Solution Structure of the Na_V1.2 C-terminal EF-hand Domain^,

Abstract

Voltage-gated sodium channels initiate the rapid upstroke of action potentials in many excitable tissues. Mutations within intracellular C-terminal sequences of specific channels underlie a diverse set of channelopathies, including cardiac arrhythmias and epilepsy syndromes. The three-dimensional structure of the C-terminal residues 1777-1882 of the human Na_V1.2 voltage-gated sodium channel has been determined in solution by NMR spectroscopy at pH 7.4 and 290.5 K. The ordered structure extends from residues Leu-1790 to Glu-1868 and is composed of four α-helices separated by two short anti-parallel β-strands; a less well defined helical region extends from residue Ser-1869 to Arg-1882, and a disordered N-terminal region encompasses residues 1777-1789. Although the structure has the overall architecture of a paired EF-hand domain, the Na_V1.2 C-terminal domain does not bind Ca²⁺ through the canonical EF-hand loops, as evidenced by monitoring ¹H,¹⁵N chemical shifts during aCa²⁺ titration. Backbone chemical shift resonance assignments and Ca²⁺ titration also were performed for the Na_V1.5 (1773-1878) isoform, demonstrating similar secondary structure architecture and the absence of Ca²⁺ binding by the EF-hand loops. Clinically significant mutations identified in the C-terminal region of Na_V1 sodium channels cluster in the helix I-IV interface and the helix II-III interhelical segment or in helices III and IV of the Na_V1.2 (1777-1882) structure.

Voltage-gated sodium channels (VGSCs)5 are molecular assemblies that span the plasma membrane of excitable cells and conduct sodium current selectively in response to depolarizing stimuli. Mutations in VGSCs underlie a variety of diseases, including the cardiac arrhythmogenic Long-QT3 and Brugada syndromes (1, 2) and neurological syndromes, such as epilepsy (3, 4).

Known components of VGSCs include a pore-forming α subunit, auxiliary β subunits, and associated modulating proteins, such as calmodulin (5, 6). The α subunit is composed of four homologous six-transmembrane helical domains connected by inter-domain linkers and N-terminal and C-terminal cytoplasmic regions. Specific α subunit isoforms are expressed differentially in skeletal muscle (Na_V1.4), cardiac muscle (Na_V1.5) and the nervous system (Na_V1.1, Na_V1.2, Na_V1.3, splice variants of Na_V1.5, and Na_V1.6-Na_V1.9) and control the rapid upstroke of action potentials (7).

VGSC activity is characterized by two open states and several inactivated states (8). Kinetics of channel inactivation occur on timescales ranging from milliseconds to seconds and determine multiple aspects of action potentials (9, 10). The molecular mechanisms of VGSC inactivation are complex and involve the α subunit, the β subunits, and calmodulin (11-13). Specific contributions to α subunit inactivation have been localized to interhelical intra-domain regions (14-16), the linker region between domains III-IV, which forms the pore occluding inactivation gate (17, 18), and the C-terminal cytoplasmic domain (CTD) (19-21).

Specific disease-causing mutations within the CTD affect channel function by altering kinetics of channel inactivation (22). The CTD is predicted by sequence analysis (23, 24) and homology modeling (25-27) to contain a paired EF-hand domain and was observed to contain a distal calmodulin binding IQ motif (4, 12, 28-31). Structural modeling also predicts that specific interactions between helix I and helix IV control channel inactivation (27, 32). A recent model, based on NMR chemical shift perturbations, fluorescence spectroscopy, and electrophysiology, suggests that inactivation is regulated by Ca²⁺ binding to the proximal EF-hand, which is strongly influenced in turn by interactions with the distal IQ motif and calmodulin (33). Nevertheless, whether Ca²⁺ binds specifically to the putative CTD EF-hand and any resultant contribution to channel regulation is controversial (12, 26, 31, 34).

