Functional characterization of NALCN by electrophysiology
We employed patch-clamp recordings in HEK293 cells to characterize the electrophysiological properties of NALCN. The current elicited in response to voltage steps was very small and appeared to be indistinguishable from the mock when NALCN channel was expressed alone in HEK293 (Supplementary Fig. 1a). By contrast, co-expression of NALCN with UNC79, UNC80 and FAM155A dramatically enhanced the current, which was largely inhibited by 1 mM verapamil, implying that the current measured was mainly mediated by NALCN (Supplementary Fig. 1b). We also clearly recorded the kinetics of voltage sensitive NALCN current: hyperpolarization voltages elicited large inward current with inactivation, while depolarization voltages activated current with an exponential course before reaching in the steady state (Supplementary Fig. 1b). These observations indicate that the auxiliary subunits are essential for a functional NALCN (Supplementary Fig. 1a), consistent with previous studies 30,31. The electrophysiological properties of NALCN suggested that it tends to maintain the RMP in a manner reminiscent the Le Chatelier’s principle in chemical equilibria. In comparison, Nav channels always show rapid current activation and inactivation for fast initiating action potential 32, while certain Kv such as KCNQ channels show slow activation kinetics to avoid fast repolarization 33.
Structure determination of human NALCN-FAM155A complex
Based on our functional characterizations, we focused on the co-expression of the four components in HEK293 cells. Unfortunately, we were unable to get a stable complex of the four components. Instead, a stable and homogeneous subcomplex of NALCN and FAM155A was obtained (Supplementary Fig. 2a). The peak fractions from gel filtration purification, were pooled and concentrated to about 8.5 mg/ml for cryo-EM sample preparation. The EM images were collected on a Titan Krios electron microscope operating at 300 kV and equipped with a K3 direct detector and a Gif Quantum energy filter. After a few rounds of two-dimensional (2D) and 3D classifications, about sixty-five thousand particles were selected, which yield a final reconstruction map with an overall resolution of 3.1 Å (Fig. 1, Supplementary Table 1, Supplementary Fig. 2, 3). Local masks of the extracellular and intracellular regions were applied to further improve the local resolutions to 2.9 Å and 3.1 Å, respectively.
The density of the final reconstruction is of high quality with evenly distributed estimated local resolution. Most side chains were clearly resolved that allow for accurate model building (Fig. 1a, 1b, Supplementary Fig. 4, 5). The model of NALCN was built with a homology model based on Cav1.1 (PDB: 5GJV), whereas FAM155A was built de novo. In total, 1295 and 182 residues were built for NALCN and FAM155A, respectively. The complex structure was confirmed by cross-linking mass spectrometry analysis (Supplementary Fig. 6). Three glycosylation sites (Asn210, Asn216 and Asn1064) and one glycosylation site (Asn217) were identified on NALCN and FAM155A, respectively (Fig. 1c, Supplementary Fig. 5c). Four disulfide bonds in the extracellular loops (ECL) of NALCN and six in FAM155A were recognized (Fig. 1c, Supplementary Fig. 5d). The glycosylation sites and disulfide bonds in turn verified the accuracy of the structure.
The overall structure of NALCN resembles those of eukaryotic Nav and Cav channels (Fig. 2). We select human Nav1.4 (PDB: 6AGF) and rabbit Cav1.1 (PDB: 6JP5) as representatives of Nav and Cav channels for comparisons with NALCN 34,35. The structures of human Nav1.7 (PDB: 6J8I), rat Nav1.5 (6UZ3) and NavPaS (6A95) from American cockroach were also picked for specific discussions 36-38. The structures of NALCN can be superimposed to the α subunit of Nav1.4 and α1 subunit Cav1.1 with root-mean-square deviation (r.m.s.d) of 2.99 Å over 760 Cα atoms and 3.894 Å over 905 Cα atoms, respectively. Except for the four homologous repeats, a C-terminal domain (CTD) after repeat IV, which was observed in the structures of Cav1.1 and NavPaS but not in human Nav channels, was also clearly resolved in the structure of NALCN (Fig. 1c). NALCN has no N-terminal domain (NTD) before repeat I, which was always observed in human Nav channels.
