LETTERS. Atomic structure of a Na 1 - and K 1 -conducting channel. Ning Shi 1, Sheng Ye 1, Amer Alam 1, Liping Chen 1 & Youxing Jiang 1

Transcription

1 Vol March 2006 doi: /nature04508 Atomic structure of a Na 1 - and K 1 -conducting channel Ning Shi 1, Sheng Ye 1, Amer Alam 1, Liping Chen 1 & Youxing Jiang 1 Ion selectivity is one of the basic properties that define an ion channel. Most tetrameric cation channels, which include the K 1, Ca 21,Na 1 and cyclic nucleotide-gated channels, probably share a similar overall architecture in their ion-conduction pore, but the structural details that determine ion selection are different. Although K 1 channel selectivity has been well studied from a structural perspective 1,2, little is known about the structure of other cation channels. Here we present crystal structures of the NaK channel from Bacillus cereus, a non-selective tetrameric cation channel, in its Na 1 - and K 1 -bound states at 2.4 Å and 2.8 Å resolution, respectively. The NaK channel shares high sequence homology and a similar overall structure with the bacterial KcsA K 1 channel, but its selectivity filter adopts a different architecture. Unlike a K 1 channel selectivity filter, which contains four equivalent K 1 -binding sites, the selectivity filter of the NaK channel preserves the two cation-binding sites equivalent to sites 3 and 4 of a K 1 channel, whereas the region corresponding to sites 1 and 2 of a K 1 channel becomes a vestibule in which ions can diffuse but not bind specifically. Functional analysis using an 86 Rb flux assay shows that the NaK channel can conduct both Na 1 and K 1 ions. We conclude that the sequence of the NaK selectivity filter resembles that of a cyclic nucleotidegated channel and its structure may represent that of a cyclic nucleotide-gated channel pore. All K þ channels contain the highly conserved signature sequence TVGYG, which is essential for K þ ion selectivity 3,4. The cyclic nucleotide-gated (CNG) channel pore shares high sequence homology with K þ channels, but is non-selective and permeable to most group IA monovalent cations 5 8. A key difference between the pores of CNG and K þ channels is that CNG channels lack a tyrosine and glycine residue from the conserved TVGYG sequence (bold) present in K þ channels. This causes structural differences in the selectivity filter and results in the loss of ion selectivity in CNG channels. In a search of the microbial genome, we identified two twotransmembrane channels from Bacillus cereus and Bacillus anthracis that have sequences very similar to the KcsA K þ channel, except for their selectivity filters, which resemble those of CNG channels with the sequence TVGDG or TVGDA (Supplementary Fig. S1). On the basis of this similarity, we hypothesized that these channels can nonselectively conduct both Na þ and K þ. This proved to be correct according to our functional assay. Here we present the crystal structure of the channel from Bacillus cereus in complex with Na þ at 2.4 Å (Table 1 and Supplementary Table S1), and we call this channel NaK (conducting both Na þ and K þ ions). NaK shares several common features with KcsA (Fig. 1a, b). It contains two membrane-spanning segments, M1 and M2, corresponding to the outer and inner helices of KcsA, and the four inner helices form an inverted teepee. The ion conduction pathway has a water-filled cavity near the centre of the membrane, with four pore helices pointing their carboxy termini towards the cavity. This overall architecture shared by NaK and KcsA presumably underlies their function to conduct cations across the cell membrane. Two notable differences distinguish NaK and KcsA from each other. First, the amino-terminal 19 amino acids of NaK form an interfacial helix (M0 helix) parallel to the membrane. Phe 4, Leu 8 and Met 11 at the N-terminal region of each M0 helix form hydrophobic contacts with Val 26, Val 29 and Leu 30 at the N terminus of helix M1 from a neighbouring subunit (Fig. 1b). The four M0 helices form a cuff that encircles the inner helix bundle crossing, and seem to be positioned appropriately to affect the opening and closing of the pore. The second structural difference between NaK and KcsA is observed in the selectivity filter (Fig. 2). The KcsA filter is formed by four extended polypeptide chains, each containing the 75 TVGYG 79 sequence (Fig. 2b, left). The backbone