Voltage clamp fluorometry: combining fluorescence and electrophysiological methods to examine the structure-function of the Na(+)/K(+)-ATPase.
This paper summarizes our recent work investigating the conformational dynamics and structural arrangement of the Na(+)/K(+)-ATPase using voltage clamp fluorometry as well as the latest biochemical, biophysical and structural results from other laboratories. Our research has been focused on combining site-specific fluorophore labeling on the alpha, beta and/or gamma subunit with electrophysiological studies to investigate partial reactions of the ion pump by monitoring changes in fluorescence intensity following voltage pulses and/or solution exchange. As a consequence of these studies, we have been able to identify a residue on the beta subunit, which following labeling with tetramethylrhodamine-6-maleimide can be used as a reporter group to monitor the conformational state of the holoenzyme. Furthermore, we have been able to delineate distance constraints between the alpha, beta and gamma subunits and to examine the relative movements of these proteins during ion transport. Concurrent to this research, significant advancements have been made in understanding the molecular mechanism of the Na(+)/K(+)-ATPase. Thus, our research will be compared with the results from other groups and future experimental directions will be proposed.
Max-Planck-Institute of Biophysics, Department of Biophysical Chemistry, Max-von-Laue-Strasse 3, D-60438 Frankfurt am Main, Germany.
Robert E
RE
Dempski
Thomas
T
Friedrich
Ernst
E
Bamberg
D000818:Animals; D018360:Crystallography, X-Ray; D055724:Electrophysiological Phenomena; D005260:Female; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase