Intersegment Contacts of Potentially Damaging Variants of Cardiac Sodium Channel

Abstract

Over 1,500 missense variants of sodium channel hNav1.5, which are reported in the ClinVar database, are associated with cardiac diseases. For most of the variants, the clinical significance is uncertain (VUS), not provided (NP), or has conflicting interpretations of pathogenicity (CIP). Reclassifying these variants as pathogenic/likely pathogenic (P/LP) variants is important for diagnosing genotyped patients. In our earlier work, several bioinformatics tools and paralogue annotation method consensually predicted that 74 VUS/NP/CIP variants of 54 wild type residues (set w54) are potentially damaging variants (PDVs). Atomic mechanisms underlying dysfunction of the PDVs are unknown. Here we employed a recent cryo-EM structure of the hNav1.5 channel with likely inactivated pore domain (PD) and activated voltage-sensing domains (VSDs), and ad hoc models of the closed and open PD and resting VSDs to explore intersegment contacts of w54 residues. We found that 44 residues from set w54 contact 84 residues with 118 disease missense variants. These include 104 VUS/NP/CIP variants, most of which are associated with the loss-of-function Brugada syndrome (BrS1) or gain-of-function long QT syndrome (LQT3). Matrix representation of the PDVs and their contact variants facilitated recognition of coupled mutations associated with the same disease. In particular, BrS1-associated coupled mutations, which disturb the P-loops region with the selectivity filter slow inactivation gate, would cause the channel dysfunction. Other likely causes of the channel dysfunction include coupled BrS1-associated variants within VSDs that would destabilize their activated states and coupled LQT3-associated variants, which would stabilize the open PD or activated VSDs. Our study proposes mechanisms of channel dysfunction for scores of BrS1- and LQT3-associated variants, confirms status for 82% of PDVs, and suggests damaging status for their contact variants, which are currently categorized as VUS/NP/CIP variants.

Introduction

Sodium channels Nav1.5 are responsible for initiation and propagation of the action potential in cardiomyocytes. The pore-forming α-subunit of the Nav1.5 channel, which is encoded by gene SCN5A, folds from a single polypeptide chain of four homologous repeat domains (I-IV). Each repeat has six transmembrane (TM) helices (S1-S6) connected by intra- and extracellular loops, including a large extracellular membrane reentering P-loop. The latter contains membrane-descending helix P1 and membrane-ascending helix P2. In each repeat, helices S1 - S4 form a voltage-sensing domain (VSD), while helices S5, S6 and the P-loop contribute a quarter to the pore domain (PD). The selectivity-filter ring DEKA (Asp, Glu, Lys and Ala) divides the ion permeation pathway into two parts: extracellularly exposed outer pore and the inner pore, which in the open channel is exposed to the cytoplasm. Sodium channels adopt various conformations, which in electrophysiological studies are categorized as resting, open, fast inactivated and slow inactivated states.

Sodium channels allow permeation of inward depolarizing current (I_Na) during phase 0 of the action potential (Amin et al., 2010; Veerman et al., 2015). Nav1.5 dysfunctions cause various hereditary and acquired arrhythmogenic syndromes. Many genetic gain-of-function mutations increase I_Na or delay cardiac repolarization, thus prolonging the action potential (Savio-Galimberti et al., 2018). Such mutations are associated with the long QT syndrome (LQT3), a congenital cardiac disorder, which is characterized by prolonged QT interval and higher risk of life-threatening arrhythmia. Clinical manifestations of LQT3 vary from life-long asymptomatic disease course to syncope and cardiac arrest due to torsades de pointes. Mutations, which diminish I_Na, may decrease cardiac excitability and reduce velocity of electrical conduction, causing various clinical phenotypes such as Brugada syndrome type 1 (BrS1), sick sinus syndrome, sudden infant death syndrome, dilated cardiomyopathy, atrial fibrillation, and arrhythmogenic cardiomyopathy. BrS1 is diagnosed via typical ECG-phenomenon with ST-segment elevation in ventricular leads without any structural alterations of the heart (Coppola et al., 2019; Skinner et al., 2019).

Thousands genetic variants of hNav1.5 and other hNav1.x channels are described in public databases, including ClinVar (Landrum et al., 2016), Swiss-Var (Mottaz et al., 2010) and Humsavar (http://www.uniprot.org/docs/humsavar). In April 2021, the ClinVar database reported 1,513 missense variants of SCN5A. Among these, only 17% are classified as benign/likely benign or pathogenic/likely pathogenic (P/LP) variants according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guideline (Richards et al., 2015). Data on pathogenicity is not provided (NP) for 200 variants, conflicting interpretations of pathogenicity (CIP) are reported for 139 variants, and 911 variants are of uncertain clinical significance (VUS).

The ACMG/AMP guideline recommends using computational predictions as a line of evidence for variant interpretation (Richards et al., 2015). Bioinformatics analysis of variants associated with BrS1 suggests that some mutations, which are currently classified as damaging, should be considered benign due to lack of experimental data on channel dysfunction (Kaltman et al., 2018). Incorrect classification of variants is an important problem in medical genetics. Indeed, in many cases a familial history or the pedigree data are lacking. Low frequency of disease-associated genetic variants in population further complicates correct data interpretation and delays diagnostics.

Earlier we employed several bioinformatics tools and the paralogue annotation method to reclassify 74 ClinVar-reported VUS/NP/CIP variants of 54 WT residues (set w54) as potentially damaging variants (PDVs) of hNav1.5 (Tarnovskaya et al., 2020). Here we used a recent cryo-EM structure of the hNav1.5 channel with likely inactivated PD and activated VSDs (Li et al., 2021), and ad-hoc models of the open and closed PD and resting VSDs to show that 44 residues from set w54 are involved in 84 intersegment contacts with residues for which 118 missense variants are reported in ClinVar, mostly as VUS/NP/CIP variants. Matrix representation of the PDVs and their contact variants facilitated recognition of coupled variants where substitution of either contact partner is associated with the same disease. We suggest that BrS1-associated variants in P-loops and VSDs would cause loss of channel function, respectively, by disturbing the selectivity-filter slow inactivation gate or destabilizing activated state of VSDs. LQT3-associated variants would cause the channel gain-of-function by increasing population of channels with the open pore. Our analysis confirms the status for most of earlier proposed PDVs, suggests that many of the 118 contact variants of the PDVs may be reclassified as P/LP variants, and proposes atomic mechanisms of loss-of-functions and gain-of function for scores of BrS1-associated and LQT3-associated mutations.

Methods

Energy Calculations and Optimization

Our methodology of molecular modeling with the ZMM program is described elsewhere, e.g., (Bruhova and Zhorov, 2010; Korkosh et al., 2014; Tikhonov and Zhorov, 2017). Briefly, we use the AMBER force field (Weiner et al., 1986) and calculate nonbonded interactions with the distance cutoff of 9 Å and a shifting function (Brooks et al., 1985). Electrostatic interactions were calculated with the distance- and environment-dependent dielectric function (Garden and Zhorov, 2010) without the distance cutoff for interactions involving ionized groups. The models were optimized by Monte Carlo energy minimizations (MCM) (Li and Scheraga, 1987) in the space of torsional angles and bond angles of prolines (Zhorov, 1981). MCM trajectories were terminated when 2,000 minimizations did not decrease energy of the apparent global minimum.