NALCN has several unique features that are distinct from both Nav and Cav channels. In particular, two lipids, lipid1 near repeat I and lipid2 near repeat III, were found to bind NALCN at the inner membrane side (Fig. 1c). Lipid2 resides in a cavity surrounded by S3, S4 and S4-5 of repeat III, while lipid1 occupies the cavity surrounded by S4I, S5I, S6I, S5II, S6II and S4-5I (Fig. 2c, Supplementary Fig. 4b). Notably, lipid1 sits in a position that occupied by S4-5 linker in Nav/Cav structures (Fig. 2c). This may have caused the conformational change of the S4-5I linker from a helix to a loop in NALCN. In addition, a short helix designated as the II-III linker, that was connected to S6II by a short loop was firstly observed in the structure of NALCN (Fig. 1c).
Specific interactions between NALCN and FAM155A
Human FAM155A contains 456 amino acids and residue 192-382 were resolved in our structure except for a short loop from residue 250 to 258 (Fig. 3a). The N-terminal and the C-terminal regions of FAM155A, including two predicted TM helices, were not resolved in current structure, probably because of their intrinsic flexibility. The resolved structure of FAM155A contains two lobes, designated as N-lobe and C-lobe, stabilized by six disulfide bonds. The N-lobe consists of three short β strands and two short α helices (H1-H2), while the C-lobe contains four α helices (H3-H6) (Fig. 3b). The overall structure of FAM155A is different from any auxiliary subunit in Nav or Cav channels (Fig. 2b, Supplementary Fig. 7a). A search of the protein data bank using the DALI server 39 revealed that the C-lobe of FAM155A contains a fold (similarity Z-score 4.3) similar to the structure of the frizzled-like cysteine-rich domain (CRD) of receptor tyrosine kinase MuSK (PDB: 3HKL), which had 84 Cα atoms aligned to FAM155A with a r.m.s.d. of 2.8 Å (Supplementary Fig. 7b). Superimposition of the CRD domain of MuSK on FAM155A shows that H3, H5 and H6 align quite well. Moreover, three disulfide bonds are structurally conserved between the two structures (Supplementary Fig. 7b).
Only the C-lobe of FAM155A is involved in the interaction with NALCN. FAM155A sits above the pore domain of repeat IV of NALCN and form extensive interactions with the ECLI, ECLIII and ECLIV of NALCN (Fig. 1c, 3c, Supplementary Table 2). The loop in FAM155A connecting H4 and H5 and a hairpin loop after H6 that is involved in binding to NALCN are designated L1 and L2, respectively (Fig. 3b). Notably, L2 of FAM155A inserts deeply into a dome formed by ECLIII of NALCN. The H3, H5, H6 helices and L2 of FAM155A resemble the palm and thumb of a hand, respectively, holding ECLIII of NALCN tightly (Fig. 3c). Detailed analysis of the interaction interfaces revealed extensive salt bridges and hydrogen bonds between NALCN and FAM155A (Fig. 3d, Supplementary Table 2). Unexpectedly, the sugar moiety linked to Asn1064 on ECLIII, also contributes to the interaction, by forming a hydrogen bond with E350 on FAM155A (Fig. 3d). Electrostatic surface analysis revealed that two surfaces between NALCN and FAM155A are electrically complementary, ensuring a favorable interaction stability (Supplementary Fig. 7c). Sequence alignment among NALCN and Nav/Cav channels revealed that the interface residues in NALCN, including Asn1064, are not conserved among other Nav/Cav channels, explaining the high binding specificity of FAM155A towards NALCN, but not other channels (Supplementary Fig. 8). The interface between FAM155A and NALCN is very different from those between the ion conducting α subunit of Nav and Cav channels and their auxiliary subunits (Fig. 2b). The key residues in the interaction interface are highly conserved among NALCN and FAM155A proteins from different species, indicating that the interaction mode between NALCN and FAM155A is evolutionarily conserved. Furthermore, the key residues mediating complex formation in FAM155A are also conserved in FAM155B, another subtype protein of the FAM155 family (Supplementary Fig. 9). Therefore, FAM155B may also be able to form a stable complex with NALCN in vivo. This notion is supported by the finding that FAM155A can be functionally substituted by human FAM155B 30.
