Neither local strain nor exposed hydrophobic surface explains pathogenicity at SCN5A Arg104, and three further candidate mechanisms are ruled out

Ethan Bradley

Independent researcher, no institutional affiliation

ORCID: 0009-0008-8925-7975

Abstract

Five candidate structural mechanisms for loss of function at SCN5A Arg104 were tested and four are refuted, in each case by a control that a plausible-looking positive result failed. Local energy redistribution on substitution is real but non-specific: it tracks side-chain volume (Spearman +0.77) and produces its largest effect in the study on a benign control, V125L. Exposed apolar surface separates R104W from R104Q decisively (p = 1.7e-7), which I first read as support for a sticky-surface mechanism, until a cross-position control substituting tryptophan at seven N-terminal-domain positions put the benign control R34C first, gaining 2.3 times more apolar area than position 104, with the dominant-negative and benign split running the wrong way (Mann-Whitney p = 1.000). Two independent protocols reached that refutation and disagreed on absolute magnitude while agreeing on the ordering. A linear degradation or endoplasmic-reticulum retention motif is ruled out at Arg104 by sequence scan, while the pathogenic control R121W does lie in one. The N-terminal domain, residues 1 to 130 of NP_932173.1, contains no N-X-S/T glycosylation sequon, so no glycosylation-mediated mechanism is available to it. What the structure does establish is that Arg104 anchors an invariant buried acidic pocket whose salt bridge to Asp84 is conserved across all nine human Nav paralogues. The observable that separates R104W from benign substitutions has not been identified, and the remaining candidates are ones a single-chain calculation cannot see.

A key to the terms used here

• SCN5A is the gene for the heart's main sodium channel; Nav1.5 is the protein. Arg104, or R104, is the arginine at protein position 104. R104Q replaces it with glutamine, R104W with tryptophan. R34C and V125L are used as benign controls, variants not expected to cause disease.
• The N-terminal domain is the first stretch of the protein, residues 1 to 130 here.
• A mechanism in this paper means a specific physical story for how a variant breaks the channel. Each is tested separately and most are eliminated.
• Local strain is stress introduced into the surrounding structure when one amino acid is swapped for another that does not fit as well.
• Hydrophobic means water-repelling. Proteins normally bury their hydrophobic parts inside. Exposed apolar surface is water-repelling surface left on the outside, which can make a protein sticky and get it flagged for disposal. SASA is the measured amount of that surface, in square ångströms.
• An ångström, Å, is a ten-billionth of a metre. A REU is Rosetta's internal energy unit.
• Repacking means letting the computer rearrange nearby side chains after a substitution; minimisation then relaxes the structure to relieve leftover stress. A rotamer is one of the positions a side chain can adopt.
• N-glycosylation is the cell's attachment of a sugar chain to a protein at a specific three-letter pattern called a sequon (N-X-S/T). No sequon means no attachment site, so that mechanism is unavailable.
• A degron is a short sequence that marks a protein for destruction; a retention motif holds it in the endoplasmic reticulum, the cell's assembly and inspection area. A protein failing inspection is destroyed rather than shipped, which is why these motifs matter here.
• A salt bridge is an attraction between a positively and a negatively charged side chain, such as Arg104 to Asp84 (aspartate at position 84). A hydrogen-bond donor is an atom offering a hydrogen to such a contact.
• Paralogues are related genes in the same organism arising from duplication; the nine human Nav alpha subunits are paralogues of each other. Conservation is how unchanged a position stays across them.
• Cryo-EM yields a 3D protein model; 8VYJ is the one used here, at 3.6 Å resolution, fine enough to place the backbone but not to fix every side chain.
• Cliff's δ measures how completely two groups separate. A confidence interval that excludes zero means the effect is distinguishable from no effect. Spearman ρ measures rank agreement.
• A control is a case whose answer is known in advance. The controls do the real work in this paper: each mechanism that looked supported was eliminated by a variant known to be harmless scoring as high.

Why eliminate mechanisms at all

A folding-stability calculation says nothing about SCN5A Arg104: a companion analysis found that folding free energy misses three of four known loss-of-function variants in this domain. The natural next move is to ask what observable does separate them. This paper reports that search, and it is mostly a record of hypotheses that did not survive their controls. I report it because the controls are the useful part. Each refuted hypothesis here produced an encouraging first result, and each was killed by asking whether a variant known to be benign scored as high.

Part 1. Local strain and exposed hydrophobic surface

1. What was computed

Nine models per frame on all 24 relaxed ensemble members (12 Rosetta density-guided, 12 restrained-OpenMM) × 3 independent repacker seeds = 1008 models, 0 failures. Position 104 substitutions Q, W, K plus A/F/Y as a volume-and-aromaticity series; benign controls R34C and V125L at their own positions.