Modeling PD in the Open and Closed States

The hNav1.5 structure with quinidine (PDB ID: 6lqa) captured PD in likely inactivated state and VSDs in the activated states (Li et al., 2021). To model PD in the activated and closed states, we used as templates the open- and closed-pore structures of the NavAb channel (PDB codes 5vb8 and 5vb2, respectively) (Lenaeus et al., 2017). The structures of Nav1.5 and NavAb were 3D aligned by minimizing root mean square deviations of C^α atoms in the P1 helices, which are the most 3D-conserved elements of P-loop channels (Tikhonov and Zhorov, 2012), from C^α atoms of sequentially matching residues in the Kv1.2/Kv2.1 channel, the first eukaryotic P-loop channel whose crystal structure was obtained at a relatively high resolution below 2.5 Å (PDB ID: 2R9R) (Long et al., 2007). The NavAb and hNav1.5 channels have rather similar folding of the transmembrane (TM) and P1 helices, but different folding of extracellular loops, especially in the PD. Therefore, we imposed distance constraints between C^α atoms in the TM and P1 helices of hNav1.5 and matching C^α atoms of the template, whereas no distance constraints were imposed to the intra- and extracellular loops. A distance constraint is a flat-bottom parabolic function that imposes the energy penalty with the force constraint of 10 kcal·mol⁻¹ Å⁻² if a C^α atom in the model deviates from the template matching atom by more than 2 Å. The 3D alignment was performed by step-wise reducing distances between the C^α atoms in the model and template. At each step, the energy was MC-minimized with all degrees of freedom allowed to vary. More methodological details on large-scale transformations of PD can be found elsewhere (Korkosh et al., 2019).

Modeling VSDs in the Resting States

To model VSDs in the resting states, we employed as a template the crystal structure of the prokaryotic sodium channel NavAb with deactivated VSDs (PDB ID: 6p6x) (Wisedchaisri et al., 2019). Methodology is similar to that used for modeling the channel with the open and closed PD.

Designations and Visualization of Residues

We designate a residue by one-letter code with superscripted residue number and superscripted prefix indicating the channel segment where the residue is located. Experimental structures and models were visualized with the PyMol Molecular Graphics System, version 0.99rc (Schrödinger, New York, NY).

Results and Discussions

Intersegment Contacts in the Cryo-EM Structure

Earlier, we employed several bioinformatics tools and paralogue annotation method to reclassify 74 missense VUS/NP/CIP variants of w54 residues in the hNav1.5 channel as PDVs (Tarnovskaya et al., 2020). Following this publication, eight additional VUS/NP/CIP variants of the w54 residues were reported in ClinVar (version April, 2021). Therefore, currently the PDV set includes 82 variants.

We used a recent cryo-EM structure of the hNav1.5 channel obtained in nanodiscs (Li et al., 2021) to find intersegment contacts of the w54 residues. We instructed PyMol to display 3D neighbor residues within 5 Å from each query residue of set w54. Since the cryo-EM structure lacks hydrogen atoms, the distances were measured between heavy atoms. We have selected those 3D neighbors, which in the amino acid sequence are at least 5 positions away from the query residue and have sidechains facing the query residue sidechain (C^α atoms of glycines were considered as sidechains). We further ensured that each 3D neighbor of the query residue has at least one ClinVar-reported missense variant. We found 64 contact residues, which satisfied the above criteria and 85 missense variants of these residues. If two WT residues form a contact, their variants are also considered to contact each other even though they may be farther than 5 Å from each other.

Table 1 shows PDVs and their contact variants in the PD. For each PDV we show location (channel segment), all contact variants, associated syndromes, and their clinical significance. Table 2 shows analogous data for VSDs. Among 85 contact variants, 74 are VUS/NP/CIP variants and clinical significance of 11 variants is P/LP. Four pairs of residues from set w54 are contacting each other (columns “e” in Table 1). Contacts involving channel segments that undergo significant transition upon channel gating are marked “S” in column “f”.

TABLE 1.

PDVs in the pore domain and their contacts ^a .

PDVs			Contacts
Variant b		Syndrome c , d	Variant	Syndrome d	Significance	e	f
IS5A242T		A	IS6Q419P	BrS1, NP	VUS		S
V		HCM
IS5L250V		BrS1	IS6V411M	LQT3, BrS1, CVP	P		S
IS5S262G		A, BrS1	IP1F358S	NS	VUS
IS5V263I		Multi	IVS4I1633V	BrS1	VUS		S
IS5-P1G274S		BrS1	IS5-P1D356Y	BrS1	LP
			N	NP/BrS1	P/LP
IS6A413E		LQT3	IIS6L935P	BrS1	NP		S
S		NS
T		A, LQT3,BrS1
IIS5G857D		A, BrS1	IIS5-P1M881I	LQT3	VUS	1
IIS5-P1M881I		LQT3	IIS6F919S	NS	LP	1
			IIS5G857D	A, BrS1	VUS
IIP1F892L		CVP	IVS6V1451D	BrS1	VUS	2
I		BrS1	L	NS, A	VUS
IIP1R893H		BrS11,3,4,8–10	IIP2E901K	BrS1	VUS	2	S
C		BrS1
IIIS5V1324I		A, BrS1, NP	IIS3W1271C	BrS1	NP
IIIS5L1342F		BrS1	IIIP1Y1409C	BrS1	NP		S
IIIP1L1410P		NP	IIIS5-P2V1400G	CVP	VUS
IIIP2I1424F		NP	IIIS5-P1F136°C	BrS1	NP
			IIIS5-P1V1400G	CVP	VUS
IIIS6V1451D		BrS11,3	IIP1F892L	CVP	VUS
L		A, NS	I	BrS1	NP
IIIS6N1463K		NP	IIS6F934S	BrS1	VUS
IIIS6F1465L		LQT3	IIIS5V1337I	A	VUS		S
			IIIS5I1334V	LQT3, BrS1, NP	CIP		S
			IVS6F176°C	BrS1	P		S
			IVS6L1761F	LQT3	NP		S
			H	LQT3	NP
IIIS6I1466T		NP, A	IIS6F934S	BrS1	VUS		S
V		NP	IVS6V1764F	BrS1	NP		S
			IVS6F176°C	BrS1	P		S
IIIS6V1468A		A, BrS1,NP	IIIS5S1333Y	LQT3	NP		S
			IIIS5V1337I	A	VUS		S
IIIS6I1469F		NP	IIIS5I1334V	LQT3, BrS1, NP	CIP		S
			IVS6L1761F	LQT3	NP		S
			H	LQT3	NP		S
			IVS6V1764F	BrS1	NP
IVS5V1667L		NP	IVS6I1758V	A, BrS1	VUS
IVS5-P1D1690N		A, BrS1, NS14,15,16	IP2R383K	BrS1	VUS
IVP1M1701I		BrS1, DCM17	IVS5M1668T	A	VUS
			IS6M390I	A	VUS
			V	BrS1, A	VUS
			IS6M394I	A	VUS
			L	A	VUS
IVS5I1660V		NP, CVP, BrS1, LQT31,3,13,14	IVS6V1764F	BrS1	NP	3	S
			IVS6L1761F	LQT3	NP		S
			H	LQT3	NP		S
			IIIS4-S5V1323G	BrS1	NP		S
			I	A, BrS1	VUS		S
			IVS6M1766I	CVP, BrS1	VUS		S
			K	NP, BrS1, SSS	VUS
			V	BrS1	VUS
			T	HCM	VUS
			L	LQT3	P
			IIIS6−IVS1F1486L	SIDS	NP
IVP1L1704H		BrS1	IVS5M1668T	A	VUS
IVP1T1709M		A, BrS1,CVP,NP1,3,18	IIIP1-P2K1419E	BrS1	NP
			IP1-P2W374G	BrS1	NP
			IP1-P2Q371R	BrS1	VUS
			IS6I397T	BrS1, LQT3, NP	CIP
IVS6M1766I		BrS1, CVP	IS6N406K	LQT3	P, LP	3	S
K		NP, BrS1, SSS	IS6N406S	BrS1	NP
V		BrS1	IVS5I1660V	A, BrS1, CVP, LQT3	CIP
T		HCM
L		LQT3

^a See Energy Calculations and Optimization Section for definition of contacts.

^bAll PDVs were listed as VUS in ClinVar-2019. In ClinVar-2021, conflicting interpretations of pathogenicity is reported for three variants (R⁸⁹⁴H, G¹²⁶²D, I¹⁶⁶⁰V) and two variants (D¹⁹⁷G, L²²⁵V) are classified as likely pathogenic.

^cSuperscripted numbers refer to clinical and experimental studies of the variants. 1. Huang et al. (2017), 2. Ortiz-Bonnin et al. (2016), 3. Kapplinger et al. (2010), 4. Walsh et al. (2014), 5. Calloe et al. (2013), 6. Liang et al. (2016), 7. Son et al. (2018), 8. Zakliaz’minskaia et al. (2013), 9. Kapplinger et al. (2015), 10. Rudic et al. (2016), 11. Shin et al. (2004), 12. Priganc et al. (2017), 13. Cordeiro et al. (2006), 14. Selga et al. (2015), 15. Núñez et al. (2013), 16. Zeng et al. (2016), 17. Bodian et al. (2017), 18. Li et al. (2018), 19. Brewer et al. (2020).

^dAbbreviations: A, Arrhythmia; AF, atrial fibrillation; BrS1, Brugada syndrome; CA, Cardiac Arrest; CIP, Conflicting interpretations of pathogenicity; CVP, Cardiovascular phenotype; DCM, Dilated cardiomyopathy; HCM, Hypertrophic cardiomyopathy; LP, Likely Pathogenic, LQT3, Long QT syndrome Type 3; M, Multiple diseases; NP, not provided; NR, Variants are not reported in ClinVar; NS, Not Specified; P, Pathogenic; PFHB-1, Progressive Familial Heart Block type 1A; PFVF1, Paroxysmal Familial Ventricular Fibrillation 1; SIDS, Sudden Infant Death Syndrome; VUS, Variant of Uncertain Significance.

^eMatching numbers show WT residues that are in contact in the cryo-EM structure.

^fstate-dependent contacts involving residues that move upon activation gating (located C-terminal to gating-hinge in KcsA).

TABLE 2.

PDVs and their contact variants in the open-PD model ^a .

PDV		Contacts
Variant	Syndrome	Variant	Syndrome	Significance
IIIS6N1463K	NP	IIS6S94°C	BrS1	VUS
		IIS6S941C	CP	VUS
		N	LQT3	P
IIIS6F1465L	LQT3	IIIS5A1330P	LQT3	NP
		T	BrS1, LQT3, More	P/LP
IIIS6I1466T	NP, A	IIS6S941C	CP	VUS
V	NP	N	LQT3	P
IIIS6V1468A	A, BrS1,NP	IIIS5A1330P	LQT3	NP
		T	BrS1, LQT3, More	P/LP

^aContacts in the cryo-EM structure (Table 1) are not shown.

Matrices of Contact Variants

To facilitate recognition of disease-related contacts, we schematically represented them in colored matrices. Each matrix represents an individual domain because inter-domain contacts of PDVs are rare. A matrix of contact variants for the inactivated PD (Figure 1) has four bottom rows, which represent PDVs, their location, and clinical significance; four left columns represent analogous characteristics of contact variants of PDVs. Variants associated with BrS1, LQT3 or non-specified arrhythmia are colored pink, green or yellow, respectively. Variants associated with other diseases are not colored. Variants, which are associated with more than one disease, are shown with two colors. Each cell in the matrix body links a PDV and its contact variant.

FIGURE 1.

Matrix of contact variants in the PD cryo-EM. Bottom rows (X1-X4) represent w54 residues and their PDVs. X1 and X2 show PD segments and UniProt residue numbers. X3 shows one-letter codes of WT residues and their variants. X4 indicates clinical significance of variants. Variants associated with BrS1, LQT3, non-specified Arrhythmias or other diseases are colored, respectively, pink, green, yellow, or not colored. If besides BrS1 or LQT3, a variant is associated with a non-specified arrhythmia, it is colored pink or green, respectively. If various diseases are reported for a variant, two colors are used. Left columns (Y1-Y4) show analogous features of contact variants of w54 residues. A colored cell in the matrix body links a PDV and its contact variant. If a WT residue has m PDVs and n contacts variants, a cluster of m x n colored cells shows all variant combinations. If PDV and its contact variant have the same color, it is applied to the cell. If a cell links pink and yellow variants, it is colored pink. If a cell links green and yellow variants, it is colored green. If a variant has color A and its contact variants colors A and B, color A is applied to the cell. Otherwise the cell is grey. If a residue is located in cytoplasmic halves of S6 helices, which move upon the channel gating, respective cell is bold-boxed.

The matrix highlighted multiple contacts where mutation of either WT residue is associated with the same syndrome. A likely cause is that a disease-related contact contributes to stabilization (or destabilization) of a particular channel state; a weakened or strengthened contact would similarly affect the channel state stability regardless of the mutated contact partner. Structural analysis of all contacts involving PDVs is beyond goals of this study. Below we propose mechanisms of dysfunction for some PDVs and their contact variants.

Contacts in P-Loops

In our models with the closed and open PD, which are described in a later section, conformations of P-loops are rather similar to those in the cryo-EM structure. And although P-loops do undergo conformational changes, e.g., upon slow inactivation, here we consider their folding as relatively state-independent. Figure 2 shows P-loops with some contacts, which apparently stabilize their folding and a matrix of respective contacts variants. Five hydrophobic residues (^IS6M³⁹⁰, ^IS6M³⁹⁷, ^IVS5M¹⁶⁶⁸, ^IVP1M¹⁷⁰¹, and L¹⁷⁰⁴) form a tight cluster (Figures 2A,B). In variants M³⁹⁰I/V, M³⁹⁴I, M¹⁶⁶⁸T, and M¹⁷⁰¹I, methionine substitutions with smaller residues (Figure 2C) would relax the cluster and increase flexibility of the smallest involved helix, IVP1. Substitutions ^IVP1L¹⁷⁰⁴H and ^IS6I³⁹⁷T would also increase flexibility of this helix. The C-end of helix IVP1 approaches the DEKA-ring alanine in the SF region (Figure 2B), which harbors the SF gate. The outer-pore of a prokaryotic sodium channel exhibits flexibility during slow inactivation (Chatterjee et al., 2018). In the Nav1.4 channel, the P-loop of domain IV is more flexible than in other domains (Tsushima et al., 1997a; Tsushima et al., 1997b). The above mutations would increase flexibility of helix IVP1, disturb the SF gate, and facilitate the channel transition to the slow-inactivated state. This proposition is supported by the fact that most substitutions in P-loops are associated with the loss-of-function syndromes, especially with BrS1 (Figure 2C).

FIGURE 2.

Residue contacts that stabilize P-loops in the cryo-EM structure. Backbones and sidechain carbon atoms in repeats I, II, III and IV are yellow, pink, green and gray, respectively. Residues w54 and their contacts are indicated with green and red labels, respectively. (A,B) Intra-membrane views of P-loops in the cryo-EM structure. Residues M³⁹⁰, M³⁹⁴, M¹⁶⁶⁸, M¹⁷⁰¹ and L¹⁷⁰⁴ form a tight hydrophobic cluster, which apparently stabilizes mutual disposition of helices IS6, IVS5 and IVP1. (C) Matrix of contact variants in P-loops. Majority of coupled variants are associated with BrS1 and arrhythmia, which also may be a loss-of-function disease. (D) Intra-membrane view of helices IIP1 and IIP2 whose mutual disposition is stabilized by salt bridge R⁸⁹³:E⁹⁰¹. Side-chain conformation of the selectivity-filter DEKA glutamate E⁹⁰¹ is stabilized by its salt bridge with R⁸⁹⁸. Helical structure of helix IIP1 is stabilized by π-stacking F⁸⁸⁸ and F⁸⁹². (E) Intra-membrane view of helices IIIP1 and IIIP2 whose mutual disposition is stabilized by a cluster of four hydrophobic residues. The selectivity-filter DEKA lysine K¹⁴¹⁹ is shown by sticks. (F) Extracellular view of salt bridges R³⁸³:D¹⁶⁹⁰, R⁸⁹³:E⁹⁰¹ and hydrophobic contacts I³⁹⁷:T1⁷⁰⁹, L¹³⁴²:Y¹⁴⁰⁹ and I¹⁴²⁴:F¹³⁶⁰.

Several other mutations involving P-loops would also increase their flexibility. For example, in the cryo-EM structure, ^IIP1F⁸⁹² and ^IIP1F⁸⁸⁸ form a stacking pair (Figure 2D) that can stabilize the α-helix (Butterfield et al., 2002). Mutation ^IIP1F⁸⁹²L affects the inter-segment contact with ^IIIS6V¹⁴⁵¹ (Table 1) and destroys the stacking pair with likely destabilization of helix IIP1. A sequential neighbor of F⁸⁹², ^IIP1R⁸⁹³ forms several strong contacts, including a salt bridge with ^IIP2E⁹⁰¹, which stabilizes mutual disposition of helices IIP1 and IIP2 (Figure 2D). Substitution of any partner in the salt bridge is associated with BrS1 (Figure 2C) likely due to destabilization of mutual disposition of helices IIP1 and IIP2. Another strong contact of ^IIP1R⁸⁹³ involves the SF glutamate ^IIP1-P2E⁸⁹⁸, which is proposed to adopt multiple conformations in a cycle of sodium ion permeation (Zhorov, 2021). The cryo-EM structure of Nav1.5 is obtained in complex with quinidine that favors inactivated channels (Li et al., 2021). However, it is unknown whether the SF gate is captured in the open or closed state. A strong electrostatic attraction to ^IIP1R⁸⁹³ may stabilize the E⁸⁹⁸ conformation in the slow-inactivated state.

Several other contacts also stabilize folding of P-loops. An example is a knob-into-hole contact ^IIIP2I¹⁴²⁴ : ^IIIP1A¹⁴¹¹ that stabilizes the mutual disposition of helices IIIP1 and IIIP2 with the selectivity-filter lysine ^IIIP1-P2K¹⁴¹⁹ (Figure 2E). Mutation I¹⁴²⁴F affects contact with A¹⁴¹¹, as well as contacts with ^IIIS5-P1F¹³⁶⁰ and ^IIIS5-P1V¹⁴⁰⁰ whose variants are associated with diseases (Table 1). Another example is a salt bridge ^IVS5-P1D¹⁶⁹⁰ : ^IP2R³⁸³ (Figures 2A,F), which is absent in BrS1-associated variants ^IVS5-P1D¹⁶⁹⁰N and R³⁸³K. In a functional study, variant D¹⁶⁹⁰N reduced I_Na and shifted the steady-state activation by +7 mV (Zeng et al., 2016).

Thus, majority of mutations affecting the P1 and P2 helices are associated with the loss-of-function BrS1. Apparently these mutations distort the SF gate and increase population of slow-inactivated channels. For comparison, two residues beyond P1 and P2 helices, ^IIS5-P1M⁸⁸¹ and ^IIS5G⁸⁵⁷, form a knob-into-hole contact that would not directly affect the SF region. Both ^IIS5-P1M⁸⁸¹I and ^IIS5G⁸⁵⁷D are PDVs (Table 1). However, unlike the above-described BrS1-associated variants that affect folding of P-helices, M⁸⁸¹I is associated with the gain-of-function LQT3, while G⁸⁵⁷D is associated with BrS1.

Contacts in the Channel Models with the Open and Closed PD

Figures 3A–C show cytoplasmic views of the PD, respectively, in the cryo-EM structure, closed state, and open state. Space-filled hydrophobic residues at the cytoplasmic ends of S6 helices highlight different dimensions of the activation gate in these states. Figure 3D shows intra-membrane view of the full-fledged channel model with the open PD and activated VSDs. PDVs and respective contact variants, which are specific for the models with the closed PD and open PD, are presented in Tables 2, 3.

FIGURE 3.

Cryo-EM structure and homology model of the hNav1.5 channel. Repeats I,II, III and IV are yellow, pink, green and gray, respectively. (A, C) Cytoplasmic views at the pore domain in the cryo-EM structure (A), and NavAb-based models of the channel states with the closed (B) and open (C) pore domain. Space-filled residues at the cytoplasmic ends of S6 helices highlight different dimensions of the activated gate. (D) Intra-membrane view of the channel model with the open PD.

TABLE 3.

PDVs and their contact variants in the closed-PD model ^a .

PDV		Contacts
Variant	Syndrome	Variant	Syndrome	Signif
IS6A413E	LQT3	IVS6N1774D	LQT3	NP
S	NS	S	BrS1	NP
T	A, LQT3,BrS1	Y	NS	CIP
IIIS6N1463K	NP	IIS5S835L	BrS1	NP
		A	BrS1	VUS
IIIS6F1465L	LQT3	IVS6I1768V	BrS1, LQT3	P
IIIS6I1469F	NP	IVS6I1768V	BrS1, LQT3	P
		IVS6L1772P	BrS1	VUS
		V	LQT3	NP
IVS6M1766I	CVP, BrS1	IVS6G1661R	BrS1	NP, CIP
K	NP, BrS1, SSS
V	BrS1
T	HCM
L	LQT3

^aContacts in the cryo-EM structure (Table 1) are not shown.

Figure 4A shows matrix of contact variants in the cytoplasmic halves of TM helices of the open PD. A hydrophobic cluster involving ^IIIS5A¹³³⁰, ^IIIS5I¹³³⁴, ^IIIS6F¹⁴⁶⁵, ^IIIS6V¹⁴⁶⁸ and ^IVS6L¹⁷⁶¹ (Figure 4B) apparently stabilizes the open PD. Substitutions of A¹³³⁰ with bigger proline or threonine, or substitution of F¹⁴⁶⁵ with more flexible leucine are associated with the gain-of-function LQT3 likely because respective variants over-stabilize the open PD. In LQT3-associated variants L¹⁷⁶¹H/F, the open state of PD would be stabilized due to π-stacking of the aromatic substitutions with F¹⁴⁶⁵.

FIGURE 4.

Open and closed conformations of PD. (A–C) Open PD model. (A) Matrix of contact variants shows only those PDVs and their contacts, which are unique in the open PD model. Most of coupled variants are associated with either BrS1 or LQT3. (B) Hydrophobic contacts L¹⁷⁶¹:F¹⁴⁶⁵:F¹⁷⁶⁰ and A¹³³⁰:V¹⁴⁶⁸:I¹³³⁴ stabilize, respectively, mutual disposition of helices IIIS6/IVS6 and IIIS5/IIIS6. (C) H-bond S⁹⁴¹:N¹⁴⁶³ stabilizes mutual disposition of helices IIS6/IIIS6. (D–F) Closed PD model. (D) Matrix of contact variants shows only those PDV and their contacts, which are unique in the closed-PD model. (E) Mutual disposition of helices IIIS6 and IVS6 in the closed PD is stabilized by hydrophobic contacts F¹⁴⁶⁵:L¹⁷⁶¹ with side chain conformations different from those in the open-PD model. In coupled LQT3-associated variants, substitutions F¹⁴⁶⁵L or L¹⁷⁶¹F/H would destabilize the closed PD. Mutual disposition of helices IVS5/IVS6 is stabilized by a hydrophobic contact M¹⁷⁶¹:I¹⁶⁶⁰ and a knob-into-hole contact M¹⁷⁶¹:G¹⁶⁶¹. (F) Mutual disposition of helices IS6/IVS6 is stabilized by a tight hydrophobic contact between A⁴¹³ and methylene group of N¹⁷⁷⁴. Replacement of any contact partner with a large residue would destabilize the closed PD, a likely cause of LQT3 associated with these mutations.

Variant ^IVS6F¹⁷⁶⁰C is associated with BrS1. In functional studies, F¹⁷⁶⁰C impaired fast inactivation, accelerated recovery from inactivation (Carboni et al., 2005; O'Leary and Chahine, 2015), and enhanced slow inactivation (Carboni et al., 2005). Mutant channel F¹⁷⁶⁰A, which is used as a model for drug binding assays, causes a ∼ 20 mV depolarizing shift of steady-state inactivation (Carboni et al., 2005). LQT3-associated mutation A¹³³⁰P increased the window current, whereas mutation A¹³³⁰T, which is associated with LQT3, BrS1 and other diseases, increased the window current, impaired inactivation and accelerated recovery from inactivation (Smits et al., 2005b).

Figure 4C shows the H-bond between ^IIIS6N¹⁴⁶³ and ^IIS6S⁹⁴¹ that is specific for the open PD. This is an example of inter-repeat H-bonds involving asparagine residues, which are exceptionally conserved in sodium and calcium channels. Earlier such H-bonds were predicted (Tikhonov et al., 2015) and demonstrated (Du et al., 2018) to stabilize the open conformations of these channels. LQT3-associated variant S⁹⁴¹N would over-stabilize the H-bond and hence the open state of PD, whereas variant S⁹⁴¹C, which is associated with a non-specific cardiovascular phenotype, may destabilize the H-bond and thus the channel open state. Variant S⁹⁴¹N was found in an infant died from the sudden infant death syndrome, the disease associated with both Nav1.5 gain-of-function and loss-of-function (Amin et al., 2010). The mutant channel S⁹⁴¹N expressed in Xenopus oocytes demonstrated the increased late current (Schwartz et al., 2000).

Contacts between cytoplasmic halves of TM helices in the closed PD are summarized in matrix shown in Figure 4D. Some open-PD contacts, e.g., ^IVS6L¹⁷⁶¹ : ^IIIS6F¹⁴⁶⁵, are also seen in the closed PD, but the residue orientations in the two models are substantially different (cf. Figures 4B, E). A knob-into-hole contact ^IVS6M¹⁷⁶⁶ : ^IVS5G¹⁶⁶¹, which is seen only in the closed-PD model (Table 3), and hydrophobic contact ^IVS6M¹⁷⁶⁶ : ^IVS5I¹⁶⁶⁰ (Figure 4E) apparently stabilize the closed PD conformation. It would be destabilized in LQT3-associated variants M¹⁷⁶⁶L and I¹⁶⁶⁰V where large hydrophobic residues are substituted with smaller ones. In functional studies, mutation I¹⁶⁶⁰V decreased the current, which was rescued by 48 h incubation at room temperature or by mexiletine; the variant was suggested to affect the channel folding, while mexiletine or room temperature stabilized the folding (Cordeiro et al., 2006). Mutation M¹⁷⁶⁶L significantly decreased the sodium channel expression, which was partially rescued by mexiletine; however, this mutation also demonstrated a 10-fold increase of the persistent late sodium current (Valdivia et al., 2002).

A tight contact between ^IS6A⁴¹³ and the methylene group of ^IVS6N¹⁷⁷⁴ is seen only in the closed-PD model (Table 3 and Figure 4F). Substitution of either contact partner with a larger residue would destabilize the closed conformation, resulting in LQT3 (Figure 4D). Mutant channel N¹⁷⁷⁴D expressed in the CHO-K1 cells demonstrated increased peak sodium current density, late current and enhanced activation (Kato et al., 2014). Similar electrophysiological changes, as well as markedly increased duration of the action potential, were found in the cardiac myocytes derived from induced pluripotent stem cells from a boy with LQT3-associated mutation N¹⁷⁷⁴D (Hirose et al., 2020). Variant N¹⁷⁷⁴S was found in a Chinese family with BrS1 (Du et al., 2005).

In the cryo-EM structure, ^IIIS6F¹⁴⁶⁵ forms hydrophobic contacts with four residues whose three variants are associated with LQT3 (Table 1). In our closed-PD model, ^IVS6I¹⁷⁶⁸ forms a tight contact with F¹⁴⁶⁵ whose substitution F¹⁴⁶⁵L is also associated with LQT3 (Table 3). Substitutions F¹⁴⁶⁵L or I¹⁷⁶⁸V would destabilize the hydrophobic contact and thus the closed conformation of PD. LQT3-associated substitution I¹⁷⁶⁸V would weaken contacts that stabilize both the inactivated and closed-state conformation of the PD. In functional studies, mutant channel I¹⁷⁶⁸V accelerated recovery from inactivation and increased the channel availability (Rivolta et al., 2002). In another study, mutant channel I¹⁷⁶⁸V accelerated recovery from inactivation, impaired slow inactivation, but did not affect steady-state activation and inactivation (Groenewegen et al., 2003).

Contacts in VSDs

PDVs and their contacts in the cryo-EM structure with the activated VSDs and models with resting PDVs are shown in Tables 4, 5, respectively. Many contacts switch upon VSD transitions between the activated-state conformations, which are captured in the cryo-EM structure, and resting-state models. We summarized these contacts in four matrices (Figures 5–7) where cells are labeled “C”, “M” or “B” depending on whether respective contacts are seen only in the cryo-EM structure, only in the resting-state model, or both in the cryo-EM structure and the model. It should be noted that ionized residues in VSDs switch contacts during channel gating. Impact of mutations in such contacts on relative stability of different channel states is difficult to predict. On the other hand, respective contacts are promising targets for further studies.

