Fluorometric measurements of intermolecular distances between the alpha- and beta-subunits of the Na+/K+-ATPase.
The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the alpha-subunit, which mediates ATP hydrolysis and ion translocation, as well as the beta-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the alpha- and beta-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the alpha- and beta-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the alpha-subunit and the beta-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the beta-subunit in relation to the alpha-subunit and suggest that the alpha- and beta-subunits move toward each other during the E2 to E1 conformational transition.
Dempski
Robert E
RE
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, D-60438 Frankfurt am Main, Germany.
Hartung
Klaus
K
Friedrich
Thomas
T
Bamberg
Ernst
E
D000818:Animals; D005452:Fluoresceins / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology; D006868:Hydrolysis; D007477:Ions; D008856:Microscopy, Fluorescence / Q000379:methods; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012235:Rhodamines / Q000494:pharmacology; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D014982:Xenopus laevis
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007477:Ions; D012235:Rhodamines; C005358:tetramethylrhodamine; C037041:fluorescein 5-maleimide; D000254:Sodium-Potassium-Exchanging ATPase