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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nature. Author manuscript; available in PMC 2014 April 8.
Published in final edited form as:
PMCID: PMC3979295
NIHMSID: NIHMS574542

Crystal structure of NavAP, an orthologue of the NaChBac voltage-gated sodium channel

Abstract

Voltage-gated sodium (Nav) channels are essential for the rapid depolarization of nerve and muscle1, and are important drug targets2. A family of bacterial Nav channels, exemplified by NaChBac (Na+-selective Channel of Bacteria)3, provides a good model system for structure-function analysis. Here we report the crystal structure of NavAP, a NaChBac orthologue from marine bacteria alpha proteobacterium HIMB114, at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr178 and Leu179 constitute an inner site within the selectivity filter (178TLSSWE183) where a Ca2+ can bind and resides in the crystal structure. The outer mouth of the Na+ selectivity filter, defined by Ser181 and Glu183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation with all the gating charges exposing to the extracellular side. We hypothesize that NavAP is captured in an inactivated conformation. Comparison of NavAP with NavAb4 reveals significant conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

Nav channels initiate and propagate action potentials in excitable cells1. Since Nav channels underlie a number of clinical disorders such as epileptic seizures and cardiac arrhythmias, they are important drug targets2. Elucidation of the structures and functional mechanisms of Nav channels will shed light on fundamental ion channel mechanisms and facilitate potential clinical applications. The eukaryotic Nav channels are comprised of a pore-forming α subunit and auxiliary subunits5. It consists of one single polypeptide chain that is organized into four repeated domains (DI–DIV) of six transmembrane-spanning (S1–S6) segments. The S5 and S6 segments from each domain form the pore region of the channel, which are flanked by four voltage sensing domains (VSDs) consisting of S1–S4. The VSD, a relatively independent structural entity69, provides the molecular basis for voltage sensing in voltage-dependent channels and enzymes.

Essential for voltage-dependent gating, VSDs contain the gating charges10 embodied in a set of highly conserved positively charged residues occurring every three residues along the S4 segment. In Kv channels, approximately 12 gating charges per channel are transferred across the membrane from the cytosolic side to the extracellular side11,12. While multiple models of voltage sensor activation have been proposed, it is generally accepted that the outward translation of S4 segments is coupled to pore opening via the interactions between the S4–S5 connecting helices and S6 segments7,8.

The activation mechanism is not fully understood. Even more bewildering is the intricate inactivation mechanism for voltage-gated channels. Fast inactivation or N-type inactivation, taking place on millisecond scale, is executed by a cytoplasmic moiety between repeats III and IV of Nav channels13, or by the N-terminus of the Shaker K+ channel1416. Also during prolonged depolarization, slow or “C-type” inactivation17 is thought to result from a conformational change of the selectivity filter18,19.

The prokaryotic homologues of Nav channels, exemplified by NaChBac3, are homo-tetramers of 6-TM subunits. Interestingly, the sequence of NaChBac is closer to that of Cav channels20. Thus, structural elucidation of NaChBac homologues is expected to provide insights into both Nav and Cav channels. To promote a deeper mechanistic understanding of Nav channels, we determined the crystal structure of a NaChBac homologue, NavAP (Supplementary Fig. 1–5, Tables S1&S2). During refinement of our structure, the atomic crystal structure of NavAb was published4. Compared to NavAb, NavAP reveals a number of distinct and mechanistically informative structural features.

As in all the known structures of voltage-gated channels, the VSD of one protomer attaches to the pore-forming unit of the adjacent protomer (Fig. 1a). The activation gate formed by S6 of NavAP is closed, although Leu219, the residue that occludes the gate, is one helical turn above the functionally equivalent Met221 in NavAb (Fig. 1b, right panel)4. Notably, the narrowest point along the pore is at Ser181, which together with Glu183 encloses the entrance to the selectivity filter vestibule (Fig. 1b, right panel). While the selectivity filter of NavAb is open and may allow the conductance of hydrated Na+, that of NavAP is closed (Fig. 1c).