EXPERIMENTAL PROCEDURES

Constructs of the Na_v1.2 CTD were designed by limited proteolysis and H/D exchange experiments. Briefly, the CTD of Na_v1.2, residues 1777-1937 with the amino acid substitutions I1877A/Q1878A and an N-terminal His₆ tag MGSSHHHHHHSSGLVPRGSHMAS (31), was subjected to proteolytic digestion with proteinase K at 4 °C for 15-60 min using a protein:protease ratio of 50:1-100:1. The termini of the protected proteolytic fragments were mapped by matrix-assisted laser desorption ionization time-of-flight time-of-flight mass spectrometry and N-terminal sequencing. H/D exchange experiments were performed by ExSAR (Monmouth Junction, NJ) and showed protection for proteolytic fragments extending from residues 1789 to 1879. The construct encompassing residues 1777-1882 of the Na_v1.2 CTD defined by the above experiments, including the N-terminal His tag, was used for structure determination by solution NMR spectroscopy.

[U-¹³C,U-¹⁵N] Na_V1.2 CTD (1777-1882) was overexpressed in Escherichia coli (BL21 DE3) transformed with a pET28 vector (EMD Biosciences) using M9 minimal media prepared with ¹⁵NH₄ Cl and [¹³C₆]glucose (35). Cultures were grown at 37 °C to A_{600 nm} = 0.7, induced with 0.5 mm isopropyl β-d-1-thiogalactopyranoside, transferred to 16 °C, and harvested after 72 h. Cells were lysed using a French press, and the Na_V1.2 CTD was purified with Ni⁺-affinity, gel-filtration (Superdex 200), and ion-exchange (Mono Q 5/50 GL) chromatography (GE Healthcare). The N-terminal tag was not removed. Sample buffer consisted of 20 mm d₁₁-Tris (pH 7.4), 100 mm d₅-glycine, 0.1 mm d₁₆-EDTA, 1 mm d₁₀-DTT, 0.02% NaN₃, and 10% D₂O. Proteins were exchanged into this buffer using centrifugal concentrators (Amicon Inc.), flash-frozen in liquid N₂, and stored at -80 °C. Samples for calcium titrations were subsequently exchanged into 20 mm d₁₁-Tris (pH 7.4), 100 mm d₅-glycine, 10 μm d₁₆-EDTA, 1 mm d₁₀-dithiothreitol, 0.02% NaN₃, and 10% D₂O. Protein concentrations of 0.5 and 0.2 mm were used for structural experiments and calcium titrations, respectively. The Na_V1.5 CTD construct, residues 1773-1878, was designed by sequence alignment to Na_V1.2, using bl2seq (36), and protein samples were prepared by the same protocol. Sample temperatures were calibrated using 99.8% MeOD to a splitting of 1.616 ppm for Na_V1.2 (290.5 K) and 1.545 ppm Na_V1.5 (298.0 K) (37).

Backbone assignments for the Na_V1.2 and Na_V1.5 CTDs were obtained with HNCO, HNCA, HNCACB, HNCOCA, HNCACO, and CBCA(CO)NH experiments; side-chain assignments for Na_V1.2 CTD were obtained with HBHA(CB-CACO)NH and HCCH-two-dimensional total correlation spectroscopy (TOCSY) experiments (38). A 10% ¹³C sample was used for stereospecific assignment of Leu and Val methyl groups (39). NOE connectivities were obtained with ¹⁵N-NOESY-HSQC (80-ms mixing time), ¹³C^aliphatic-NOESY-HSQC (100 ms), and ¹³C^aromatic-NOESY-HSQC (80 ms). Residual dipolar coupling constants were measured in a sample containing 15 mg/ml Pf1 phage (Asla Biotech) using two-dimensional In-phase/Antiphase ¹H,¹⁵N HSQC for ¹H,¹⁵N (40), three-dimensional HNCO for ¹³C′-¹³C^α (41), quantitative three-dimensional HNCO for ¹⁵N-¹³C′ (42), and HCACO for ¹H^α-¹³C^α residual dipolar coupling constants (43). Fitting of the ¹⁵N-¹³C′ coupling constants was performed with Mathematica v5.2 (Wolfram Research, Inc.). Chemical shifts were referenced using DSS (44).

An initial structure of Na_V1.2 CTD was calculated from dihedral angle and NOE distance restraints, with several iterations to resolve ambiguity using ARIA 2.2 (45) and CNS 1.2 (46). The structure was refined with XPLOR-NIH 2.18 using dihedral angle, NOE distance, and residual dipolar coupling constants restraints (47, 48). Dihedral angle restraints were derived from chemical shifts using TALOS (49). Distance restraints were obtained from NOE intensities corrected for multiplicity of the ¹H spins. NOE connectivities were categorized into three classes (50). Class I contains all intra-residue H^N-H^α and all intra-residue, sequential, and medium range H^β-H^X NOEs, where X is not a methyl proton. Class III contains all NOEs involving a methyl group, and class II includes all other NOEs. A calibration factor, k_I, was obtained by equating the average class I intensity to 3.4 Å. The class II calibration factor k_II = k_I/2.4². The class III calibration factor k_III = k_II/2. Class I was averaged with a 1/6 order exponent, whereas classes II and III were averaged using a 1/4 exponent (50, 51). A standard 10% error term was applied to the upper bound of each restraint. All distances were constrained to the range (1.8, 5.5 Å). Pseudo atom corrections were applied to upper distance restraints for geometric considerations (52).

The ¹H,¹⁵N, ¹⁵N-¹³C′, and ¹H^α-¹³C^α residual dipolar coupling constants were included in the structure calculations. The residual dipolar coupling magnitude and rhombicity were set to -12.5 Hz and 0.55, respectively, during the initial minimization and were refined in the final all-atom minimization. The final average residual dipolar coupling magnitude and rhombicity are -12.8 ± 0.23 Hz and Rh = 0.56 ± 0.01, respectively, for the 200 conformers.

Structural quality statistics refer to residues Leu-1790—Glu-1868 of the 15 lowest-energy structures of 200 total structures calculated. NOE completeness was determined with aqua3.2 (53). The Pearson correlation coefficient (R) and the quality factor (Q) were computed with PALES (54) from 64 C′-C^α dipolar couplings that were not included in the structure calculation. MolProbity scores were calculated for the lowest energy structure (55). Average root mean square deviation values were calculated to the average coordinates with VMD (56). Interhelical distances and angles (rounded to the nearest degree) were computed using interhlx.6 Structural alignments were performed with CE (57), and structure figures were prepared with VMD (56) and MOLMOL (58).

RESULTS

The isolated Na_V1.2 CTD (1777-1882) and Na_V1.5 CTD (1773-1878) constructs each contain the region just after their respective predicted IVS6 transmembrane helix and extend to a region highly conserved among all VGSCs just before the IQ motif. Assignments of ¹H,¹⁵N resonances for the Na_V1.2 CTD and the Na_V1.5 CTD are, respectively, 99 and 97% complete. Notably, Asn-1835 could not be assigned in the ¹H,¹⁵N HSQC of Na_V1.2. The resonances for Asn-1831 (the homologue of Asn-1835) and Gln-1832 were not assigned, and the resonance for Ile-1833 appears broadened in ¹H,¹⁵N HSQC of Na_V1.5. Moreover, homologous resonances Leu-1855 in Na_V1.2 and Met-1851 in Na_V1.5 have liminal intensities in ¹H,¹⁵N HSQC spectra. These observations suggest conserved dynamics between isoforms. For the Nav1.2 CTD (1777-1882), ¹³C^α and ¹³C^β assignments are 100% complete, ¹³C′ assignments are 97.1% complete, ¹H aromatic assignments are 89.1% complete, and non-aromatic ¹H assignments are 97.7% complete. The Na_V1.2 CTD construct contains six proline residues, of which Pro-1789, Pro-1807, Pro-1827, and Pro-1845 are in a trans conformation, whereas Pro-1828 and Pro-1834 are in a cis conformation. The cis conformation is evidenced by stronger X-Pro H^α-H^α than X-Pro H^α-H^δ NOE contacts and differences of C^β-C^γ chemical shifts of 9.4 and 8.5 ppm, respectively (59, 60). Medium range ¹H-¹H NOEs, steady-state {¹H}-¹⁵N NOE, and ¹³C^α secondary chemical shifts for Na_V1.2 indicate that the CTD forms a well folded domain between residues Leu-1790 and Glu-1868, with a less well ordered region between residues Ser-1869 and Arg-1882 and a disordered N-terminal region between residues Gly-1777 and Pro-1789 (Fig. 1 and supplemental Fig. S1). Secondary chemical shifts indicate that the Na_V1.5 CTD has a similar secondary structural architecture as Na_V1.2 CTD (Fig. 1C).

FIGURE 1.

Sequence alignments and NMR data for Na_V1.2 and Na_V1.5 CTDs. A, sequence alignment of Na_V1.2 (1777-1882) and Na_V1.5 (1773-1879) CTDs, with 83% identity and 93% similarity. Non-conservative substitutions are shown in bold type. B, medium range ¹H-¹H NOEs. C, secondary structure elements predicted from chemical shifts using TALOS (49) are shown as bars for α-helices and arrows for β-strands. {¹H}-¹⁵N steady-state NOE (D) and secondary ¹³C^α chemical shifts for Na_V1.2 CTD (E) indicate a well folded domain encompassing residues Leu-1790—Glu-1868. F, ¹H,¹⁵N HSQC (right panel) with expansion of the central region (left panel) of Na_V1.2 (1777-1882). The W1802^ε1 resonance is aliased in the ¹⁵N dimension from 131.5 ppm.

The structure of Na_V1.2 CTD is presented in Fig. 2 and supplemental Fig. S1 with statistical details of the calculation presented in Table 1. The structure contains four α-helices and two short anti-parallel β-strands, consistent with homology models based on structures of paired EF-hand domains (25, 26). Comparison of interhelical angles of helices I and II of Na_V1.2 CTD and the N-terminal lobe of the prototypical EF-hand protein calmodulin suggests that the isolated Na_V1.2 CTD most closely resembles the canonical apoEF-hand conformation (Fig. 3D and Table 2). The hydrophobic interface between helices I and IV predicted through mutational analysis (27) is observed with direct NOE contacts between residues Phe-1795, Phe-1798, and Tyr-1799 in helix I and Leu-1855, Ile-1857, and Leu-1858 in helix IV.

FIGURE 2.

Solution structure of the ordered core EF-hand motif of the Na_V1. 2 CTD, residues Leu-1790-Glu-1868. Structural statistics are presented in Table 1; the structure of the construct from residues Gly-1777 to Arg-1882, including the N- and C-terminal regions, is shown in supplemental Fig. S1. Traces through the backbone heavy atoms of the 15 lowest energy conformers described in Table 1 are superposed in panel A. Phenylalanine side chains are superposed in panel B. Phe-1859 is not shown because the aromatic chain is not well ordered in the ensemble. A ribbon diagram of the lowest-energy structure is presented in panel C. The structural alignment of the N-terminal EF-hand motifs of Na_V1.2 (blue ribbon) and apoCa²⁺ calmodulin PDB code 1CFD (green ribbon) is shown in panel D. The structural alignments to Ca²⁺-bound calmodulin PDB code 1EXR (orange ribbon) is shown in panel E. Pentagonal bipyrimidal coordination (dashed lines) of calcium by the N-terminal calmodulin (PDB code 1EXR) EF-Hand (orange) is shown with the corresponding residues of Nav1.2 in panel F. Coordination by T28 (green dashed line) occurs through a water molecule.

TABLE 1.

Structural statistics for Na_v 1.2 CTD

Quality statistics are given for residues Leu-1790—Glyu-1868 of the 15 lowest energy structures out of 200 total structures calculated. Errors shown are S.D. for the ensemble. r.m.s.d., root mean square deviation.

Quantity	Value
Unique NOE distance restraints	1772
Intra-residual	699
Sequential	442
Medium range (2 ≤ i ≤ 5)	321
Long range (i > 5)	310
Residual violations >0.3 Å per structure (n = 15)	1.2 ± 1.1
Maximum violation (Å)	0.27 ± 0.2
NOE completeness per shell
2.0-2.5 Å (%)	87
2.5-3.0 Å (%)	69
3.0-3.5 Å (%)	57
3.5-4.0 Å (%)	44
TALOS dihedral restraints (ϕ, ψ)	65, 65
Residual dipolar coupling restraints
H-N	41
N-C′	64
Hα-Cα	44
R-value; Q-factor (64 C′-Cα couplings)	0.93 ± 0.02; 0.38 ± 0.05
PROCHECK
Most favored (%)	82.2 ± 1.6
Allowed (%)	15.8 ± 1.7
Generously allowed (%)	1.41 ± 0.8
Disallowed (%)	0.5 ± 0.9
MolProbity score; all-atom clash score (percentiles)	3.09 (20th); 20.33 (31th)
Average backbone r.m.s.d. (Å)	0.80
Average all atom r.m.s.d. (Å)	1.29

FIGURE 3.

Ca²⁺ titration of Na_V1. 2 (1777-1882) (panel A) and Na_V1.5 (1773-1878) (panel B). The plots show joint ¹H,¹⁵N chemical shift deviations from resonance assignments in 0 mm Ca²⁺. The titration was performed by serial addition of Ca²⁺ obtaining the following concentrations: 0 (red), 0.1 (orange), 0.5 (maroon), 1.5 (magenta), 2.5 (cyan), 3.5 (blue), and 4.5 mm (green) for Na_V1.2 (panel A) and (0 (red), 0.1 (orange), 0.5 (maroon), 2.5 (magenta), 3.5 (cyan), 4.5 (blue), and 5.5 mm (green) for Na_V1.5. Insets show resonances Phe-1812—Ile-1813 and Phe-1808—Ile-1809 for Na_V1.2 and Na_V1.5, respectively. Titration curves are shown in supplemental Fig. S2. In panel C the joint ¹H,¹⁵N chemical shift changes for Na_V1.2 (1777-1882) at 4.5 mm Ca²⁺ are mapped onto the lowest energy structure, interpolated between 0 ppm (blue) and 0.1 ppm (red).

TABLE 2.

Comparison of helix orientations in EF-hand proteins

Interhelical angles are shown in degrees with interhelical distances shown in Å in parentheses. Calculations refer to the following structures. Ca-CaM is Ca²⁺ -loaded target-free calmodulin (PDB code 1EXR, 1.00 Å) (67), IQ-Ca-CaM is Ca²⁺ -loaded calmodulin bound to the voltage-gated Ca²⁺ channel Ca_v1.2 IQ-motif (PDB code 2F3Y, 1.45 Å) (81), and apoCaM is apoCa²⁺ target-free calmodulin (PDB code 1CFD, NMR) (82), with helix I defined as residues 6-18, helix II as residues 29-38, helix III as residues 45-54, and helix IV as residues 65-74. Interhelical angles for the Na_v1.2 CTD are the averages with S.D. for the structural ensemble.

Molecule	Helix II	Helix III	Helix IV
Helix I
Ca-CaM	85 (20.4)	−134 (25.5)	91 (14.6)
IQ-Ca-CaM	92 (18.3)	−161 (21.9)	117 (10.1)
ApoCaM	136 (12.9)	−93 (21.2)	126 (11.9)
Nav1.2 CTD	152 ± 2 (10.9 ± 0.1)	−103 ± 6 (20.9 ± 0.3)	143 ± 1 (15.7 ± 0.2)
Helix II
Ca-CaM		83 (10.1)	−20 (16.7)
IQ-Ca-CaM		107 (11.2)	−41 (18.6)
ApoCaM		125 (11.9)	−49 (12.9)
Nav1.2 CTD		100 ± 5 (10.8 ± 0.4)	−46 ± 2 (13.4 ± 0.1)
Helix III
Ca-CaM			65 (15.3)
IQ-Ca-CaM			80 (16.5)
ApoCaM			129 (14.1)
Nav1.2 CTD			98 ± 6 (14.4 ± 0.4)

Helices I and IV contribute to the hydrophobic core of the protein, with a majority of aromatic side chains contributed from helix I. The segments between Gln-1811—Glu-1814 and Arg-1851—His-1853 participate in an anti-parallel β-sheet. An additional anti-parallel β-sheet contribution from residues Met-1846—Val-1847 is not present in all conformers of the structural ensemble. The helix II-III interhelical segment, delimited by two cis proline residues, Pro-1828 and Pro-1834, is well ordered in the structural ensemble. The conformation of residues Asp-1826—Leu-1829 is consistent with a type VI tight-turn (61), also called a βα_R turn (62). The unique di-proline-leucine motif, Pro-1827—Leu-1829 extends the helix II-III interhelical segment by forming a small handle at the base of helix II (Figs. 3, D and E). The absence of long-range NOE contacts for residues Ser-1848 and Gly-1849 is represented by disorder of this region in the ensemble.

The segment from residues Ser-1869 to Arg-1882 is predicted to have residual helical content based on secondary ¹³C chemical shifts and characteristic d_αN(i, i+3) and d_αβ(i, i+3) NOEs (Fig. 1). A short helix V is observed in the final ensemble extending from Gly-1870 to Arg-1876 with a backbone root mean square deviation of 0.59 Å when superposed on itself (supplemental Fig. S1). However, the reduced magnitudes of the secondary ¹³C chemical shifts and the {¹H}-¹⁵N NOEs for helix V compared with helices I to IV, suggest that the helical conformation is not fully populated in solution. Furthermore, helix V does not exhibit residual dipolar couplings or long-range NOE contacts and, hence, is not well defined relative to the core EF-hand domain structure (supplemental Fig. S1). Additional interactions present in longer constructs of the CTD or in complexes with other components of the VGSC may stabilize helix V.

Binding of Ca²⁺ by the Na_V1.2 (1777-1882) and Na_V1.5 (1773-1878) CTDs was assessed by monitoring ¹H,¹⁵N chemical shifts as a function of Ca²⁺ concentration (0-4.5 mm). Chemical shift perturbations exhibit titration behavior suggesting that the interaction occurs on a fast-exchange timescale with equilibrium constants of 1.65 ± 0.03 mm for Na_V1.2 CTD and 3.28 ± 0.13 mm for Na_V1.5 CTD (Fig. 3 and supplemental Fig. S2), consistent with a previous report for the Na_V1.5 CTD (33). However, resonance assignments were not obtained previously, and the structure of Na_V1.2 CTD now reveals that chemical shift perturbations >0.05 ppm are localized to residues in the N terminus of helix I, the linker between helices II and III, the C terminus of helix IV and the partially structured helix V. Thus, this weak Ca²⁺ binding site is distal to the canonical EF-hand loop motifs. In contrast, the average chemical shift change between the end points of the titration is <0.01 ppm in the N-terminal EF-hand loop (residues 1806-1817) and in the C-terminal EF-hand loop (residues 1842-1853) for the Na_V1.2 CTD. Respective values <0.02 ppm were obtained for corresponding residues 1802-1813 and 1832-1849 in the Na_V1.5 CTD. In comparison, the average chemical shift changes of the N-terminal EF-hand loop between apoCa²⁺ and Ca²⁺-loaded calmodulin are 0.59 and 0.65 ppm in the N-terminal and C-terminal domains, respectively (63, 64). In particular, canonical Ca²⁺ binding by an EF-hand would require coordination of a Ca²⁺ atom by the backbone carbonyl atoms of Phe-1812 in Na_V1.2 and Phe-1808 in Na_V1.5, leading to significant chemical shift changes for inter-residual and sequential amide resonances (65, 66). In opposition, chemical shift changes less than 0.02 ppm were observed for backbone amide resonances for residues Phe-1812-Ile-1813 and Phe-1808—Ile-1809 of Na_V1.2 and Na_V1.5, respectively (Fig. 3). A structure-based sequence alignment of calmodulin and Na_V1.2 and a comparison of Ca²⁺-induced chemical shift changes are shown in supplemental Fig. S3.

DISCUSSION

The solution structure determined by NMR spectroscopy for the Na_V1.2 CTD (1777-1882) exhibits a core-ordered domain from residues Leu-1790 to Glu-1868, with four α-helices and two short anti-parallel β-strands arranged in tandem helix-sheet-helix motifs characteristic of paired EF-hand domains. Structural alignment of the Na_V1.2 CTD and calmodulin reveals that the structure is more similar to apo-Ca²⁺ calmodulin than to peptide target and/or Ca²⁺-loaded calmodulin. The Na_V1.5 CTD (1773-1878), which shares 83% identity with the Na_V1.2 CTD, adopts a similar secondary structure and, likely, tertiary structure.

Titrations monitored by NMR chemical shift perturbations demonstrate that the canonical EF-hand loops of the Na_V1.2 CTD (1777-1882) and Na_V1.5 CTD (1773-1878) do not bind Ca²⁺; rather, Ca²⁺ binds weakly at a site distal to the canonical loops near the N terminus of helix I, the linker between helices II and III, the C terminus of helix IV, and the partially structured helix V. The high resolution crystal structure of calmodulin identified an additional Ca²⁺ binding site in the homologous region corresponding to the helix II-III linker, but the authors judged this site to be non-physiological (67).

