Abstract
The Na(+)/K(+) ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na(+)/K(+) ATPase sequence. These five normally functional LBT-Na(+)/K(+) ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na(+)/K(+) ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na(+)/K(+) ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies.
Highlights
Ouabain binds at the permeation pathway of the Naϩ/Kϩ ATPase
lanthanide-based resonance energy transfer (LRET) Measurements between External Linkers of the Naϩ/Kϩ ATPase and Bodipy-Fl Ouabain—Upon a short 9-ns excitation pulse (266 nm) to an oocyte expressing Naϩ/Kϩ ATPases with lanthanide binding tag (LBT) inserted within the fifth external loop, Tb3ϩ bound luminescence in the absence of acceptor decayed with a D of 2.52 ms (Fig. 1A)
We have made LRET distance determinations from Tb3ϩ bound to LBTs inserted in the Naϩ/Kϩ ATPase and a fluorescent ouabain
Summary
Ouabain binds at the permeation pathway of the Naϩ/Kϩ ATPase. Results: We have identified two binding sites for ouabain along the ion conductive pathway of the Naϩ/Kϩ pump that are mutually exclusive and differ in their affinities by about an order of magnitude. Homology models of the five Naϩ/Kϩ ATPase constructs with LBT were generated to find possible dye positions compatible with the set of measured distances For both affinity binding sites, the most likely positions for ouabain deduced from all available data situate the molecule along the permeation pathway of the Naϩ/Kϩ ATPase, as seen in the crystal structures [21, 22]. Our results suggest that the ouabain molecule sits deep in the channel In both positions the rhamnose moiety faces the intracellular end of the permeation pathway of the Naϩ/Kϩ ATPase, rather than the outwardly facing orientation shown in the crystal structures. We propose a sequential two-step binding mechanism that unifies all previous structural and functional data with our proposed binding site
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