Fig. 1
The structure of NavAP exhibits a closed conformation

Both the pore domain and the VSDs of NavAP exhibit structural variations among the four protomers, resulting in an asymmetric tetramer. The selectivity filter of NavAP (178TLSSWE183) connects P1 (corresponding to the P-helix in K+ channel) and P2 helices (Fig. 2, Supplementary Fig. 6). The side groups of Ser180, Ser181, and Glu183, as well as the carbonyl oxygen atoms of Thr178 and Leu179 constitute the electronegative vestibule of the selectivity filter (Fig. 2a). The entrance to the selectivity filter is negatively charged owing to the side groups of Glu183. The side groups of Ser181 adopt distinctive conformations among the four protomers, leading to the asymmetry of the selectivity filter (Supplementary Fig. 6).

Fig. 2
A Ca2+ ion is bound in the asymmetric selectivity filter of NavAP

Notably, two residues in the selectivity filter of (NavAP Ser180/Glu183 vs. Glu178/Ser181 of NavAb) are swapped in the primary sequences. However, structural superimposition shows that the carboxylate groups of NavAP-Glu183 in are positioned similarly to those of Glu178 in the adjacent protomer of NavAb despite their distinct backbone locations (Fig. 2b). Therefore, NavAP-Glu183 and NavAb-Glu178 appear to be functional equivalents. This structural observation provides a basis to begin to understand the function of the negatively charged residues in the eukaryotic Nav channels that seem to be located at different positions in the selectivity filter (Fig. 2b, left panel).

Like many other bacterial channels, NavAP did not yield measurable ion currents when heterologously expressed in insect or mammalian cell lines, or when expressed in E.coli BL21(DE3) purified, reconstituted into lipids (POPE:POPG 3:1 mass ratio) and fused into bilayers with lipid composition of either POPE:POPG (3:1 mass ratio) or DPhPC. In order to test the selectivity of the NavAP channel, we generated a chimera by replacing the selectivity filter of NaChBac with that of NavAP (Supplementary Fig. 7). The chimeric channel was Na+-selective when expressed in HEK-293 cells and measured under voltage-clamp (Fig. 2c). Similar to observations with other NaChBac pore mutations21,22, the chimera’s (NaChBac/NavAP-filter) voltage dependence of activation was shifted (+49 mV; Fig. 2d) with an altered rate of inactivation (1.6× increase; Fig. 2d). Similar to NaChBac, the chimera was blocked by the Nav channel antagonist, lidocaine, and the Cav antagonist, nifedipine (Supplementary Fig. 7d), but remained insensitive to tetrodotoxin.

During structure refinement, a spherical electron density appeared in the selectivity filter (Supplementary Fig. 8a). We conclude that the electron density is from a calcium ion since: 1) addition of 100 mM CaCl2 was indispensible for obtaining well-diffracting crystals; 2) crystals were also obtained for proteins purified in solutions with RbCl instead of NaCl, and diffracted X-rays at the high remote wavelength for Rb+. After structural refinement, no anomalous signal for Rb+ was observed whereas the omit electron density persisted, suggesting that the electron density was from Ca2+.

When Ca2+ was built into the 3.05 Å structure and further refined, the 2Fo-Fc electron density at 1.5 σ had an elongated tail on the side of the ion facing the central cavity (Supplementary Fig. 8b). A water molecule was then built into the appendage that was approximately 2.4 Å away from the ion, fulfilling the geometric restraint of the interaction between water and Ca2+ (Fig. 2e). The Ca2+ ion is caged by the eight carbonyl oxygen groups from Thr178 and Leu179. The distances between Ca2+ and the eight carbonyl oxygen atoms are in the range of 3.5 to 4.6 Å (Fig. 2e). For direct coordination of a Ca2+ by carbonyl oxygen atoms, the distance is usually between 2.3 – 2.5 Å23. The well-defined electron density suggests that the ion is properly stabilized and thus the Ca2+ should be in a fully, or mostly, hydrated state. However, there was no distinguishable electron density for the surrounding water molecules, perhaps due to the moderate resolution of the structure and/or the intrinsic motility of those water molecules. Ca2+ and Na+ effective ionic radii are practically identical (1.00 Å vs. 1.02 Å), but with the primary hydration shell the radii are 2.7 Å for Ca2+ and 2.2 Å for Na+, respectively24. The observation that the inner binding site of the selectivity filter is spacious enough to accommodate a hydrated Ca2+ or Na+ thus provides structural evidence for the hypothesis that Nav channels allow the passage of mostly hydrated Na+ 25.

Since the chimera was impermeant to Ca2+ (Fig. 2c), we hypothesized that Ca2+ might block Na+ permeation26. Indeed, the Na+ currents from NaChBac and the chimera were substantially blocked by mM concentrations of extracellular Ca2+ and µM concentrations of Cd2+ (Fig. 2f). We speculate that divalent ions are able to enter the channel and occlude the pore at the Leu179/Thr178 site.

Compared to the subtle conformational variations of the filter residues among the four protomers, the divergences of VSDs are more prominent, particularly for S3–S4 linkers (Fig. 3a, Supplementary Fig. 9). Unlike the VSDs of Kv channels, in which the C-terminal segment of S3 and the N-terminal half of S4 form a paddle-like structure68, the C-terminal fragments of S3 in both NavAP and NavAb are unwound. We name the four NavAP protomers Mol A through Mol D. S3–S4 linkers in Mol A and C are not resolved, while those in Mol B and D show distinct conformations; neither is similar to that of NavAb. The flexibility of the S3–S4 linker may allow the movement of the S4 segment during voltage sensing.

Fig. 3
The VSDs of NavAP exhibit a depolarized conformation

Transmembrane segments S1 – S4 of the four VSDs can be superimposed with RMSD (root-mean-squared deviation) values within 0.9 Å over 71 to 81 Cα atoms (Fig. 3b). Consistent with a 0 mV field during crystallization, all four conserved Arg residues on the S4 segment point extracellularly, representing a depolarized (“up”) conformation (Fig. 3b). The external negative clusters stabilize the gating charges through two invariant interactions: R4 interacts with Asp48 (known as anion 1or An1) on S2 and R3 is H-bonded to the carbonyl oxygen of Ile90 on S3 (Fig. 3c). In addition to these invariant interactions, there are additional stabilizing contacts specific to individual VSDs. In Mol A, an extra H-bond is between R2 and the carbonyl oxygen of Asn25 on S1. In Mol B, R3 binds to An1. In Mol C, R3 is further H-bonded to the carbonyl oxygen of Ser88.

The structure of NavAP has a closed inner gate and VSDs in a depolarized (i.e. open) conformation. Similar features were described for NavAb, which was proposed to be in the pre-open conformation4. While the structure of NavAP may also represent a pre-open state, an alternative interpretation is that NavAP is in an inactivated state. The following lines of evidence support this speculation: 1) NaChBac homologues and NaChBac/NavAP-filter chimera undergo inactivation on a millisecond to second time scale (Fig. 2d)17 and since purification and crystallization of the proteins occurs over days at 0 mV, we assume this should favor complete inactivation of the channel; 2) There is little interaction between S4–S5 connecting helices and S6 segments (Fig. 3a), indicating a loss of coupling between the voltage sensor and the inner gate. Furthermore, the structure of NavAP is consistent with a possible form of inactivation discussed by Schmidt et al to account for the gating properties of KvAP27; when S4–S5 linkers release their constriction of the S6 helices, the inner gate may close even if the VSDs are still in the up conformation; 3) In both Nav and Shaker K channels, mutagenesis analyses suggested that the selectivity filter residues are involved in C-type inactivation17,19. In rat Nav1.4, residues Glu403, Glu758, Asp1241, and Asp1532, which correspond to Glu183 in NavAP (Fig. 2b and Supplementary Fig. 1) are important for inactivation28. In our structure of NavAP, Glu183 and Ser181 collectively close the outer mouth to the selectivity filter (Fig. 1c), supporting the reported functional significance of the outer negative charges in the inactivation process. Based on the above analyses, we speculate that the structure of NavAP shown here represents an inactivated conformation.

Superposition of the pore domains of NavAP and NavAb reveal prominent conformational changes of the VSDs. Viewed from the cytoplasm, the VSDs of NavAP are rotated counter-clockwise around the pore axis by ~30° and the relative positions of the VSDs in NavAP are more like those in the depolarized and open conformation of Kv1.27 (Fig. 4a). When the individual VSDs of NavAP and NavAb were compared by superimposing the S4–S5 linkers, (Supplementary Fig. 10a), the VSDs diverge from each other suggesting that the VSD and S4–S5 linker do not move as a single unit. Asp48 (An1), Phe55, and Glu58 (An2) on the S2 segment constitute the charge transfer center (CTC)29 in NavAP. Superimposing the VSDs of NavAP and NavAb relative to the CTC, it is clear that the other transmembrane segments now are discordant, indicating a significant intra-domain rearrangement within the VSD (Supplementary Fig. 10b).

Fig. 4
Molecular basis of charge transfer of VSDs

Gating charges are transferred in response to a change in transmembrane voltage. Superimposition of NavAP and NavAb VSDs relative to the CTC unambiguously shows that there is a one helical turn shift of NavAP-S4 toward the extracellular side (Fig. 4b). That is, for each NavAP VSD, one more charge is transferred than for each NavAb VSD. Interestingly in NavAb, S4 exists as a 310-helix from R1 to R44. In NavAP, however, while the segment from R3 to R4 forms a 310-helix, the preceding segment is relaxed into an α-helix (Fig. 4b). We also compared the voltage sensors of NavAP to those of Kv1.2 and the paddle chimera, in which only the C-terminal halves of S4 segments containing R3 to R5 adopt a 310-helix whereas the segments containing R0 to R2 are relaxed into α-helices. However, when the CTCs are superimposed, the relative positions of the gating charges, exemplified by R4 of NavAP are located between those of Kv1.2 and the paddle chimera (Fig. 4c).

The availability of voltage sensor structures with unique positions of the gating charges relative to the CTC provides evidence that supports our structure based animation of gating charge transfer (Fig. 4d and Supplementary movie S1).animation (Fig. 4d and Supplementary movie S1). The movie illustrates how R3 and The movie illustrates how R3 and R4 are stabilized sequentially by An1 and An2 to lower the energy barrier during the transfer of R4 across the occluding Phe residue. It also shows the secondary structure transition between 310- and α-helices, concurrent with the translational motion of S4 segment relative to CTC, which exemplifies the ‘concertina effect’ discussed for Kv channels8 and is consistent with the disulfide cross-linking experiments of NaChBac30. It is noteworthy that the flexibility of the S3–S4 linker may help lower the energy barrier during the motion and the secondary structural transition of S4 segment (Fig. 3a). The complementary studies of NavAP and NavAb thus provide an important framework for future functional and mechanistic investigations of voltage-gated ion channels.

Materials and methods

Protein preparation

The cDNAs of NaChBac homologs, whose sequences were codon optimized for E.coli expression, were cloned into bacteria expression vectors and the recombinant proteins were over-expressed in E. coli BL21(DE3). After screening several dozens of homologs, only alpha proteobacterium HIMB114 (NavAP) yielded crystals. The proteins from all homologs were purified without protease inhibitors, which we surmise helps in the selection of the most compact and stable targets31. The full-length NavAP was cloned into pET21b vector (Novagen). The NavAP mutants were generated using two-step PCR and were subcloned, overexpressed and purified in the same way as wild-type (WT) protein. Overexpression of NavAP was induced in E. coli BL21 (DE3) by 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) when the cell density reached O.D600 nm 1.5. After growth at 30 °C for 12 h, the cells were harvested, resuspended in a buffer containing 25 mM Tris-HCl, pH 8.0, and 150 mM NaCl, and disrupted by sonication. Cell debris was removed by centrifugation at 27,000 g for 10 min. The supernatant containing the membrane was collected and applied to ultracentrifugation at 150,000 g for 1 h. The membrane fraction was collected and incubated with 1.6% (w/v) n-dodecyl-β-D-maltopyranoside (DDM, Anatrace) for 2 h at 4 °C. After an additional ultracentrifugation at 150,000 g for 30 min, the supernatant was loaded to Ni2+-nitrilotriacetate affinity resin (Ni-NTA, Qiagen). Subsequently, the resin was rinsed ×3 with 10 ml buffer containing 25 mM Tris-HCl, pH 8.0, 500 mM NaCl, 50 mM imidazole-HCl, pH 8.0 and 0.02% DDM. The protein was eluted from the affinity resin with wash buffer supplemented with 400 mM imidazole-HCl, pH 8.0. The proteins were concentrated to about 15 mg/ml before applying to gel-filtration chromatography (Superdex-200 10/30, GE Healthcare), which was equilibrated in the buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.4% n-nonyl-β-D-glucopyranoside (β-NG, Anatrace). The peak fractions of the protein (~8 gm/ml) were collected and incubated with 0.1 mg/ml lipids POPC:POPE:POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine:1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1-glycerol, Anatrace) at mass ratio 3:1:1 for crystallization trials.

Crystallization

Crystals were grown at 18 °C by the hanging-drop vapor diffusion method. To improve resolution, multiple steps of construct modification, crystal growth optimization, and post-crystallization manipulation were explored. In the beginning, the C-terminal His6 tagged, WT proteins yielded cubic-shaped crystals in the buffer containing 0.7 M MgSO4, 0.1 M MES-NaOH, pH 6.0. The crystals diffracted to ~ 8Å at BL41XU, Spring-8, Japan. Removal of His6-tag improved the crystal quality significantly. Crystals of the WT, non-tagged protein appeared overnight in the buffer containing 16% PEG 400 (v/v), 100 mM MES-NaOH, pH 6.5, 100 mM CaCl2, and diffracted to 4.0 Å at the synchrotron radiation resource. However, it was difficult to scale the data sets to a specific space group. A single point mutation G208S improved the data quality. The space group was ultimately assigned as P41212 using the data sets obtained for NavAP-G208S. Further improvement was achieved with a new crystallization condition. Crystals appeared in the buffer containing 5% PEG 8,000 (v/v), 100 mM HEPES-NaOH, pH 7.0, 100 mM CaCl2, 10% Glycerol and 20% 1,4-Butandiol in 2 d, and grew to 50 µm × 50 µm × 100 µm tetragonal rods in 5 d. The crystals, in the space group of P42, were able to break the 4.0Å diffraction limit but with poor reproducibility and a high mosaicity value (>5). Finally, the best crystals were obtained through dehydration manipulation, by gradually increasing the precipitant concentration in crystallization buffer to 15% PEG 400 (v/v), 20% PEG 8,000 (v/v). The crystals were flash frozen in liquid nitrogen, and diffracted beyond 3.05 Å at SSRF (Shanghai Synchrotron Radiation Facility) beamline BL17U. Mercury derivatives were obtained by soaking the crystals for 3 h in the dehydration solution plus 10 mg/ml methylmercury chloride (CH3HgCl) as the final concentration.

Data collection and processing

All data sets were collected at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U, except for the native data in the space group of P41212, which were collected at the SPring-8 beamline BL41XU. All were integrated and scaled with HKL200032. Further processing was carried out using programs from the CCP4 suite33. Data collection statistics are summarized in Table S1.

Experimental phasing and structure refinement

The mercury positions in the Hg-derived crystal of the P42 space group were determined using the program SHELXD34. The identified heavy-atom sites were refined and the initial phases were generated in the program PHASER35 with the SAD experimental phasing module. Cross-crystal averaging combined with solvent flattening, histogram matching and NCS averaging in DMMulti36 gave rise to electron density maps of sufficient quality for model building, using the data sets in Table S1. An initial model was built into the high-resolution P42 native data using COOT37. The structure was refined with PHENIX38. All structure figures in the manuscript were prepared with PyMol39. The surface electrostatic potential presented in the manuscript was calculated with PyMol. The pore radii were calculated with the program “HOLE”40.