As our structure was captured as a subcomplex between NALCN and FAM155A, we then asked whether co-expression of NALCN and FAM155A only was also able to recapitulate measurable NALCN current. Interestingly, we have readily recorded large current in response to voltage steps when NALCN and FAM155A was co-transfected (Supplementary Fig. 1c). The current-voltage curve of NALCN co-expressed with either all three auxiliary subunits or with FAM155A only was non-linear (Supplementary Fig. 1d). Compared with NALCN co-expressed with three auxiliary subunits, NALCN with FAM155A exhibited similar voltage sensitivity in current as the apparent gating charge (Zapp) was similar (Supplementary Fig. 1d, Zapp: 1.25 ± 0.55 e0 and 1.22 ± 0.18 e0, respectively). However, the G-V curve of NALCN co-expressed with three auxiliary subunits was largely shifted towards the hyperpolarization voltage as compared to NALCN with FAM155A (V1/2: -44.66 ± 1.91 mV and -65.07 ± 2.11 mV, respectively) (Supplementary Fig. 1e). These findings are distinct from other recent studies 40,41. Moreover, we observed no inactivation of the steady state current by setting the prepulse potential to different levels when NALCN channel was co-expressed with auxiliary subunits (Supplementary Fig. 1f and 1g), which is consistent with the physiological role of NALCN being a leaky sodium channel.
Structural basis for the voltage dependence of NALCN
Our structural study has provided an opportunity to understand the voltage sensitivity of NALCN. Detailed analysis of the voltage sensing domains (VSDs) of NALCN suggest that they possess several key features shared among functional VSDs in Nav/Cav channels. For instance, each VSD preserves the gating charges (GCs) in a pattern of occurring every three amino acids in the S4 segments. As usual, we define the position of last gating charge in each S4 as R6 42. NALCN has two to four GCs in each repeat distributed at positions R2-R6 (Fig. 4a). The last GC of repeat IV is at a position that has one residue shift to R6, a phenomenon also observed in other channels 42. Moreover, the residues of charge transfer center (CTC) consists of a negatively charged residue (An2) on S2, an aromatic occluding residue (F) on S2, and a negatively charged residue on S3 are highly conserved in NALCN except for the occluding residue on VSDIII. The An1 sites that are seven residues ahead of the occluding residues on S2 segments of NALCN are mainly negatively charged or polar residues, similar to those of Nav/Cav channels (Fig. 4a, b). In addition, previous structural studies on Nav channels identified several negatively charged or polar residues on S1 that may also play roles in gating charge transfer 34. These residues were also observed on NALCN (Fig. 4a). When the four VSDs are superimposed relative to An1 and CTC, all R4 residues are above the occluding residues on S2, reminiscent of an up or depolarized state of the four VSDs (Fig. 4b, Supplementary Fig. 10).
