linked from the ENA page of SGD http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=ENA1

A boolean network was created using booleannet (after experimenting with Squad and CellNetAnalyzer). This network can be simulated and visualized using additional software components that will be part of the pyMantis CMS that is developed by the Translucent project.

A interaction network analysis tool (currently based on the BioGrid - PSICQUIC web services) was created that helps to discover interactions of Yeast proteins. The tool will at some point be freely available on the www as part of the pyMantis CMS created within the Translucent project.

Elucidation of protein networks involved in the regulation of cation homeostasis using protein interaction datasets.

Development of bioinformatic tools to investigate the role of transcription factors and 14-3-3 proteins in the regulation of genes involved in cation homeostasis.

Based on a kinetic model a description of the potassium current is achieved. Its properties with respect to changes in membrane potential and potassium concentrations are derived.

Proton fluxes ensue a change in the membrane potential to which the potassium uptake responds. The membrane potential changes depend on the extrusion of protons, buffering capacities of the media and experimental parametes.

Analytical methods and computational analyses (regression, fitting) will be employed to find properties of the Trk system under different external conditions.