carbonyl oxygen atoms from the TVGY residues, along with the hydroxyl oxygen atom from Thr 75, point towards the centre of the pathway and form four equivalent binding sites (numbered 1 to 4) for dehydrated K þ ions. The eight oxygen atoms surrounding each site mimic the hydration shell of a K þ ion. Four additional carbonyl oxygen atoms from Gly 79 sit at the perimeter of the filter entrance and point directly into the extracellular solution, generating an electronegative environment that stabilizes a half-dehydrated K þ ion. The side chains from the four Tyr 78 residues form specific packing interactions with neighbouring aromatic residues from the pore helices that surround the filter. In comparison, the NaK filter has a sequence of 63 TVGDG 67, with the hydroxyl oxygen atom from Thr 63 and backbone carbonyl oxygen atoms from Thr 63 and Val 64 forming two ion-binding sites equivalent to sites 3 and 4 in KcsA (Fig. 2b, right). For comparative purposes, these two sites are also numbered 3 and 4 in Fig. 2b. Sites 1 and 2 of KcsA do not exist in NaK owing to the Table 1 Refinement statistics Na þ complex K þ complex Resolution (Å) R work /R free (%) 23.5/ /28.0 Number of atoms Protein 1,648 1,655 Ligand/ion 5 5 Water B-factors Protein Ligand/ion Water r.m.s deviations Bond lengths (Å) Bond angles (8) Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas , USA. 570

2 NATURE Vol March 2006 different backbone conformations adopted by the GDG residues at these two positions. The carbonyl groups from Gly 65 and Asp 66 align tangentially to the ion conduction pathway, and therefore their oxygen atoms do not point towards the centre to coordinate ions. Furthermore, the Ca of Asp 66 is directed away from the centre and its side chain protrudes upward and is exposed to the extracellular solution, creating a vestibule where ions can diffuse but not bind very specifically. At the entryway to the filter from the extracellular solution, the main chain at Gly 67 pinches inwards so that its carbonyl oxygen points towards the pore axis, forming an ionbinding site, which, as we discuss below, can have important physiological consequences. Superimposition of the NaK filter with that of KcsA gives rise to a root-mean-square deviation (r.m.s.d.) of 1.6 Å in their main-chain positions. These differences are caused mainly by the last three residues, which have an r.m.s.d. of 2.1 Å. The observed differences between NaK and KcsA are not due to crystal packing, as the NaK filter is in the centre of the channel tetramer and is not involved in protein packing in the crystal. In KcsA, K þ ions are required to stabilize the selectivity filter: the structure of KcsA in conditions of high Na þ, low K þ reveals significant changes at the filter region 9. In contrast, the structure of NaK does not depend on the ion composition: we determined a structure in KCl (rather than NaCl) at 2.8 Å and found no apparent differences in the filter structure (Fig. 3a c). Thus, NaK retains its proper conformation for ion conduction in both Na þ and K þ. Electron density maps of NaK show that ions can bind at the extracellular entrance, along the selectivity filter, and in the central cavity of the ion conduction pore (Fig. 2a). Ion-omit maps calculated with data from crystals grown in NaCl (Fig. 3a) or KCl (Fig. 3b) show that these ions bind at all the same sites in the pore. The crystallization solutions also contained 200 mm CaCl 2 in addition to 100 mm NaCl or KCl. To deduce whether it is the monovalent or divalent cations that bind at specific sites within the pore, we carried out soaking experiments in which the NaK crystals were soaked in stabilization solutions containing various monovalent and divalent salts at the same concentrations as those in the crystallization solutions. To minimize non-isomorphism, a native reference crystal was subjected to the same soaking procedures in a stabilization solution containing CaCl 2 and NaCl (Supplementary Table S2). Difference electron density maps using Fourier coefficients (F soak F reference ) were calculated with phases from the model (Fig. 3d h). The electron density difference between crystals soaked with Na/Ca and Cs/Ca, Na/Ca and Rb/Ca, or Na/Ca and Tl/Ca all give rise to three strong peaks at sites 3, 4 and the central cavity, indicating the binding of Cs þ,rb þ and Tl þ at these sites (Fig. 3d f, red mesh). The