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The ion pathway through the opened Na+,K+-ATPase pump

Abstract

P-type ATPases pump ions across membranes, generating steep electrochemical gradients that are essential for the function of all cells. Access to the ion-binding sites within the pumps alternates between the two sides of the membrane1 to avoid the dissipation of the gradients that would occur during simultaneous access. In Na+,K+-ATPase pumps treated with the marine agent palytoxin, this strict alternation is disrupted and binding sites are sometimes simultaneously accessible from both sides of the membrane, transforming the pumps into ion channels (see, for example, refs 2, 3). Current recordings in these channels can monitor accessibility of introduced cysteine residues to water-soluble sulphydryl-specific reagents4. We found previously5 that Na+,K+ pump-channels open to the extracellular surface through a deep and wide vestibule that emanates from a narrower pathway between transmembrane helices 4 and 6 (TM4 and TM6). Here we report that cysteine scans from TM1 to TM6 reveal a single unbroken cation pathway that traverses palytoxin-bound Na+,K+ pump-channels from one side of the membrane to the other. This pathway comprises residues from TM1, TM2, TM4 and TM6, passes through ion-binding site II, and is probably conserved in structurally and evolutionarily related P-type pumps, such as sarcoplasmic- and endoplasmic-reticulum Ca2+-ATPases and H+,K+-ATPases.

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Figure 1: Alternative routes for ions through the Na + ,K + -ATPase transmembrane domain.
Figure 2: Effects of MTSET + on current through palytoxin-bound Na + ,K + pump-channels with cysteines in TM5 or the TM5–TM6 loop.
Figure 3: Effects of MTSET + on current through palytoxin-bound Na + ,K + pump-channels with cysteines in TM1, TM2 or the TM1–TM2 loop.
Figure 4: Structural model and characteristics of ion pathway through the palytoxin-bound Na + ,K + -ATPase.

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Acknowledgements

We thank N. Fataliev for help with molecular biology, the late R. F. Rakowski for cDNAs encoding Xenopus α1 and β3 Na+,K+-ATPase subunits, and P. Nissen, B. Vilsen and J. V. Møller for providing atomic coordinates before their publication. The work was supported by a grant from the NIH (to D.C.G.) and a fellowship from the Vicente Trust (to P.A.); N.R. is presently a Jane Coffin Fund Fellow. We dedicate this paper to the memory of our colleague R. F. Rakowski.

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Correspondence to David C. Gadsby.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8 with Legends. (PDF 3687 kb)

Supplementary Movie

This movie file shows a 360° view of ion pathway through palytoxin bound Na+,K+-ATPase. Rotating Na+,K+-ATPase TM domain homology model (based on the E2·BeF3-structure of SERCA) in which atoms of MTSET+-reactive positions are shown as red spheres, and the rest as cartoons, as in Supplementary Fig. 2c. TM helices 3 and 7-10 are coloured grey; the others are TM1 (pale blue), TM2 (magenta), TM4 (blue), TM5 (purple) or TM6 (green). (MOV 26320 kb)

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Takeuchi, A., Reyes, N., Artigas, P. et al. The ion pathway through the opened Na+,K+-ATPase pump. Nature 456, 413–416 (2008). https://doi.org/10.1038/nature07350

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