The voltage sensitivity of NALCN, however, seems to be weaker than that of Nav/Cav channels (Supplementary Fig. 1). Several unique structural features of NALCN may lead to its relative weak voltage sensitivity. First, the occluding residue on S2 in repeat III is replaced by a methionine (Met929), instead of a conserved phenylalanine or tyrosine (Fig. 4c). The occluding residue of the CTC is crucial for the gating transfer during voltage sensing. A previous study shows that mutations of the occluding residue from bulky hydrophobic residue to methionine on a voltage-gated potassium channel diminish its voltage sensitivity 43. Second, the S4 of VSDs are usually formed as 310 helices, whereas in repeat IV the S4 segment largely relax to regular α helix. The only 310 helix turn in S4IV, which is at position of R5, is a serine instead of a GC. These structural observations are also consistent with reported experimental data. For example, it was reported that repeat III and IV contribute little to the voltage sensitivity of NALCN, probably due to their lack of an occluding residue on S2, and lack of GCs and 310 helices on S4, respectively. In contrast, neutralization of the GCs R146+R152 in repeat I or R481+R484 or R481+K487 in repeat II lead to significantly changes in voltage sensitivity 30. Third, the upper gating charges (R2-R4) in repeat I-III form more extensive hydrogen bonding interactions with surrounding negatively charged or polar residues, compared with their counterparts in Nav/Cav channels (Fig. 4c). During the gating charge transfer process, the GCs need to break old interactions with surrounding residues and form new interactions with other residues, accompanying with a sliding movement along the S4 segment. The extensive interactions of the GCs and surrounding residues in NALCN may need the VSDs to overcome a higher energy barrier to undergo conformational changes thus resulting in a weaker voltage sensitivity. Fourth, the S4-5I linker unexpectedly forms a flexible loop instead of a juxtamembrane helix seen in other repeats and other reported Nav/Cav structures. Meanwhile, S5I is extended and elongated to the cytoplasmic region with three helix turns. The unique structure of S4-5I and S5I results in a cavity that accommodate a lipid, that may contribute to the regulation of NALCN (Fig. 2c, Supplementary Fig. 4b). As S4-S5 is supposed to be a key transducer from VSDs to the ion conducing pore during voltage sensing gating in VGICs 44, its relaxation to a flexible linker may hinder the effective transduction of electromechanical coupling of repeat I in NALCN. Altogether, these structural variations in NALCN may contribute to its weak voltage sensitivity.
EEKE selectivity filter in NALCN
Like all reported Nav/Cav structures, the pore domain of NALCN is formed by S5 and S6 helices of the four repeats and the selectivity filter (SF) of NALCN was supported by two pore helices (P1 and P2) that intervene between S5 and S6 (Fig. 5a). The key residues in the SF of NALCN responsible for the ion selectivity are E280/E554/K1115/E1389 (EEKE), distinct from DEKA in Nav channels and EEEE/EEDD in Cav channels. Sequence alignment of the P1-SF-P2 among NALCN, Cav1.1 and Nav1.4 reveals an invariant tryptophan in the first residue of P2 and a highly conserved residue (Thr/Ser/Cys) in the last residue of P1, while other residues are not very conserved (Fig. 5b). The overall structure of P1-SF-P2 in NALCN is more closely related to that of Cav1.1 than to that of Nav1.4, with r.m.s.d. of 0.848 Å over 110 Cα atoms and 1.602 Å over 107 Cα atoms, respectively. Both the pore helices and the SF of NALCN align well with those of Cav1.1, whereas the P2s are not aligned well in superimposition between NALCN and Nav1.4 (Fig. 5c). Despite its structural similarity with Cav channels, NALCN was reported to be mainly selective for monovalent cations and could be blocked by extracellular divalent cations 30. In addition, NALCN is responsible for the background sodium leak conductance in hippocampal neurons, indicating that NALCN is selective for Na+ ion in vivo 5.