difference map between Na/Ca- and Na/Mg-soaked crystals reveals only one Ca 2þ -binding site, at the extracellular entrance to the filter (Fig. 3g, green mesh). This suggests that only monovalent cations bind at sites 3, 4 and the central cavity, and that divalent cations can bind at the external site outside the selectivity filter. On the basis of these results, looking back at the ion omit maps we can conclude that it is Na þ and K þ, but not Ca 2þ, inside the selectivity filter and central cavity (Fig. 3a, b). The Ca 2þ -binding site formed by the four carbonyl oxygen atoms from the Gly 67 residues may not be specific for divalent cations Ca 2þ binding might have prevailed simply because it is at a higher concentration in the crystallization solutions. A 2F o F c map of a native crystal soaked in a stabilizing solution containing only NaCl still shows reasonably strong electron density at this position (data not shown), possibly owing to Na þ binding. Monovalent cation binding at this position probably occurs as ions conduct through the pore. Divalent cations might bind at this position to block the conduction of monovalent cations through the pore, a property relevant to CNG channels, for which a divalent cation block of monovalent currents is important for physiological channel function The difference map between Na/Ca and Na/Ba soaked crystals reveals two Ba 2þ -binding sites, one at the filter entrance (equivalent to the Ca 2þ -binding site) and another at site 3 in the selectivity filter (Fig. 3h, green mesh). Ba 2þ can block a K þ channel 16,17 by binding to site 4 in the selectivity filter 18.Ba 2þ might also serve as a blocker for the NaK channel by binding either to site 3 (instead of site 4 as it does in KcsA) of the selectivity filter or at the external entrance. Indeed, our functional assay showed that Ba 2þ reduces the Rb þ flux at micromolar concentrations (data not shown). Neither the ion-omit maps of native crystals nor the difference maps between various soaked crystals reveal any specific ion binding in the vestibule of the filter. The 2F o F c maps of native crystals (Fig. 2a) and the difference map between Na/Ca- and Tl/Ca-soaked crystals reveal weak electron density in the vestibule, indicating the presence of an ion with low occupancy. Ion binding in the vestibule can be stabilized by the electronegative environment generated by the surface lining main-chain carbonyl groups from Gly 65 and Asp 66. However, such binding is not as specific as that of sites 3 and 4, and the bound ion distributes over a larger volume, giving rise to weak electron density on both 2F o F c and difference maps. Based on the premise that NaK is capable of conducting Rb þ (among other monovalent cations), we performed an 86 Rb flux assay 19 using NaK channels reconstituted into liposomes in order Figure 1 Overall structure of NaK. a, b, Ribbon representation of NaK viewed from the membrane in a (with front and rear subunits removed), and from the intracellular side in b. Green spheres represent ions in the channel. Each subunit is individually coloured. Green ball-and-stick representations show side chains from residues involved in hydrophobic contacts between M0 and neighbouring M1 helices. 571

3 NATURE Vol March 2006 to study channel ion-selectivity properties. We also reconstituted a truncated form of the channel, NaKND19, in which the N-terminal M0 helix-forming residues were removed, as we suspected that the four M0 helices around the helix bundle might lock the channel in a closed form. A Na þ concentration gradient was established across the liposome membrane (high concentration of NaCl inside) so that any outflow of Na þ through NaK would leave a deficit of positive charge inside and drive the influx of external radioactive 86 Rb, which could be monitored using a scintillation counter. Figure 4a shows the timedependent accumulation of 86 Rb inside liposomes, confirming that NaK is capable of conducting both Na þ and Rb þ. As a much higher flux rate was observed for NaKND19, this truncated form of NaKwas used in all other flux assays. As expected, the flux rate was negligible for control liposomes (containing no reconstituted protein) and for liposomes reconstituted with KcsA, which is a K þ -selective channel. Addition of gramicidin A, a Group 1A metal ionophore, to the control liposomes increases Rb þ influx, confirming that it is the outflow of Na þ that causes Rb þ influx. We performed the same flux assay using liposomes loaded with KCl instead of NaCl. As shown in Fig. 4b, liposomes reconstituted with KcsA or NaKND19 both show 86 Rb accumulation in a timedependent manner, indicating the conduction of K þ in both channels. The difference in flux rates of NaKND19 in the presence of Na þ or K þ could be the result of different batches of liposomes used for each set of experiments. We then measured 86 Rb influx in the presence of external cations at different concentrations in addition to 86 Rb. NaKND19-containing liposomes were loaded with NaCl and flux was allowed to proceed for 10 min before radioactivity levels in the liposomes were measured. Any of the test ions that the channel was able conduct were expected to compete with 86 Rb and decrease its influx into liposomes. The data for these competition assays are shown in Fig. 4c and indicate that the channel is able to conduct both Na þ and K þ, which result in lowering of the 86 Rb signal compared to control ( 86 Rb only, in a background of,35 mm NaCl). Li þ and NMG þ (N-methyl-D-glucamine) do not lower the 86 Rb signal and hence we conclude that the channel is unable to conduct these cations. Competition assays repeated in liposomes with KCl yielded the same pattern of results (data not shown). From the competition assays, it seems that K þ has a greater effect on 86 Rb flux than Na þ, and that the channel might therefore conduct K þ ions better than Na þ ions. However, we are reluctant to draw such conclusions before a more thorough and quantitative functional analysis of the channel is performed. What is clear from the data is that the NaK channel is not very selective among Na þ,k þ and Rb þ. This result is consistent with our crystallographic studies showing that these ions all bind similarly inside the selectivity filter and central cavity of the NaK channel. One of the more intriguing conclusions we draw from the NaK channel has to do with ion-binding sites 3 and 4. Chemically, these Figure 2 Structural comparison of the selectivity filters in NaK and KcsA. a, Stereo view of a 2F o F c map at 1j contour (blue mesh), showing the selectivity region of NaK with the front subunit removed. Green spheres represent ions in the filter and cavity. b, Structural details of the selectivity filters from KcsA (left) and NaK (right). The front and rear subunits have been removed for clarity. Only discrete ion-binding sites in the filter are numbered (1 4 from the extracellular side in KcsA, 3 and 4 in NaK). 572

4 NATURE Vol March 2006 Figure 3 Ion binding in the NaK channel. a, b, 2F o F c ion-omit maps of Na þ (a) and K þ (b) complexes of NaK contoured at 6j (red mesh). c, Superimposition of NaK selectivity filters in Na þ - (green) and K þ - (yellow) bound states. d h, F soak F reference difference maps between the reference crystal and crystals with various soaking conditions reveal the binding of cations (labelled underneath each panel) in NaK. All maps are contoured at 10j except for the density of Ca 2þ binding, which is contoured at 6j. Densities for monovalent and divalent cations are coloured red and green, respectively. sites are indistinguishable from the corresponding positions in the KcsA K þ channel, yet their ion-binding properties are clearly different from those observed in K þ channels. Specifically, in K þ channels, Na þ is never observed to bind at position 3, whereas in the NaK channel, Na þ binds there without difficulty. On the basis of these experimental data, we conclude that structural differences in otherwise chemically identical sites must account for their different ion-binding properties. These structural differences must arise from differences in amino acid packing around the binding site. Our structural study of NaK also addresses a fundamental issue concerning ion selectivity in K þ channels. A theory-based computational assay has suggested that K þ selectivity in K þ channels originates not from geometric constraints provided by the protein, but rather from electrostatic repulsion between carbonyl oxygen atoms the repulsion was said to prevent the oxygen atoms from approaching close enough to each other to form a cage small enough to coordinate the smaller Na þ ion 20. Comparison of NaK and KcsA provides a clear experimental demonstration that