Assays

What is the relationship between the activity of Trk1,2 system and cell volume? Is cell volume affected by lack of the main potassium uptake systems? Comparison of cell volume in wild type and TRK mutants.

To estimate the differnces of internal pH between the wild type, single trk1 and trk2 or double trk1 trk2 mutants, the pH-dependent variant of GFP pHluorin was expressed in cells from a multicopy plasmid, cells were grown under various conditions, and the cell fluorescence was monitored.

To estimate the relative level of plasma-membrane potential in the wild type, single trk1 and trk2 or double trk1 trk2 mutants, the diSC3(3) fluorescent probe was added to cells grown under various conditions and the levels of cell fluorescence were monitored.

Is protein content of trk1,2 mutants affected? Determination of proteins in wild type and TRK mutants

Is internal potassium affected by mutations in the Trk1,2 system? Under which conditions? Potassium measurements in wild type and TRK mutants grown and/or incubated under several external conditions.

The current-voltage relation for Trk1,2 will be determined according to the conditions of Kuroda et al.

2D Gels in wild type and mutants grown or incubated under several conditions: starvation, potassium limiting or different growth phases.

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.