TABLE 4.

PDVs and their contacts in activated VSDs^a.

PDVs		Contacts
Variantb	Syndromec	Mutation	Syndromed	Significance	e	f
IS1M138I	AF	IS4T229S	BrS1	VUS		S
T	DCM1	IS4-S5V232I	A, BrS1, CVP	CIP		S
IS1I141N	BrS1	IS4T229S	BrS1	VUS		S
F	NS	IS4R225Q	BrS1, LQT3, More	CIP		S
		W	BrS1, LQT3, More	P/LP		S
		IS4A226D	BrS1	VUS		S
		V	BrS1, LQT3, More	CIP
		IS4V232I	A, BrS1, CVP	CIP
IS2R179P	BrS1	IS1L128P	BrS1	VUS	4
Q	BrS1	IS2-S3T187S	BrS1	VUS
		I	BrS1	NP
		A	BrS1, CA	CIP
IS2-S3T187S	BrS1	IS2R179P	BrS1	VUS	4
I	BrS1, NP	Q	BrS1	VUS
A	BrS1
IS3D197G	BrS1	IS2K175D	BrS1	NP
H	BrS1	IS2E171G	BrS1	VUS
Y	DCM
IIS3E795K	BrS1	IIS4L807P	BrS1, NP	VUS		S
IIS4-S5L825P	BrS1	IIS4F816L	A	VUS		S
		Y	LQT3	NP		S
		IIIS5V1340L	CVP	VUS
		I	NP, BrS1, A	VUS
IIS5I848F	A,BrS1, LQT31	IS4A226V	BrS1, LQT3, More	CIP		S
		D	BrS1	VUS		S
		IS4I230T
IIIS1A1221V	A, BrS1	IIIS2L1239P	BrS1	NP
IIIS4R1306L	CVP	IIIS2D1243E	BrS1	VUS		S
H	NP, BrS1	NG	BrS1, More	VUS		S
C	A	IIIS2E1225K	BrS1	VUS		S
S	BrS1		LQT3, BrS1, A	VUS		S
IIIS4R1316L	BrS1	IIIS1E1208K	A, NP	VUS		S
Q	BrS1
IVS2E1574K	BrS11,3	IVS1I1534S	LQT3	VUS		S
IVS2C1575Y	A, BrS1	IVS3F1596I	LQT3, BrS1, More	VUS
S	A
IVS3N1592Y	BrS1	IVS4R1638P	NP	LP
IVS4-S5I1643L	NP	IVS3I1637T	NS	VUS

^a-e footnotes to Table 1.

TABLE 5.

PDVs and their contacts in resting VSDs ^a .

PDV		Contacts
Variant	Syndrome	Variant	Syndrome	Signif.
IS1I141N	BrS1	IS4V223A	A	LB
F	NP	L	BrS1	NP
IS2R179P	BrS1	IS2-S3H184Q	BrS1, CVP	CIP
Q	BrS1	IS2-S3A185T	BrS1, LQT3, A	CIP
		V	BrS1, A	VUS
IS4R219C	A, BrS1	IS2E161Q	BrS1	NP
H	BrS1, DCM	K	BrS1	CIP
P	LQT3, BrS1, More
IIS3E795K	BrS1	IIS4R808P	LQT3	NP
		C	A	VUS
		H	LQT3, A	VUS
IIS4-S5L825P	BrS1	IIS4A819P	BrS1	VUS
IIS5I848F	A, BrS1, LQT3	IS4V223A	A	LB
		L	BrS1	NP
		IS4L227P	BrS1	CIP
IIIS4R1306L	CVP	IIIS3I1278N	LQT3	NP
H	NP, BrS1
C	A
S	BrS1
IVS2C1575Y	A, BrS1	IVS3D1595H	BrS1, LQT3, More	LP
S	A	N	BrS1, More	P
IVS4-S5I1643L	NP	IS5D252V	BrS1	VUS
		IVS3I1637T	NS	VUS

^aContacts in the cryo-EM structure are shown in Table 4.

FIGURE 5.

VSD-I in the activated-state structure and resting-state model. (A,B) Intra-membrane views at VSD-I in the activated (A) and resting (B) states. Side chains of some w54 residues and their contacts are indicated with green and red labels, respectively. Dashed lines indicate displacement of K²²⁸ upon activation of VSD-I. (C) Matrix of contacts variants. To indicate contacts, which may switch upon activation of VSD-I, each cell is labeled “C”, “M” or “B” depending on whether the contact is found only in the cryo-EM structure, only in the resting-state model, or both in the cryo-EM structure and the model. Overwhelming majority of PDVs and their contact variants are associated with BrS1. See section VSD-I for more details.

FIGURE 7.

Matrices of contact variants and structures of VSD-III and VSD-VI. (A) Matrix of contact variants of VSD-III. (B,C) VSD-III in the activated-state cryo-EM structure (B) and resting-state model (C). Residues from set w54 and their contacts are indicated with green and red labels, respectively. See section VSD-III for more detail. (D) Matrix of contact variants of VSD-IV. (E) Activated-state structure of VSD-IV. Residues from set w54 and their contacts are indicated with green and red labels, respectively. See section VSD-IV for more details.

VSD-I

Figure 5 shows VSD-I in the activated (A) and resting (B) states with w54 residues and their contacts. Dashed lines indicate displacement of ^IS4K²²⁸ upon transition of the voltage-sensing helix IS4 between the resting and activated states. In the activated VSD-I, R²¹⁹ lacks specific inter-segment contacts, while in the resting VSD-I, R²¹⁹ forms a salt bridge with ^IS2E¹⁶¹ (Figure 5B). BrS1-associated substitutions R¹⁹C/H/P or E¹⁶¹Q/K would eliminate the salt bridge, likely destabilizing the resting VSD-I. Mutation E¹⁶¹K, which was found in patients with the sick sinus syndrome, BrS1, or cardiac conduction disease, reduced current density and markedly impaired activation (Smits et al., 2005a).

Variant R²¹⁹H is associated with mixed arrhythmia and dilated cardiomyopathy, likely due to the proton leak current, which was observed in biophysical studies (Gosselin-Badaroudine et al., 2012; Moreau et al., 2018). Mutations R²¹⁹P and E¹⁶¹K would destroy the salt ridge and destabilize the resting state, the effect apparently inconsistent with BrS1. However, the same mutations are also associated with LQT3 (Figure 5C).

In the activated VSD-I, ^IS2R¹⁷⁹ is H-bonded with ^IS2-S3T¹⁸⁷ and forms a hydrophobic contact with ^IS1L¹²⁸. Variants of these residues are associated with BrS1, indicating that respective mutations destabilize the activated VSD-I. In the resting VSD-I, R¹⁷⁹ retains the H-bond with T¹⁸⁷ and interacts with ^IS2-S3H¹⁸⁴ and ^IS2-S3F¹⁸⁸. Substitutions R¹⁷⁹P/Q and T¹⁸⁷S/A/I would eliminate the contacts, thus affecting flexibility of loop IS2-S3. Contact between R¹⁷⁹ and T¹⁸⁷ is seen in both activated and resting states of VSD-I, whereas variants of these residues are associated with BrS1, implying that flexibility of loop IS2-S3 is important for activation of VSD-I.