electrostatic repulsion between carbonyl oxygen atoms is not the origin of K þ over Na þ selectivity in K þ channels, and that protein atoms surrounding the ion-binding site must confer size-selectivity through geometric constraints. We also find that the external site in the NaK selectivity filter is distinct from the ion-binding site just outside the selectivity filter of K þ channels (Fig. 2b). In K þ channels, the external site does not bind divalent cations, whereas in NaK it can bind divalent cations that could serve to block monovalent cation conduction. This difference can be understood on the basis of their different structural properties. In the K þ channel this region of the selectivity filter appears to be very constrained. An apparent greater flexibility of the main chain in this region of the NaK selectivity filter probably allows the carbonyl oxygen atoms to conform to an ion with more degrees of freedom. Figure 4 86 Rb flux assay. a, Time-dependent 86 Rb influx into liposomes prepared in NaCl. The liposomes contain NaK, NaKND19, KcsA or no protein (as a control). Arrow indicates 86 Rb influx into control liposomes upon addition of 10 mgml 21 gramicidin A. b, Time-dependent 86 Rb influx into KcsA or NaKND19-containing liposomes prepared in KCl. c, Competition assay showing 86 Rb influx in the presence of externally added monovalent cations. Three final concentrations of each cation (0.1, 0.5 and 1.0 mm) were tested in the assay. No external cations were added in the control experiment. c.p.m., counts per minute. 573

5 NATURE Vol March 2006 Divalent cation binding at the external site in the NaK selectivity filter reveals the structural basis of external divalent cation blockage in CNG channels. The physiological significance of this blockage to visual transduction was first revealed nearly 20 years ago 21. Here for the first time we observe its chemical basis in the form of a precise binding site at the entryway. METHODS Detailed experimental methods are provided in the Supplementary Information. The NaK channel from Bacillus cereus was cloned into the pqe60 vector and expressed in E. coli XL1-Blue cultures. The protein was purified as a tetramer in n-decyl-b-d-maltoside with NaCl or KCl as the monovalent salt. Crystals were grown by sitting-drop vapour diffusion at 20 8C by mixing equal volumes of protein solution at mg ml 21 and reservoir solution containing 36 42% polyethylene glycol 400 (PEG400), 200 mm CaCl 2, 100 mm Tris HCl ph , 4% t-butanol. The crystals were of space group C222 1 with cell dimensions a ¼ 81.5 Å, b ¼ 85.5 Å, c ¼ Å, a ¼ b ¼ g ¼ 908, and contained two subunits per asymmetric unit. The four-fold axis of the channel tetramer coincides with one of the crystallographic dyads. All data were collected at the Advanced Photon Source (APS) and processed using HKL The structure was determined by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. Hg binding sites were determined with SHELXD 23 and initial phases were improved by solvent flattening in n-decyl-b-d-maltoside 24. The model was constructed in O 25 and was refined in CNS 26 to 2.4 Å with R work ¼ 23.5% and R free ¼ 26.0%, and contained residues in one subunit, in the other. The K þ -bound NaK structure was determined by molecular replacement and was refined to 2.8 Å with R work ¼ 24.1% and R free ¼ 28.0%. For soaking experiments, crystals of Na þ complexes were soaked in a stabilization solution of 40% PEG400, 100 mm Tris HCl ph 8.0, 20 mm n-decyl-b-d-maltoside, 4% t-butanol, 100 mm XCl and 200 mm YCl 2, where X and Y represent a monovalent cation (Na þ,rb þ or Cs þ ) and a divalent cation (Ca 2þ, Mg 2þ or Ba 2þ ), respectively. For Tl þ -soaking, 100 mm TlNO 3 and 200 mm Ca(NO 3 ) 2 were used. All channel proteins used in the flux assay were reconstituted into lipid vesicles composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine and 1-palmitoyl-2-oleoyl-phosphatidylglycerol using the same method as previously described 27. The 86 Rb flux assay was performed as described 19. For competition assays, the tested ions (Li þ,na þ,k þ or NMG þ ) were added directly into the flux buffer. Received 28 July; accepted 5 December Published online 8 February Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination and hydration revealed by a K þ channel -Fab complex at 2.0 Å resolution. Nature 414, (2001). 