A structure-based sequence alignment with calmodulin also suggests that the canonical EF-hand loops of Na_V1.2 CTD do not bind Ca²⁺ (Table 3, Figs. 2, D and E, and supplemental Fig. S3). Chelation of Ca²⁺ requires an acidic residue, such as Glu or Asp, at sequence position 1817, corresponding to position 12 in a canonical EF-hand calcium binding motif (68), rather than the Lys residue present in Na_V1.2. Mutation of the corresponding residue, Glu to Lys, in Drosophila melanogaster calmodulin abolishes Ca²⁺ binding, although this mutation may mimic a Ca²⁺-bound state in the context of certain targets (69, 70). Lys is found at position 12 in the non-canonical Ca²⁺ binding loop of scallop myosin essential light chain; however, coordination of Ca²⁺ is accomplished by an acidic residue at position -2, the backbone carbonyl group at position +2, and a water molecule (71). In Na_V1.2 the residue at position +2 is Pro, and the residues at positions -3 and -2 are Glu and Lys. The latter two residues have chemical shift changes less than 0.05 ppm after the addition of 4.5 mm Ca²⁺.

TABLE 3.

Structure-based sequence alignment

N-terminal calcium binding loop of CaM aligned with the respective N-terminal EF-hand loop sequences from Na_v1.2 and Na_v1.5 CTD sequences, based on a structural alignment between apo-CaM and the Na_v1.2 CTD.

Ca2+ coordination	X		Y		Z		−Y		−X			−Z
Position	1	2	3	4	5	6	7	8	9	10	11	12
Nav1.2	D	P	D	A	T	Q	F	I	E	F	A	K
Nav1.5	D	P	E	A	T	Q	F	I	E	Y	S	V
CaM	D	K	D	G	D	G	T	I	T	T	K	E

Higher affinity Ca²⁺ binding has been reported for longer constructs of Na_V1.5 CTD, residues 1773-1920 and residues 1773-1925 that include the IQ motif, and binding is abolished by mutation of the IQ motif (33). However, the resonance assignments obtained for Na_V1.5 indicate that chemical shift perturbations for key EF-hand canonical loop residues Phe-1808—Ile-1809 are not larger in these longer constructs (comparing the inset of Fig. 3B with supplemental Fig. 5D of Ref. 33), suggesting that higher affinity binding of Ca²⁺ also does not involve the canonical EF-hand loops.

The solution structure of Na_V1.2 CTD can be used to predict the effect(s) of clinical mutations in VGSCs (Fig. 4) because of the high degree of homology between VGSC CTDs. Generally, clinically significant mutations that map in the CTD can be divided into two classes, with some overlap for several sites (supplemental Table SI). Mutations in Na_v1.5 associated with the Long QT variant 3 (LQT3) cardiac arrhythmia phenotype and a subset of mutations in Na_v1.1 associated with certain epilepsy syndromes lead to persistent current during maintained depolarization. A second set of mutations in Na_v1.1 associated with multiple epilepsy syndromes and mutations in Na_v1.5 associated with the Brugada syndrome cardiac arrhythmia led to decreased current, resulting from loss of function or enhanced inactivation kinetics.

FIGURE 4.

NMR structure of Na_V1. 2 (1777-1882) CTD with functionally significant mutations observed in Nav1.1 and Nav1.5 channels. The lowest energy structure of the calculated ensemble is shown. Mutations leading to persistent current cluster in helices I and IV (show in red) and the helix II-III segment (shown in orange), whereas a position (1842) at which mutation (M1852T) leads to decreased current is shown in blue. Position 1799 at which substitutions lead to increased or decreased inactivation is shown in violet, and residue Cys-1854 is shown in green. The putative β subunit interaction site is shown in pink.

Multiple mutations in Na_V1.1 and Na_V1.5 associated with an increased persistent current are observed at positions clustering in the corresponding helix I of the Na_V1.2 CTD. The F1808L mutation associated with intractable childhood epilepsy with generalized tonic clonic seizures in Na_V1.1 may destabilize the protein core because the aromatic ring of Phe-1798 in Na_V1.2 contacts residues in helix IV and the helix II-III interhelical segment (4, 72). The insertion of an Asp residue at position 1795, Y1795insD, leads to both LQT3 and Brugada syndrome phenotypes in Na_V1.5 and potentially disrupts helix I by shifting the register of helical interactions (73).