In the activated VSD-I, ^IS3D¹⁹⁷ is salt-bridged with ^IS2K¹⁷⁵ and ^IS4K²²⁸ (Figure 5A). In the resting VSD-I, D¹⁹⁷ retains the salt bridge with ^IS2K¹⁷⁵ and approaches ^IS4K²²⁸ and ^IS4R²²⁵ (Figure 5B). Substitutions D¹⁹⁷G/H/T, K¹⁷⁵N, and R²²⁵Q/W would eliminate or weaken these contacts in both states. The fact that D¹⁹⁷G/H/T and K¹⁷⁵N are associated with BrS1 (Figure 5C) suggests that salt bridge D¹⁹⁷G : K¹⁷⁵ contributes to stabilization of the activated VSD-I. Notably, most of variants in VSD-I are associated with BrS1, but four variants are associated with both BrS1 and LQT3 (Figure 5C). The structural cause of the later ambiguity is unclear.

VSD-II

The matrix of contact variants in VSD-II (Figure 6A) shows four w54 residues, including ^IIS5I⁸⁴⁸, which forms state-dependent interdomain contacts with four residues in helix IS4, and ^IIS4-S5L⁸²⁵ that forms interdomain contact with ^IIS5V¹³⁴⁰. Most residue substitutions in the interdomain contacts are associated with BrS1, but three variants are also associated with LQT3. Homozygous mutation ^IIIS5V¹³⁴⁰L was found in two female patients with atrial standstill, while in three unaffected relatives the mutations was heterozygous. In a functional study, V¹³⁴⁰L reduced the current density and impaired channel activation (Tan et al., 2018). Another mutation, V¹³⁴⁰I, did not alter biophysical characteristics at room temperature, but at 40°C it decreased the current density, caused hyperpolarizing shift of steady-state inactivation, and accelerated recovery from the fast inactivation (Samani et al., 2009).

FIGURE 6.

VSD-II in the activated-state structure and resting-state model. (A) Matrix of contact variants, in addition to contacts within VSD-II, slows contacts IIS4-5/IIIS5 and IIS5/IS4 (B,C) VSD-II in the activated-state cryo-EM structure (B) and resting-state model (C). Residues from set w54 and their contacts are indicated with green and red labels, respectively. VSD-II Section for more details.

The activated VSD-II is stabilized by a salt bridge ^IIS4R⁸⁰⁸ : ^IIS1E⁷³⁷, an H-bond ^IIS4R⁸¹¹ : ^IIS2N⁷⁵³, a hydrophobic contact ^IIS3E⁷⁹⁵ : ^IIS4L⁸⁰⁷, and hydrophobic contacts involving ^IIS4F⁸¹⁶, ^IIS4A⁸¹⁹ and ^IIS4-S5L⁸²⁵ (Figure 6B). The latter contacts are also seen in the resting VSD-II, but the downshift of helix IIS4 significantly rearranged contacts involving R⁸⁰⁸ and R⁸¹¹ (Figure 6C). Salt bridges R⁸⁰⁸ : E⁷³⁷, R⁸¹¹ : E⁷⁹⁵ and an H-bond R⁸⁰⁸ : E⁷⁹⁵ apparently stabilize the resting VSD-II. Mutations R⁸⁰⁸P/H would eliminate the salt bridges and destabilize the resting state of VSD-II stronger than its activate state in which R⁸⁰⁸ forms only one salt bridge. This model-based prediction agrees with the fact that variants R⁸⁰⁸ P/H are associated with LQT3 (Figure 6A).

VSD-III

The matrix of contact variants in VSD-III shows seven PDVs and seven their contact variants (Figure 7A). Activated VSD-III is stabilized by a salt bridge ^IIIS4R¹³⁰⁶ : ^IIIS1E¹²²⁵, which is seen only in the cryo-EM structure, two salt bridges seen in both the cryo-EM structure and resting-state model (R¹³⁰⁶ : ^IIIS2D¹²⁴³ and ^IIIS4R¹³¹⁶ : ^IIIS1E¹²⁰⁸) and the hydrophobic contact ^IIIS2L¹²³⁹ : ^IIIS1A¹²²¹ (Figure 7B). Elimination of the salt bridges in these variants is usually associated with BrS1 (Figure 7A). In the cryo-EM structure, ^IIS1R¹¹⁹⁵ and ^IIS2-S3G¹²⁶² are farther than 5 Å from each other (Figure 7B), but PDV G¹²⁶²S (Table 6) may form an H-bond with R¹¹⁹⁵ and decrease flexibility of linker IIS2-S3.

TABLE 6.

PDVs whose missense contact variants in the cryo-EM structure or models are not listed in ClinVar.

#	PDV		Exposure	Comment
	Varianta	Syndromeb,c
1	IS6V396L	BrS11,3,4	Lipids	Tight contacts withIS5F264 IP1L365 and IP1L368
2	IIS1L732P	BrS1
3	IIS1-S2L736P	BrS1	Extracellular	Loop Flexibility
4	IIS1-S2H738P	BrS1	Extracellular	Loop Flexibility
5	IIS3M794T	NP	Lipids
6	IIS4-S5G833R	LQT-3,NP,A, BrS17	Lipids
7	IIS5V924F	NP	Lipids
8	IIIS1R1195C	BrS1	Cytoplasm	R1195 may reach G1262 “
9	IIIS1R1195H	“	Cytoplasm
10	IIIS2-S3G1262S	BrS1, LQT-3, PFHB1,3,11,12	Cytoplasm	Loop flexibility
11	IIIS6I1448L	A, BrS1, CVP1,3,4	Lipids

^a-e See footnotes to Table 1.

The resting state of VSD-III is stabilized by a hydrophobic contact of I¹²⁷⁸ with methylene groups of R¹³⁰⁶ (Figure 7C). Variant I¹²⁷⁸V, which was found in a Thai patient with congenital complete heart block, is considered disease-causative because this variant was not found in unaffected members of the patient’s family (Thongnak et al., 2016). Substitution I¹²⁷⁸N would destabilize the resting state in agreement with the fact that this variant is associated with LQT3 (Figure 7A).

VSD-IV

The matrix of contact variants in VSD-IV shows five PDVs and seven their contacts variants (Figure 7D). Several contacts at the cytoplasmic half of VSD-IV stabilize the activated state (Figure 7E); weakening or elimination of these contacts in BrS1-associated PDVs would destabilize the activated VSD-IV. In contrast, LQT3-associated mutation ^IVS1I¹⁵³⁴S would stabilize the activated state due to H-bond between ^IVS2E¹⁵⁷⁴ and ^IVS1I¹⁵³⁴S. In both activated and resting states of VSD-IV, ^IVS3F¹⁵⁹⁶ forms a hydrophobic contact with ^IVS2C¹⁵⁷⁵ (Figure 7D). Variants C¹⁵⁷⁵S/Y are associated with BrS1, whereas variant F¹⁵⁹⁶I is associated with both BrS1 and LQT3. In functional studies, F¹⁵⁹⁶I did not change peak current, but enhanced steady-state inactivation and accelerated recovery from inactivation (Olesen et al., 2012; Hoshi et al., 2015).

