Imaging studies of cell physiology with particular reference to Na,K-ATPase function

The membrane protein Na,K-ATPase is well known for its critical function of transporting sodium out of the cell and potassium into the cell, thereby creating a fundamental electrochemical gradient upon which several other important cell functions are dependent. In the current study we have investigated novel aspects of Na,K-ATPase function in cell physiology. In order to study this, a considerable part of the present thesis has involved methodological development of different microscopic techniques. It is well established that Na,K-ATPase is important for maintaining cell volume, membrane potential and reabsorption of electrolytes in the kidney. The new findings in this study are: 1) A role for Na,K-AT'Pase in the regulation of cell adhesion. Partial inhibition of Na,K-ATPase activity significantly reduced cell attachment to fibronectin. The results suggest that this effect is mediated by perturbation of normal Ca2+ signaling and a reduction of focal adhesion kinase activity. These findings indicate the importance of Na,K-ATPase during development and differentiation. 2) A major role of Na,K-ATPase activity in regulatory volume decrease (RVD). A direct link between Na,K-ATPase activity and the ability of COS-7 cells to perform RVD was demonstrated. With site directed mutagenesis of the a subunit of Na,K-ATPase it was demonstrated that Na,K-ATPase may have both negative and positive effects on the rate of RVD. Further knowledge about the interaction between ouabain and NaK-ATPase was achieved by demonstrating that a Leu-799 to Cys substitution in the alpha subunit of rat Na,K-ATPase produced complete ouabain resistance. This residue belongs to the extracellular loop between transmembrane segment 5 and 6 of the enzyme which is of importance for ion occlusion and ion transport. The same mutant has been demonstrated to have a positive effect on the rate of RVD. This thesis was also concerned with the rigorous use of fluorescence lifetime imaging to investigate intracellular pH. We made theoretical predictions concerning sensitivity and noise which were supported by experimental results. Our study on the influence of probe binding on pH based fluorescent lifetime imaging indicates that the method is not a straightforward approach to measure pH in the absence of correction for the effect of probe binding. We find it likely that other fluorescent ion probes have similar probe-binding-sensitive fluorescence lifetimes. However, the overall effect is difficult to pr