Each model was scored in two tiers:

The control that makes this interpretable. A wild-type pose keeps its input rotamer through IncludeCurrent; a mutant cannot. Comparing mutant-to-WT therefore charges every mutant for the repacker's inability to rediscover the input conformation. So every variant is compared against a self-mutation control, MutateResidue(104, "ARG") on the wild-type pose, then the identical repack/minimise protocol. Arg→Arg is chemically null; everything it registers is method noise. All effect sizes below are frame- and seed-matched differences against that control, with 95% paired-bootstrap CIs (20 000 resamples) and Cliff's δ.

Metrics: per-residue ref2015 energy for all 8 Å-shell residues (target excluded, its own energy changes trivially with residue type); Rosetta HBondSet donor counts and an independent geometric donor count at the Asp84/Asp82 carboxylates; BuriedUnsatHbondFilter (vsasa burial); FreeSASA side-chain and apolar SASA; compute_residue_packing_scores (packstat) for cavity formation; Cα displacement over the 82-92 loop and 100-110 strand; DSSP secondary structure over B78-115.

2. Result 1: local energy redistribution is real, and it is not specific

Excess local |ΔE| over the 8 Å shell, relative to the matched self-mutation control (OpenMM prep, n = 36 models each):

R104W does rank first. But V125L, a benign control with no ClinVar entry, correctly predicted neutral by the global method, ranks fourth of eight and has the single largest effect size in the table (δ = +0.79, and δ = +1.00 in the Rosetta prep). A metric that fires this hard on a benign control cannot be the missing discriminator.

Hypothesis A's exact predicted signature appears in the wrong variant. The prediction was "significant local redistribution with near-zero total energy." V125L delivers precisely that: local |ΔE| = +3.99 REU (CI excludes zero) while total energy change is +0.27 REU (CI -0.39 to +1.09, includes zero). R104W's total is also indistinguishable from control (+1.75, CI -0.47 to +4.06), but so is a benign variant's. The signature is not diagnostic.

Ranking test, explicitly. Local disruption at position 104 anti-correlates with global ΔΔG (Spearman ρ = -0.77, n = 6, p = 0.072) and correlates with side-chain volume at exactly the same magnitude (ρ = +0.77). The local ranking is W > Y > F > A > Q > K. Clinically, R104W is Pathogenic/Likely pathogenic and R104Q is Conflicting; R104Y and R104F have no ClinVar entries and no functional data, so the local ranking places two uncharacterised variants above the one variant of clinical interest. Volume, not pathogenicity, is what this axis measures.

3. Result 2: the fixed-backbone strain signal is repacker noise

Tier 1 (no relaxation) appears to show a large R104W effect: local |ΔE| = 26.4 REU, total ΔE = +38.0 REU, roughly 3× any other variant. That signal has a standard deviation equal to its own mean (SD 26.1 REU across 36 models; coefficient of variation 0.99, the highest of any variant, control included). Cartesian relaxation removes it: R104W's local |ΔE| falls from 26.4 to 12.4 REU and its total ΔE from +38.0 to +2.1 REU.

This is the confound the task flagged, and it is quantitatively confirmed: the apparent fixed-backbone strain of the bulkiest substitution is dominated by the repacker's failure to place a large side chain, not by physical strain. Any local-strain claim built on fixed-backbone repacking alone would have been an error of method, not a finding. Reported here rather than debugged away.

The self-mutation controls put a floor on everything: chemically-null Arg→Arg registers local |ΔE| = 6.37 ± 4.61 REU (OpenMM) and 0.72 ± 1.34 REU (Rosetta). The Rosetta ensemble's ~9× smaller noise floor is the conformational degeneracy already documented for that prep (SD 0.005 Å in side-chain distances); it makes the Rosetta prep look more precise without making it more informative, which is why OpenMM is the primary prep here.

4. Result 3: the acidic pocket is dismantled by every substitution

H-bond donors within 3.5 Å of the Asp84 carboxylate (Rosetta HBondSet, tier 2):

Every substitution except lysine drops Asp84 to essentially zero donors (all CIs exclude zero, δ ≈ -0.76 to -1.00, both preps). R104W and R104Q are indistinguishable on this metric (paired W-Q difference +0.03 donors, p = 0.66). Only R104K retains partial coordination, 0.78-0.97 donors, which is consistent with the prior finding that lysine cannot reach Asp84 but can still contribute at the pocket periphery.

This reproduces the project's prior numbers (WT ~2.5 → R104Q ~1.0 by the older geometric count; the absolute values differ with detection method, the direction and magnitude do not) and confirms the microenvironment note's framing. But it also settles a question: loss of Asp84 coordination cannot explain the R104W/R104Q clinical difference, because both lose it identically.

Buried-unsatisfied-polar counts inverted from expectation, and the reason is instructive: removing arginine exposes Asp84 to solvent rather than leaving it buried and unsatisfied. Shell-summed unsatisfied polars go down in every mutant (Δ = -0.3 to -2.6). The "orphaned buried charge" picture overstates the defect, as the microenvironment note already concluded from a different direction, because the carboxylate gains solvent access when its partner leaves.

5. Result 4: the backbone barely moves, and secondary structure never changes

R104W's loop displacement exceeds control (+0.05 Å, CI +0.01 to +0.10) and exceeds R104Q (+0.03 Å, p = 0.019). These are hundredths of an ångström, far below the ~3.6 Å resolution of the parent structure and below any threshold at which a backbone claim is meaningful. Statistical significance here reflects 36 matched replicates, not physical magnitude.

DSSP over B78-115 is identical to the matched wild-type in 100% of Rosetta models (all 396) and 83-94% of OpenMM models. The residual OpenMM variation is method noise, not mutation effect: the chemically-null Arg→Arg control scores 0.861, below R104W's 0.944, and the lowest score of any variant belongs to the benign control V125L (0.833). The 101-106 β-strand and the 82-89 loop survive every substitution intact. No variant unzips the sheet. This is the expected result given the prior finding that R104 occupies the non-H-bonded cross-strand position, so its side chain is not load-bearing for sheet register, and it independently confirms that framing.

Packstat detects no cavity: shell-averaged change is ≤0.05 for every variant in both preps, including the R104A "cavity-creating" case.

6. Result 5: what R104W actually does differently

One metric separates R104W from R104Q decisively, and it is not strain. Exposed apolar side-chain surface, normalised to each residue's Gly-X-Gly extended-reference maximum (removing the trivial size dependence):

R104W and R104Q move in opposite directions, and this is the largest, most reproducible separation in the whole analysis: paired W-Q difference = +0.150 relative units, p = 1.7 × 10⁻⁷, consistent in both preps. R104W adds ~28 Ų of exposed apolar surface at a position that is 11.6% solvent-accessible in the assembled channel; R104Q removes ~13 Ų. Both benign controls move slightly negative (V125L -1.1 Ų) or, in R34C's case, positive at an already solvent-exposed position, R34 measures 52.4% RSA in this carve-out (V125 is 5.3%, R104 15.1%), so R34C adds apolar area to an already-wet surface rather than creating a buried hydrophobic patch. R34 sits three residues from a carve-out chain terminus, so its exposure is an upper bound.

This looked like hypothesis B, and I initially reported it as support for it. That was wrong, and the control that shows why is in §6a. The observation itself stands: tryptophan at 104 presents an apolar face where arginine presented a polar one, without destabilising the fold. What it does not do is explain pathogenicity.

6a. The control that refutes hypothesis B: a cross-position test

The §6 comparison holds position fixed and varies the substitution. That design cannot detect a metric which is simply large wherever a big hydrophobic residue is introduced. The missing control is the transpose: substitute tryptophan at seven different NTD positions and ask whether apolar gain tracks phenotype. Same tier-2 protocol, same self-mutation controls, 24 OpenMM models per position:

The benign control ranks first, gaining 2.3× more apolar area than position 104, and position 104 is third of seven. Splitting the phenotyped positions into dominant-negative (87, 104, 121) versus benign (34, 125) gives means of 21.0 vs 32.6 Ų, the wrong direction, Mann-Whitney p = 1.000. R121W, a demonstrated dominant-negative, gains little.

Hypothesis B is therefore refuted as an explanation of pathogenicity. An independent parallel analysis in this work (reported below) reached the same verdict via a different protocol, Bio.PDB ShrakeRupley, 10 Å shell, repack-plus-minimise, and reported position 34 first and position 121 last on the same test. Two protocols, same refutation. My §6 numbers differ from theirs in absolute magnitude (+30.0 vs +13.7 Ų at position 104) because the shell definition, SASA implementation and reference state differ; the ordering, which is what the claim rests on, agrees.

What survives. Two narrower statements, both still useful. (i) R104W and R104Q change the chemical character of the position-104 surface in opposite directions (p = 1.7 × 10⁻⁷), so whatever distinguishes them, it is not a shared surface property, they are candidates for mechanistically distinct variants at the same residue. (ii) A folding-ΔΔG method is blind to surface chemistry by construction, so the negative result is not evidence that R104W is structurally uneventful, only that it is thermodynamically uneventful. Neither statement licenses a hydrophobic-association mechanism.

7. Which hypothesis do the data support?

Not A. Local perturbation is measurable but non-specific: it tracks side-chain volume (ρ = +0.77), fires with the largest effect size in the study on a benign control (V125L, δ = +0.79 to +1.00), and produces a ranking (W > Y > F > A > Q > K) that places two uncharacterised variants above the variant of clinical interest. R104W and R104Q are statistically indistinguishable on the Asp84 coordination loss that would have been hypothesis A's mechanism. Local strain is not the thing global ΔΔG was averaging away.

Not B either. Exposed apolar surface is the one axis on which R104W and R104Q separate decisively (§6), and I initially read that as support for hypothesis B. The cross-position control (§6a) removes it: benign position 34 gains 2.3× more apolar area on tryptophan substitution than position 104 does, dominant-negative position 121 gains little, and the DN-versus-benign split runs in the wrong direction (p = 1.000). The metric measures "a large hydrophobic residue was introduced at a partly-exposed site", not pathogenicity. Both hypothesis A and hypothesis B are refuted by the same class of control, a benign variant that scores at or above the pathogenic one.