Domain Distribution of PDVs and their Contact Variants

Table 7 shows domain distribution of PDVs, their contact variants, and clinical significance of the latter variants. In the PD, 27 WT residues with a total of 38 PDVs are engaged in contacts with 45 WT residues, which have a total of 63 missense disease variants. In the four VSDs, 17 WT residues with a total of 33 PDVs contact 39 WT residues with a total of 55 missense disease variants. While majority of PDVs and their contact variants are seen in the cryo-EM structures, models of the open/closed PD and resting VSDs revealed an additional PDV and 34 contact variants. Thus, in the considered channel states, w54 residues with 82 PDVs form contacts with 84 WT residues that have a total of 118 disease variants. Current number of PDVs exceeds the published number (Tarnovskaya et al., 2020) because new variants of w54 residues were recently reported in ClinVar. For majority of the published PDVs clinical significance is still VUS/NP/CIP, but two variants, ^IS5L²⁵⁰V and ^IVS6M¹⁷⁶⁶L, were recently reclassified as P/LP. As many as 104 contact disease variants are categorized as VUS/NP/CIP variants. Further bioinformatics analysis is required to predict potentially damaging effect of these variants, but the fact that they are coupled with earlier predicted PDVs suggests that a large portion of the 104 variants would be reclassified as a P/LP variants.

TABLE 7.

Domain distribution of PDVs and their contact variants.

PDVs				Contacts		Clinical significance of contact variants
Domain	State	WT	Variants	WT	Variants	VUS	P/LP	NP	CIP
PD	Inactivated a	27	38	37	49	29	7	11	2
	Open b			3	5	2	2	1
	Closed b			5	9	2	1	5	1
VSD-I	Activated a	5	13	11	15	8	1	2	4
	Resting c	1	3	3	6	2		2	2
VSD-II	Activated a	4	5	8	10	8			2
	Resting c			6	10	6		2	2
VSD-III	Activated a	3	7	4	6	4		1	1
	Resting c			1	1			1
VSD-IV d	Activated a	4	5	4	4	3	1
	Resting c			2	3	1	2
Total		10	11
		54	82	84	118	65	14	25	14
Set e		w54	PDV	c84	cv118

^aCryo-EM structure.

^bWT residues that have contacts variants in the inactivated PD (cryo-EM structure) are not counted.

^cWT residues that have contacts variants in the activated VSD (cryo-EM structure) are not counted.

^dLocation is not shown for WT residues that do not form contacts with disease-associated residues.

^eNames of the sets are shown in bold.

Table 8 shows domain distribution of disease variants. Most of these are associated with BrS1, followed by LQT3 and unspecified arrhythmia. A total of 32 variants are associated with other diseases, which are specified in Tables 1–5. In the contact variant matrices respective variants are not colored. It should be noted that most of “other” diseases are associated with the Nav1.5 loss-of-function. The cases when variants associated with “other” loss-of function diseases are coupled with BrS1-associated variants provide additional examples of contacts in which mutation of either partner destabilizes the open channel.

TABLE 8.

Domain distribution of disease variants.

Location	State	BrS1		LQT3		Arrythmias		Other diseases
		PDV	Contacts	PDV	Contacts	PDV	Contacts	PDV	Contacts
PD	Inactivated a	21	30	6	10	3	6	10	7
	Open b		2		3				1
	Closed b		6		3				1
VSD1	Activated a	10	14		2			3	2
	Resting c	3	5	1	2		1
VSD2	Activated a	3	7	1	3		1	2	1
	Resting c		4		2		2		2
VSD3	Activated a	5	5		1	1	1	1
	Resting c				1
VSD4	Activated a	4	2		2			1	1
	Resting c		3		1
Total		46	78	8	30	4	11	17	15

^a-c See footnotes to Table 7.

Only eleven of 82 PDVs (13%) are not coupled with other disease variants (Table 6). One PDV (^IS6V³⁹⁶L) is involved in tight intra-PD contacts, while 10 PDVs are exposed to lipids, cytoplasm or extracellular space. Three variants (^IIS1-S2L⁷³⁶P, ^IIS1-S2H⁷³⁸P, and ^IIIS2-S3G¹²⁶²S) affect flexibility of VSD linkers. The fact that 87% of PDVs are coupled with disease variants supports bioinformatics-based predictions of PDVs (Tarnovskaya et al., 2020) and suggests that many contact variants of PDVs, which are currently annotated as VUS/NP/CIP, may be reclassified as P/LP variants.

Limitations of the Approach

In the absence of cryo-EM structures of the hNav1.5 channel with the open and closed pore domain and resting VSDs, we modeled these states using as templates crystal structures of prokaryotic sodium channel NavAb (see Methods). Some of the proposed intersegment contacts in these states require future validation in functional studies. Ionizable residues in VSDs are involved in multiple state-dependent electrostatic interactions. Structural interpretation of respective mutations may be ambiguous.

Concluding Remarks

Only a small fraction of known missense variants of the hNav1.5 channel are classified as P/LP variants. Prediction of damaging effects of VUS/NP/CIP variants would improve diagnostics of increasingly large number of genotyped cardiac patients. Here we employed a recent cryo-EM structure of the Nav1.5 channels and ad-hoc homology models of the channel with the open or closed PD and resting VSDs to explore intersegment contacts of 54 WT residues whose VUS/NP/CIP variants are reclassified as PDVs (Tarnovskaya et al., 2020). We found that 44 of the 54 WT residues form intersegment contacts with 84 WT residues for which 118 disease variants are reported. Overwhelming majority of the contact variants is categorized as VUS/NP/CIP. We propose that if two WT residues, which are engaged in an intersegment contact, have VUS/NP/CIP variants, these are P/LP variants. Validation of this proposition requires bioinformatics analysis of a large set of contacts involving disease variants and electrophysiological testing of some contacts (Smith and Goldin, 1997; Bénitah et al., 1999). In many contacts, variants of either residue are associated with the same syndrome likely because they similarly affect stability of a particular channel state. To facilitate recognition of such coupled variants, we presented them in colored matrices. Structural analysis suggested atomic mechanism of the channel dysfunction in scores of coupled variants. These include BrS1-associated mutations in P-loops that would destabilize the selectivity-filter slow inactivation gate, BrS1-associated variants in VSDs that would destabilize their activated states, and LQT3-associated mutations in PD that would stabilize the channel states with the open pore (or destabilize states with the closed pore). Understanding these mechanisms may assist structure-based predictions of pathogenic variants and contribute to general physiology of ion channels.

Author Contributions

VK, Molecular modeling, manuscript writing; AZ, Collecting literature on channelopathies, manuscript writing; AK, Project administration, manuscript editing; BZ, Conceptualization, supervising, manuscript writing and editing, funding acquisition.

Funding

This study was supported by grants to BSZ from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2020-07100) and Russian Science Foundation (17-15-01292-Π).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, orclaim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2021.756415/full#supplementary-material

Abbreviations

BrS1, brugada syndrome type 1; CIP, conflicting interpretations of pathogenicity; LQT3, long QT syndrome type 3; MC, monte carlo; MCM, MC-minimizations; NP, data on pathogenicity are not provided; P1 and P2, membrane-descending and membrane-ascending pore helices in P-loops; PD, pore domain; P/LP, pathogenic/likely pathogenic; PDV, potentially damaging variant (P/LP and PDV are used, respectively, for database-listed variants and those predicted computationally); SF, selectivity filter; TM, transmembrane; VSD, voltage-sensing domain; VUS, variant of uncertain clinical significance; w54, 54 wild-type residues for which PDVs are predicted; WT, wild type.