Substitution at position Tyr-1795 in Na_V1.5 differentially leads to decreased inactivation for Y1795C in LQT3 or enhanced inactivation kinetics for Y1795H in Brugada syndrome, whereas both substitutions lead to sustained current during maintained depolarization and negative shift of voltage dependence of inactivation (27, 74). The Y1795C mutation has been suggested to form an intra-molecular disulfide bond with Cys-1850 in Na_V1.5 (32). The average C^β-C^β distance of the corresponding residues in the Na_V1.2 CTD structural ensemble is 9.6 ± 0.4 Å. The C^β-C^β distance in cysteine disulfide bonds ranges from 3.4 to 4 Å (75); thus, the proposed disulfide bond may be intermolecular or require structural rearrangement on the order of several angstroms between helix I and IV (Fig. 4) if it is formed. Furthermore, although Tyr-1795 in Na_V1.5 was predicted to contribute to the hydrophobic interface between helices I and IV (27), the corresponding residue Tyr-1799 in Na_V1.2 is found in a position closer to the surface; the total side-chain exposed surface area is 103 ± 10 Ų for the conformers in Table 1. Hence, mutations at position Tyr-1799 may also affect interactions with other components of the intact channel. On the other hand, the conserved Trp-1802, corresponding to Trp-1798 in Nav1.5, is not completely accessible as observed previously (27); the total side-chain exposed surface area is 9 ± 5 Ų for the conformers in Table 1.

The L1825P mutation associated with LQT3 and the R1826H mutation associated with sudden infant death syndrome in Na_V1.5 occurs in the helix II-III interhelical segment (76, 77). The L1825P mutation results in significant persistent current and slows kinetics of inactivation. Interestingly, the L1825P mutation in Na_V1.5 introduces a di-proline motif, as is observed in wild type Na_V1.1, Na_V1.2, Na_V1.3, and Na_V1.7, but shifted by one residue. The residue corresponding to Arg-1826 in Na_V1.2 is Leu-1830, and some local difference in conformation probably exists. Like L1825P, the R1826H mutation leads to persistent current in Na_V1.5, further suggesting that the helix II-III interhelical segment is critical to channel inactivation.

Two mutations implicated in interactions with other components of the sodium channel cluster in helices III and IV. The D1866Y mutation in Na_V1.1, associated with generalized epilepsy and febrile seizures plus, leads to persistent current and decreased fast inactivation kinetics in the presence of the β subunit (78). The corresponding position Asp-1856 in Na_V1.2 is at the start of helix IV and may disturb a putative surface for interaction with the β subunit, as interaction with the β1 subunit and the CTD is suggested to occur through the second helix-sheet-helix motif by yeast-two-hybrid analysis of residues Lys-1846—Arg-1886 in Na_V1.1 (78). Additionally, the M1852T mutation in Na_V1.1, also associated with generalized epilepsy and febrile seizures plus, results in decreased current (loss of function). This phenotype can be rescued by co-expression with β subunits or calmodulin (79). Proposed to be a folding/trafficking defect, this mutation may destabilize helix III, further suggesting that the second helix-sheet-helix motif may be important for interaction with other components of the sodium channel.

The notable exception to the above patterns is the LQT3 mutation D1790G in Na_V1.5, resulting in a relative negative shift in the voltage dependence of inactivation in the presence of the β subunit (19, 80). D1790G corresponds to position D1794 in helix I of Nav1.2 and may disrupt the helix by introduction of a glycine residue, with the effect of propagating to helices III and IV.

The mechanisms and extent of Na_V1 CTD function in binding the IQ motif and the specific role of calmodulin as well as Ca²⁺ in multiple phases of inactivation remains to be elaborated. Interactions with the IQ motif may be more complicated than present models and may involve additional components (33). Previous evidence shows that Ca²⁺-dependent regulation of VGSC is mediated by calmodulin (31), with the exact mode of interaction yet to be determined. The solution structure of the Na_V1.2 C-terminal domain and chemical shift assignments of Na_V1.5 (1773-1878) are initial steps in elucidating the mechanism of inactivation, extended to other isoforms by virtue of high degrees of homology. The current work provides a template to begin probing specific interactions between the C-terminal domain and other components that play a role in inactivation of voltage-gated sodium channels.