Not C. Nothing here suggests the ΔΔG calculations were wrong. Both benign controls behave correctly, the two preps agree on every conclusion that clears its own noise floor, self-mutation controls are near-null where they should be, and the one metric that looked dramatic (tier-1 strain) failed its own reproducibility check and was discarded rather than reported. The negative ΔΔG result stands as a true statement about folding stability, the mechanistic explanation is that folding stability is the wrong observable, not that it was measured badly.

Why the global ΔΔG method missed R104W, stated precisely and within what the data support: the perturbation R104W causes is not a stability perturbation at all, not globally, and not locally. It leaves fold stability, secondary structure, backbone geometry, packing quality and carboxylate coordination in the same state a benign substitution does. Restricting the radius of a stability calculation therefore cannot recover it, and neither can the one surface observable tested here. The observable that separates R104W from benign substitutions has not yet been identified in this work. That is a weaker and more honest conclusion than either hypothesis A or B would have given, and it redirects effort: the remaining candidates are properties a monomer calculation cannot see at all, α-α interface geometry, glycosylation-state processing, or CaM-region effects, none of which is measurable in the assay this analysis used.

8. What would falsify this

1. A solvent-exposure measurement showing R104W does not add apolar surface in the assembled channel. The 28 Ų figure is measured on a multi-segment carve-out (NTD 49-140 plus chains 12-37, 166-192, 229-242, 422-433) that reproduces the local burial environment. A calculation on the complete 1395-residue channel, or on a cryo-EM structure of R104W, could revise it. If R104W's apolar surface is occluded in the full assembly, hypothesis B loses its structural basis here.
2. A co-immunoprecipitation or FRET titration showing R104Q associates with wild-type Naᵥ1.5 as avidly as R104W does. Hypothesis B predicts R104W > R104Q in α-α association propensity, since R104Q reduces exposed apolar surface. Equal association would break the link between apolar exposure and the dominant-negative effect. Note the Iamshanova caveat: this must not be done in an overexpression system.
3. A local-strain metric that separates V125L from R104W. If some better-chosen local observable cleanly ranks benign below pathogenic at these positions, hypothesis A returns. The specific failure documented here is that energy redistribution, carboxylate coordination, backbone displacement, packstat and buried-unsat all fail to do so.
4. A phenotype pattern that rescues apolar exposure. The cross-position control (§6a) kills it on two counts: benign position 34 ranks first and dominant-negative position 121 ranks fifth of seven. If R34W were found to be pathogenic and R121W's dominant-negative status were overturned, Wang 2020 already disputes the latter, the control would weaken. Both would have to go the same way, which is unlikely but is the specific evidence that would reopen hypothesis B.

9. Honest limitations

• A repacked-and-minimised model is not a physical ensemble. Every energy here is ref2015 on a locally-relaxed model, with the confound the task named. The self-mutation control quantifies it rather than removing it: the OpenMM noise floor is 6.4 REU of local |ΔE| for a chemically null mutation, which is why R104Q's +2.70 REU excess is reported as above-noise-but-small and R104K's +1.72 is reported as not resolved.
• No sampling of alternative backbone conformations. Cartesian minimisation relaxes the shell around the input backbone; it does not explore whether R104W would prefer a different local fold. A short MD run per variant would test that and was not performed.
• The apolar-exposure result is a static-structure measurement. It says a tryptophan at 104 presents apolar surface in this conformational ensemble. It does not measure association free energy, does not model a second Naᵥ1.5 protomer, and cannot distinguish genuine interface formation from non-specific stickiness, a distinction Iamshanova et al. show is experimentally fraught in this exact protein.
• Gly-X-Gly normalisation is a convention. Absolute Ų values are reported alongside the normalised fractions so any other reference state can be substituted.
• The carve-out is not the full channel. R104 reads 15.1% RSA in the ensemble carve-out versus 11.6% in the complete 1395-residue chain A, the DI-region segments retained (166-192, 229-242, 422-433) reproduce most but not all of the body-donated burial. Apolar exposure for R104W is therefore a slight over-estimate in absolute terms; the W-vs-Q direction, which is the claim, is unaffected because both are measured in the same environment.
• R104F/R104Y/R104A have no clinical or functional data. They are included as a chemical series to separate volume from chemistry, and they succeed at that. They are not answer-key members and are not treated as such.
• Statistical significance ≠ physical significance, and this is load-bearing for the backbone result. 36 matched replicates make a 0.03 Å Cα difference formally significant. It is not structurally meaningful at 3.6 Å parent resolution and is not claimed to be.
• n = 6 for the position-104 rank correlations. ρ = -0.77 has p = 0.072, i.e. not significant at the conventional threshold. The claim rests on the benign-control failure (V125L) and the volume correlation, which are independent of that correlation's significance.
• Single energy function. All local energies are ref2015. The prior project finding that ThermoMPNN and RaSP disagree on R104W's sign is a warning that method choice matters; a second local-energy model was not run.