Electrophysiology

Whole-cell voltage-clamp experiments were performed at 22°C in transiently transfected HEK-293 cells. Transfected cells were seeded onto glass coverslips and placed in a perfusion chamber for experiments in which extracellular conditions could be exchanged. Unless otherwise stated, the extracellular solution contained (in mM): NaCl 150; CaCl2 1.5; MgCl2 1; glucose 10; and HEPES 10; pH 7.4, and the intracellular (pipette) solution contained (in mM): CsF 105; EGTA 10; NaCl 35; MgCl2 4; and HEPES 10; pH 7.3. For experiments shown in the left panel of Figure 2c, representative current traces elicited by 0.5-s depolarizations from −140 mV (holding potential) to 0 mV. Na+ was substituted by the ions indicated (150 Cs+, K+; 110 Ca2+, Ba2+). Normalized current magnitudes plotted as a function of time as Na+-containing solution is exchanged for solutions with the indicated ions (colored boxes). For experiments shown in the right panel of Figure 2c and 2d, “±” indicates SEM.; n = 4 – 5 each. Current-voltage relationships were fit to (V−VRev)/{1 + exp[(V−V1/2)/k]}, where VRev is the extrapolated reversal potential, V1/2 is the half-activation voltage, and k is a slope factor equal to RT/zF (z is the apparent gating charge, R the ideal gas constant, and F is Faraday’s constant). Half-inactivation voltages were derived from fits to 1/{1+exp(V−V1/2)/k} to derive steady-state inactivation curves. Inactivating currents during 500 ms pulses were fit to C + A(e−t/τ), where τ is the time constant, A, the amplitude, and C, the baseline. For experiments shown in the left panel of Figure 2f, decay in response to a 500-ms pulse to the indicated potentials was fit to a single exponential. The half maximal inhibitory concentration (IC50) was estimated by fitting the average percent of inward Na+ blocked at each concentration to: % block of the current amplitude = 1/{([D]/IC50)n +1}, where n is the Hill coefficient and [D] is the respective drug or divalent concentration.

Animation

In order to generate the morph to visualize the conformational change of the S4 segments between NavAP and NavAb, the homology-based model of NavAP was generated using the online SWISS-MODEL workspace4143 with the structure of NavAb (PDB code: 3RVY, Chain A) as the model. The resulting structure was then superimposed on that of NavAP relative to the CTC. The shifted coordinates of the modeled structure and the original coordinates of NavAP were used as the initial and end states, respectively, for morph generation. The intermediate morphs were obtained with the multiple-chain morphing script44,45 for Crystallography & NMR System (CNS)46,47. The animations were finally produced using PyMol.

Supplementary Material

Supplementary Information

Acknowledgements

We thank R. MacKinnon at Rockefeller University for critical discussions and critical reading of the manuscript. We thank L. Feng at Rockefeller University for help. We thank S. Huang and F. Yu at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U. K. Hasegawa acknowledges Spring-8 for proposal 2011A2039. This work was supported by funds from the Ministry of Science and Technology (grant numbers 2009CB918802, 2011CB910501, and 2011CB911102), Projects 31125009 and 91017011 of the National Natural Science Foundation of China, and funds from Tsinghua University.

Footnotes

Author Contributions X.Z., W.R., P.D., X.T., D.C., and N.Y. designed all experiments. X.Z., W.R., P.D., C.Y., X.T., L.T., J.W., K.H., T.K., J.H., and J.W. performed the experiments. X.Z., W.R., P.D., C.Y., X.T., J.W., D.C., and N.Y. analysed the data. X.Z., P.D., X.T., C.Y., J.W. and D.C. contributed to manuscript preparation. N.Y. wrote the manuscript.

Author Information The atomic coordinates of NaVAP have been deposited in the Protein Data Bank under accession code 4DXW.

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