2. Doyle, D. A. et al. The structure of the potassium channel: molecular basis of K þ conduction and selectivity. Science 280, (1998). 3. Heginbotham, L., Abramson, T. & MacKinnon, R. A functional connection between the pores of distantly related ion channels as revealed by mutant K þ channels. Science 258, (1992). 4. Heginbotham, L., Lu, Z., Abramson, T. & MacKinnon, R. Mutations in the K þ channel signature sequence. Biophys. J. 66, (1994). 5. Yau, K. W. & Baylor, D. A. Cyclic GMP-activated conductance of retinal photoreceptor cells. Annu. Rev. Neurosci. 12, (1989). 6. Kaupp, U. B. & Seifert, R. Cyclic nucleotide-gated ion channels. Physiol. Rev. 82, (2002). 7. Matulef, K. & Zagotta, W. N. Cyclic nucleotide-gated ion channels. Annu. Rev. Cell Dev. Biol. 19, (2003). 8. Zagotta, W. N. & Siegelbaum, S. A. Structure and function of cyclic nucleotidegated channels. Annu. Rev. Neurosci. 19, (1996). 9. Zhou, Y. & MacKinnon, R. The occupancy of ions in the K þ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates. J. Mol. Biol. 333, (2003). 10. Haynes, L. W., Kay, A. R. & Yau, K. W. Single cyclic GMP-activated channel activity in excised patches of rod outer segment membrane. Nature 321, (1986). 11. Stern, J. H., Knutsson, H. & MacLeish, P. R. Divalent cations directly affect the conductance of excised patches of rod photoreceptor membrane. Science 236, (1987). 12. Colamartino, G., Menini, A. & Torre, V. Blockage and permeation of divalent cations through the cyclic GMP-activated channel from tiger salamander retinal rods. J. Physiol. (Lond.) 440, (1991). 13. Zimmerman, A. L. & Baylor, D. A. Cation interactions within the cyclic GMPactivated channel of retinal rods from the tiger salamander. J. Physiol. (Lond.) 449, (1992). 14. Frings, S., Seifert, R., Godde, M. & Kaupp, U. B. Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels. Neuron 15, (1995). 15. Zufall, F., Firestein, S. & Shepherd, G. M. Cyclic nucleotide-gated ion channels and sensory transduction in olfactory receptor neurons. Annu. Rev. Biophys. Biomol. Struct. 23, (1994). 16. Armstrong, C. M. & Taylor, S. R. Interaction of barium ions with potassium channels in squid giant axons. Biophys. J. 30, (1980). 17. Armstrong, C. M., Swenson, R. P. Jr & Taylor, S. R. Block of squid axon K channels by internally and externally applied barium ions. J. Gen. Physiol. 80, (1982). 18. Jiang, Y. & MacKinnon, R. The barium site in a potassium channel by X-ray crystallography. J. Gen. Physiol. 115, (2000). 19. Heginbotham, L., Kolmakova-Partensky, L. & Miller, C. Functional reconstitution of a prokaryotic K þ channel. J. Gen. Physiol. 111, (1998). 20. Noskov, S. Y., Berneche, S. & Roux, B. Control of ion selectivity in potassium channels by electrostatic and dynamic properties of carbonyl ligands. Nature 431, (2004). 21. Haynes, L. & Yau, K. W. Cyclic GMP-sensitive conductance in outer segment membrane of catfish cones. Nature 317, (1985). 22. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, (1997). 23. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, (2002). 24. Collaborative Computational Project, Number 4, The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, (1994). 25. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, (1991). 26. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, (1998). 27. Heginbotham, L., LeMasurier, M., Kolmakova-Partensky, L. & Miller, C. Single Streptomyces lividans K þ channels: functional asymmetries and sidedness of proton activation. J. Gen. Physiol. 114, (1999). Supplementary Information is linked to the online version of the paper at Acknowledgements We thank R. MacKinnon for discussion and critical review of the manuscript. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the US Department of Energy, Office of Energy Research. We thank the beamline staff for assistance in data collection. This work was supported by grants from the David and Lucile Packard Foundation (to Y.J.) and the Searle Scholars Program (to Y.J.). Author Contributions S.Y. and A.A. contributed equally to this work. S.Y. helped with the structure determination and A.A. performed the 86 Rb flux assay. Author Information Atomic coordinates of the Na þ and K þ complexes of the NaK channel have been deposited in the Protein Data Bank with accession numbers of 2AHY and 2AHZ, respectively. Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to Y.J. (youxing.jiang@utsouthwestern.edu). 574