Part 2. An independent test of the hydrophobic hypothesis, by a different protocol

Why this was worth testing

Andrew Glazer, who measured R104Q's dominant-negative effect directly, described the mechanism as probably involving "multi-Nav1.5 complexes that are formed and deleterious interactions between wildtype and misfolded mutant Nav1.5's." That puts misfolding upstream of the interaction.

My own data contradict that at this residue. R104W is the least destabilising of all nineteen substitutions at position 104, and it is the one classified Pathogenic/Likely pathogenic with nine concordant ClinVar submitters. R104Q is clearly destabilising and is only Conflicting.

One reconciliation would have explained everything: what if tryptophan does not destabilise the fold, but instead exposes a hydrophobic surface patch that promotes subunit-to-subunit association? A dominant-negative effect with no misfolding required. The idea was independently plausible because Iamshanova et al. (Sci Rep 2026, PMID 42082654) document that overexpressed Nav1.5 is predisposed to hydrophobic stickiness that mimics genuine interaction.

A prediction was recorded before any measurement: that the ring would bury rather than expose, filling the cavity the arginine vacated, and that the hypothesis would fail on that basis. That prediction was wrong. The hypothesis failed anyway, for a different and better reason.

Method

Twelve OpenMM-relaxed frames of 8VYJ chain A in assembled-channel context (chains A-E present; the NTD is chain B, residues 49-140). At each target position: PyRosetta MutateResidue, repack the 10 Å CA shell with RestrictToRepacking and IncludeCurrent, then minimise side chains and backbone in that shell (LBFGS, two repack/minimise passes). Solvent-accessible area from Bio.PDB ShrakeRupley at atom level, hydrophobic defined as carbon plus sulphur, side chain defined as excluding N, CA, C and O.

Validation before use. ShrakeRupley reproduced this work's independently measured accessibilities on the same structure: R104 13.4% against 11.6%, D82 8.0% against 8.5%, across 171 residues with a median of 45.5% and a wide spread. This check exists because a hand-rolled accessibility routine written earlier in the project returned near-zero for every residue, a consequence of unfiltered hydrogens where the PDB element column is blank.

The clash that nearly produced a false positive

The first pass used repacking only, without minimisation, and appeared to support the hypothesis strongly: aromatics exposed far more hydrophobic carbon than wild type or the polar substitutions, surviving size normalisation, with W versus WT at +15.80 Ų (paired Wilcoxon p = 0.0005).

Then the check for that clash:

A repulsive term of 197 is a severe steric clash, not a comfortably surface-exposed ring. Side-chain repacking alone cannot accommodate an aromatic at position 104; the rotamer is shoved outward and left clashing, and the "exposed area" was largely that clash. Those numbers were discarded.

This is a reusable protocol warning, though it needs care in how it is stated. Running the same test at seven positions shows that after full repack plus minimisation the residual strain from tryptophan is modest everywhere, and lowest of all at position 104 (excess fa_rep +0.70, against +4.21 at position 125, +3.30 at 84, +2.97 at 124). So the +197 was a packing failure of the fixed-backbone protocol, not a physical impossibility: once the backbone can relax, tryptophan is accommodated comfortably at 104. The warning is about protocol, not about the position.

That distinction cuts against an appealing but wrong inference. It would be tempting to read the clash as evidence that R104W is structurally disruptive after all, and therefore that ThermoMPNN's near-zero score is a packing blindness. The control says otherwise: position 104 accommodates tryptophan better than five of the six comparison positions. ThermoMPNN's low score for R104W looks correct, not naive.

After repack plus minimisation, fa_rep returns to baseline: Trp 1.58, Phe 1.46, Tyr 1.24 against wild type 0.84. Only these clash-verified poses were measured.

What the corrected measurement shows

The effect survives the clash correction. It also survives the obvious size objection, and the cleanest framing does not need normalisation at all: total exposed side-chain area is nearly identical across wild type, Gln and Lys (34.2, 37.5 and 34.0 Ų). What changes is the chemical character of the exposed face.

Position 104 is partially exposed in every case. Wild type and the polar substitutions present a polar face to solvent; the aromatics present a carbon face of similar total size.

This refutes my own earlier explanation for R104W's low ΔΔG. I had attributed it to aromatic bulk refilling the vacated cavity (introduces-aromatic r = -0.639, p = 0.0057). A cavity-filling ring would be less exposed than the arginine. The tryptophan is more exposed: side-chain relative accessibility 31.9% against 16.6%. That explanation should be retired or heavily qualified wherever it appears.

The control that kills the hypothesis

The same protocol at seven real N-terminal positions, ranked by carbon area gained on substitution to tryptophan, against what is known about each position:

Two failures, either of which is sufficient:

The benign control ranks first. Position 34 gains three times more exposed hydrophobic area than position 104. If exposed hydrophobic surface drove pathogenicity, position 34 would be the most dangerous site in the domain. It is the one position known to tolerate substitution in healthy people.