To better understand Na+ binding and selectivity in the SF, we performed canonical MD simulation to investigate the interaction of membrane-embedded pore domain in 150 mM NaCl. In three independent trials of 200 ns equilibration, we observe three potential ion binding sites, designated Site1-3, in proximity to SF (Fig. 5d). In addition to the glutamic acid residues in SF, the Na+ binding sites are formed by several negatively changed residues from the pore helices, including E275, D558, D561 and D1390. Among the three binding sites, Site1 is most stably located at the middle of SF, while the other two are transient and presumably cation attractants (Fig. 5d, Supplementary Movie 1). The simulation results were reproducible in three parallel runs. Probability statistics suggest that there is an average of two Na+ ions within 4 Å range of the SF residues through time (Fig. 5e). Notably, Site3 formed by E280/E554/D558 (designated EED site) is spatially close to the Na+ binding site formed by a DEE motif in Nav channels (Fig. 5f) 34. However, the sidechains of the EED motif in NALCN are not as closely spaced as the DEE site in Nav channels and do not form a favorable Na+ binding site. On the other hand, Site1, the most occupied binding site, is spatially close to a putative Ca2+ binding site in Cav1.1 and Cav3.1, suggesting that NALCN and Cav channels share similar ion selectivity mechanisms 35,42 (Fig. 5f). The replacement of glutamic acid (E) or aspartic acid (D) with lysine (K) in repeat III from Cav channels to NALCN has shifted the ion selectivity from Ca2+ to monovalent ions. Like Nav channels, the lysine in NALCN favors Na+ ion and blocks Ca2+ permeation through the selectivity filter. Meanwhile, the EEKE SF may still preserve the ability to bind Ca2+ as the structure of SF in repeat I/II/IV is almost identical to that of Cav channels (Fig. 5c). In this sense, extracellular Ca2+ may compete with Na+ in binding to the SF, explaining why Ca2+ is a blocker of NALCN as shown by previous study 30.
Closed intracellular gate of NALCN
The ion permeation path below the SF in NALCN is enclosed by the S6 tetrahelical bundle, a structural feature that is shared in all reported Nav/Cav structures (Fig. 5a, 6a). However, due to the existence of FAM155A, ions can only enter the permeation path from one side, that is above the pore domain of repeat II (Fig. 6a, left). We used HOLE 45 to calculate the radius along the permeation path and compared it with Nav1.4 and Cav1.1, whose intracellular gates are in open and closed state, respectively. Surprisingly, although NALCN is supposed to conduct a ‘leak’ current, the intracellular gate is closed, even tighter than the closed Cav1.1. The narrowest region, sealed by two layers of hydrophobic residues Val, Ile and Leu, is about 10 Å in length along the permeation path with a radius less than 1 Å (Fig. 6a, right). Notably, the lower gate is fully blocked by the III-IV linker, causing the permeation path to enter the cytosol only from the side. Detailed analysis reveals that the III-IV linker is stabilized by extensive hydrogen bonds or polar interactions with S6I and the II-III linker (Fig. 6b). Most of the involved residues are not conserved among Nav/Cav channels, indicating that the local interactions among S6I, the II-III liner and the III-IV linker are highly specific to NALCN, which implies distinct gating mechanism of NALCN (Supplementary Fig. 7).
It is intriguing that the position of the III-IV linker in NALCN is distinct from those of both Nav1.4 and Cav1.1, in different ways. NALCN does not have an IFM motif, a highly conserved motif in the III-IV linker of all Nav channels that is crucial for fast inactivation through an allosteric inhibition mechanism 44,46. In Nav1.4, the III-IV linker is away from the inner pore, and its helix rotates counterclockwise by about 750 but at a similar vertical height compared to that of NALCN (Fig. 6c, top). The helix in the III-IV linker, however, shows a similar orientation but distinct vertical height in Cav1.1. Compared to Cav1.1, the helix in the III-IV linker of NALCN is about 6 Å up shifted towards the extracellular side, probably due to a shorter loop connecting S6III and the helix in the III-IV linker (Fig. 6c, bottom). The II-III linker, which is part of the elongated S6II in Cav1.1, bends towards the center pore. These structural features in NALCN make S6I, II-III linker and III-IV linker form a close contact that tightly seal the inner gate.
Notably, S5II and S6II are much closer to the center pore in NALCN compared with Nav1.4/Cav1.1. The adjacent S4-5II also displays significant conformational changes in NALCN (Fig. 6c). The dramatic differences in the pore domain of repeat II are closely related to the binding of a lipid molecule (Fig. 2c). The lipid, only observed in NALNC, may play an important role in regulating the gating of NALCN.