A demonstrated dominant-negative variant gains nothing. R121W exposes essentially no additional carbon. The mechanism is not general even across the arginine-to-tryptophan variants it was invented to explain.

Position 104 is mid-pack, fourth of seven.

A second control, which sharpens the negative result

Because the clash raised the question of whether position 104 is unusually intolerant of aromatic substitution, the residual strain after full relaxation was measured at every control position:

Position 104 accommodates tryptophan better than four of the six comparison positions. There is no hidden steric penalty. This independently supports the negative result rather than undermining it: R104W really is a structurally benign substitution at this site, by physical packing as well as by the learned predictor, and it is nonetheless the variant with nine concordant pathogenic submissions.

Independent replication

This question was worked twice, in parallel and without shared code: once as described above (side-chain carbon area, 12 OpenMM frames, 8 Å patch definition), and once by a separate agent using its own protocol, its own patch definition, and full apolar surface rather than side-chain carbon only. The two runs agree on the refutation.

Spearman ρ = 0.750 (p = 0.052) between the two rankings. Both put the benign control at rank 1 and a demonstrated dominant-negative variant near the bottom. The two failure modes that kill the hypothesis are reproduced independently.

The one substantial disagreement is instructive rather than troubling. At position 124 the native residue is alanine with a single side-chain carbon, so substituting tryptophan adds nine. A metric counting side-chain carbons registers that differently from one measuring total apolar surface. Neither is wrong; they answer different questions, and the phenotype mismatch survives either.

The independent run also extended the analysis to the free N-terminal domain as well as the assembled channel, which matters because pre-assembly is when inter-subunit association would have to initiate. Position 104's rank improves there (2 of 7 rather than 4 of 7), which is the reading least favourable to refutation, and it was reported rather than buried. Even so, the benign control still ranks first in that context too (+80.9 Ų against position 104's +33.2), so the hypothesis fails in both contexts.

Verdict

Hypothesis refuted. Not by the size objection and not by the clash, both of which were controlled for. It fails the phenotype test, which is the one that matters.

The structural observation stands on its own terms: aromatic substitution at position 104 converts a polar solvent-facing surface into a partly hydrophobic one, and cannot be accommodated without backbone relaxation. Neither of those facts explains pathogenicity.

Two things worth keeping. The magnitude was never large enough to carry the claim: roughly 17 Ų is a sub-hotspot patch, where a real protein-protein interface buries 600 to 1000 Ų per side and a single hot-spot residue contributes 50 to 200 Ų. And frame-to-frame variation was 50 to 60% of the mean, so the exposure is strongly conformation-dependent rather than a fixed structural feature.

What would actually test the association hypothesis

Solvent accessibility on a monomer cannot answer it. The question requires the α-α interaction interface itself, and per Iamshanova et al. (Sci Rep 2026, PMID 42082654) that interface cannot be mapped in heterologous overexpression systems, because overexpressed Nav1.5 is predisposed to hydrophobic stickiness that mimics genuine interaction. Native-tissue crosslinking or a structure of the complex would be needed.

Which leaves the local-strain reconciliation, or the possibility that something in my own calculations is wrong. Both remain open.

Data: HYDROPHOBIC_EXPOSURE_RELAXED.csv, POS104_SIDECHAIN_RSA.csv, POS104_MIN_ENERGETICS.csv, POSITION_SPECIFICITY_CONTROL.csv, TRP_EXPOSURE_VS_PHENOTYPE.csv, ctrl_energetics.csv. Twelve frames for position 104, six for the control positions. Statistical error bars only; single relaxation protocol.

Part 3. No linear degradation or retention motif at Arg104

• A negative with a built-in control.*

Why this branch mattered

Gütter 2013 listed five candidate mechanisms for R104Q loss of function. this work has closed two of them. "Enhanced degradation" is one of the three that survive, and it is the one most directly testable from sequence: many degradation and endoplasmic-reticulum-retention signals are short linear motifs, so a variant that creates or destroys one has an obvious mechanism.

Result: R104 is not in any canonical motif

Scanning the canonical protein (NP_932173.1, 2016 aa, residue 104 = Arg, verified) for motifs spanning residue 104:

R104 is an isolated arginine. Its local sequence is NKGKTIF-R-FSATNAL, flanked by hydrophobics, not by basic residues. Since Arg→Gln can only destroy arginine-dependent motifs, and there are none to destroy, R104Q cannot act through this class of signal.

The scan was sanity-checked rather than trusted: the same regex finds 8 RxxL occurrences elsewhere in the protein, so the absence in this window is real rather than a broken pattern.

The control that makes this decisive

R121W is the project's other ΔΔG false negative, also an arginine, also in this domain, also pathogenic. If a linear retention motif explained pathogenicity here, the two should look alike.

They do not. R121 sits inside a dibasic pair (FHPI-RR-AAVKIL, R121-R122), which is a canonical retention-signal context. R104 sits alone.

So the two pathogenic arginines of this domain have opposite motif contexts, yet behave the same way: both pathogenic, both false negatives for folding ΔΔG, both with reduced current. A linear-motif mechanism cannot explain the pair, which argues against the class rather than just against R104.

What this does and does not close

Argues against: R104Q acting through a linear degron or a linear endoplasmic-reticulum retention signal.

Does not close the degradation branch. Degradation can be triggered by conformational quality control, misassembly recognised by chaperones, with no linear motif involved anywhere. That is in fact what the intracellular-dimer reconciliation would predict, since quality control there acts on an assembly state rather than a sequence feature. This result removes the simple version of the degradation hypothesis and leaves the conformational version untouched.

Running score on Gütter's five-way fork:

Four of five branches have been narrowed or closed, three of them, all from sequence and published data. The surviving branch is the one the coupled-gating literature already points at.

Sequence NP_932173.1. Motif consensus definitions from standard published forms; this is a sequence-level screen, not a proteomics measurement, and a real degron could exist without matching a canonical consensus.

Part 4. The N-terminal domain has no glycosylation sequon

• A clean negative with a real consequence.*

Why this was asked

Two 2026 papers converge on glycosylation as central to Naᵥ1.5 α-α association:

• Iamshanova et al. 2026 (PMID 42082654) report that heterologous Naᵥ1.5 interactions "depend on nascent N-linked glycosylation" and are supported by non-native intermolecular disulfide bonds.
• Li & Schmalzing 2026 (PPR1279120) report an intracellular antiparallel homodimer whose surface form is monomeric, with mature Golgi-processed glycosylation confirmed by Endo H / PNGase F.

If the dimer that quality control acts on depends on N-linked glycosylation, and if R104Q sits in or near a glycosylation determinant, that would be a direct mechanistic link from the variant to the dimer. It is a sequence question, answerable in minutes.

Result: there is no site to affect

The canonical N-glycosylation sequon is N-X-S/T where X ≠ P. Scanning residues 1-130 of the canonical sequence (NP_932173.1, 2016 aa, matching transcript NM_000335.5):

Four asparagines, zero sequons. The Naᵥ1.5 N-terminal domain carries no N-glycosylation site at all. Substituting position 104 with Gln, Trp or Lys neither creates nor destroys one.

What this rules out, and what it does not

Ruled out: any mechanism in which R104Q alters dimer behaviour by changing N-linked glycosylation of the N-terminal domain itself. There is nothing there to change.

Not ruled out, and this is the more interesting reading. Both papers report strong binding between full-length channel and the isolated N-terminal domain peptide. If that association is glycosylation-dependent yet the N-terminal domain is itself unglycosylated, then the glycosylation determinant must lie on the partner, the transmembrane regions, which carry the known Naᵥ1.5 glycosylation sites. That makes the interaction asymmetric: an unglycosylated N-terminal domain binding a glycosylated partner surface.

That asymmetry is a testable prediction rather than a conclusion, and it is not something this analysis establishes.

Why the negative is worth recording

It closes a plausible-sounding hypothesis in under an hour and prevents a longer investigation into "does R104Q disrupt a glycosylation signal." It also constrains the fourth reconciliation: whatever couples R104Q to the intracellular dimer, it is not a glycosylation-site effect in the domain where the variant lives.

Methods note, including one error caught

The first attempt used NP_000326.2, which returned 2015 residues rather than the expected 2016. That is isoform b, which lacks Q1077. Re-run against NP_932173.1 (isoform a, 2016 aa, matching the transcript whose CDS was verified as 210..6257 = 2016 codons). The two isoforms are identical across residues 1-130, so the result is unaffected, but the mismatch was checked rather than assumed, because a silent isoform substitution is exactly the kind of error that propagates.

Sequences from NCBI (NP_932173.1, NP_000326.2). Sequon definition N-X-S/T, X ≠ P. Companion sources: PMID 42082654; Europe PMC PPR1279120 (abstract only, closed access).

Part 5. The empagliflozin site and Arg104 are structurally independent

• A structural test of the empagliflozin lead, run on 8VYJ. Cheap, decisive, and it makes the therapeutic argument sharper in both directions.*

The question

Empagliflozin raises peak sodium current by binding Y1767, the local-anaesthetic site, the same site mexiletine uses, and mutating it abolishes both drugs' effect. R104Q sits in the N-terminal domain. Whether the drug could help depends on whether the two sites are coupled.

The site is intact, and it is far away

Checked against the 8VYJ cryo-EM structure (1,395 modelled residues, 12-1882):

The local-anaesthetic site is present and correctly composed. R104 to Y1767 is 50.7 Å Cα-Cα.

They share no structural neighbourhood at all

Residues within 12 Å of each site:

• R104 shell: 36 residues, segments 58-61, 78-84, 88-96, 101-107, 125, 128-129, 178-179, 185-188
• Y1767 shell: 32 residues, segments 398-410, 1466-1470, 1653-1660, 1760-1774
• Overlap: zero.

Why this is good news for the lead

The drug's mechanism cannot be blocked by the variant. Empagliflozin acts through a site that shares no contact surface with R104, so the N-terminal defect cannot interfere with binding or with the conformational change that follows. Whatever is wrong at position 104, the local-anaesthetic site is intact and available.

That is the best possible structural answer for transferability: the drug does not need the N-terminal domain to be functional in order to work.

Why it is also the problem

By the same argument, the drug cannot repair the defect. It has no contact with the damaged region. It would act by increasing delivery of whatever channel protein exists, mutant and wild-type alike.

In a heterozygote, that means more mutant subunit at the membrane. If the mechanism is dominant-negative interference occurring at the membrane, more mutant protein is the wrong direction. The structural independence that makes the drug robust to the variant is exactly what prevents it from being selective.

An incidental structural result

R104's 8 Å shell contains only its own domain (residues 60, 92-95, 102-106). Its nearest out-of-domain contacts sit at 8-12 Å: A178, R179, A185, F186, T187, F188, the start of the channel body.

So R104 is not buried in an isolated appendage, but nor is it in direct contact with the pore-forming regions. It sits near the domain boundary with second-shell reach into the channel body. That is consistent with the earlier finding that this residue is not N-terminal-domain-internal, and it is the structural basis for how a defect there could influence the whole channel without touching it directly.

What would falsify these conclusions

Each refutation rests on a control, and each control is the thing to attack.

The local-strain refutation would be overturned by a strain metric on which the benign controls R34C and V125L score below the dominant-negative variants. Mine did not; a better-constructed metric might.

The hydrophobic-surface refutation would be overturned by a cross-position test in which apolar gain does track phenotype, or by evidence that position 34 is not in fact benign. Both are checkable. The benign status of R34C rests on its appearance in unrelated healthy individuals, including in the report that first described R104Q (PMID 11960580).

The degron refutation would be overturned by a non-canonical or structurally-defined retention determinant at Arg104 that a linear motif scan cannot see, which is a real possibility and the reason I state the claim as being about linear motifs specifically.

The glycosylation conclusion would be overturned by a glycosylation site outside the canonical N-X-S/T sequon, or by evidence that the relevant sequon lies on an interaction partner rather than in this domain.

The drug-independence conclusion would be overturned by an allosteric path between the two sites, which a distance and shell analysis cannot exclude. Fifty Angstroms of separation with no shared shell residue argues against direct competition, not against long-range coupling.

Data availability

All primary data are public. The structure is Protein Data Bank entry 8VYJ, chain A, with cryo-EM map EMD-43662 for density-guided relaxation. The reference protein sequence is RefSeq NP_932173.1 and the transcript NM_000335.5. Paralogue sequences are the nine human Nav alpha subunits retrieved from RefSeq, accessions as listed in the relevant section. Variant classifications came from ClinVar via NCBI E-utilities. Published functional data are cited by PMID throughout. Solvent accessibility was computed with Bio.PDB ShrakeRupley; relaxation used Rosetta and OpenMM. Tool version numbers are not recorded in my working notes for every step and I report that gap rather than reconstruct it.

All derived tables are deposited as a single archive with a permanent identifier. The identifier is recorded in DATA_DOI.txt alongside this manuscript and should be cited as the data source. They comprise the per-model accessibility measurements, the seven-position tryptophan scan, the substitution profiles and the shell residue lists.

Competing interests

I am a heterozygous carrier of SCN5A p.Arg104Gln, the variant at the centre of this analysis, and I have a clinical diagnosis of Brugada syndrome. No funding was received.

Nothing here is clinical guidance for any person, including me. In particular, the empagliflozin section reports a structural relationship and is not a statement about whether any person should or should not take that drug. That decision belongs to a treating clinician.

References

1. Clatot J, et al. Dominant-negative effect of SCN5A N-terminal mutations through the interaction of Nav1.5 alpha-subunits. Cardiovasc Res 2012;96(1):53-63. PMID 22739120.
2. Wang Z, et al. Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5. Channels (Austin) 2020;14(1):268-286. PMID 32815768.
3. O'Neill MJ, et al. Dominant negative effects of SCN5A missense variants. Genet Med 2022;24(6):1238-1248. PMID 35305865.
4. Moreau A, et al. Mexiletine differentially restores the trafficking defects caused by two Brugada syndrome mutations. Front Pharmacol 2012;3:62. PMID 22529811.
5. Levy-Nissenbaum E, et al. Genetic analysis of Brugada syndrome in Israel: two novel mutations and possible genetic heterogeneity. Genet Test 2001;5(4):331-334. PMID 11960580.
6. Gutter C, Benndorf K, Zimmer T. Characterization of N-terminally mutated cardiac Na+ channels associated with long QT syndrome 3 and Brugada syndrome. Front Physiol 2013;4:153. PMID 23805106.
7. Protein Data Bank entry 8VYJ, human Nav1.5 cryo-EM structure, with map EMD-43662.
8. RefSeq NP_932173.1 (SCN5A protein) and NM_000335.5 (transcript).

Additional PMIDs cited at the point of use in the text are not repeated here.

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