The cardiac sodium channel is post-translationally modified by arginine methylation

The α subunit of the cardiac sodium channel (Na(v)1.5) is an essential protein in the initial depolarization phase of the cardiomyocyte action potential. Post-translational modifications such as phosphorylation are known to regulate Na(v)1.5 function. Here, we used a proteomic approach for the study of the post-translational modifications of Na(v)1.5 using tsA201 cells as a model system. We generated a stable cell line expressing Na(v)1.5, purified the sodium channel, and analyzed Na(v)1.5 by MALDI-TOF and LC-MS/MS. We report the identification of arginine methylation as a novel post-translational modification of Na(v)1.5. R513, R526, and R680, located in the linker between domains I and II in Na(v)1.5, were found in mono- or dimethylated states. The functional relevance of arginine methylation in Na(v)1.5 is underscored by the fact that R526H and R680H are known Na(v)1.5 mutations causing Brugada and long QT type 3 syndromes, respectively. Our work describes for the first time arginine methylation in the voltage-gated ion channel superfamily.     

A novel mutation in the SCN5A gene is associated with Brugada syndrome

Brugada syndrome (BS) is an inherited cardiac disorder associated with a high risk of sudden cardiac death and is caused by mutations in the SCN5A gene encoding the cardiac sodium channel alpha-subunit (Na(v)1.5). The aim of this study was to identify the genetic cause of familial BS and characterize the electrophysiological properties of a novel SCN5A mutation (W1191X). Four families and one patient with BS were screened for SCN5A mutations by PCR and direct sequencing. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in tsA201 cells, and the sodium currents (I(Na)) were analyzed using the whole-cell patch-clamp technique. A novel mutation, W1191X, was identified in a family with BS. Expression of the WT or the mutant channel (Na(v)1.5/W1191X) co-transfected with the beta(1)-subunit in tsA201 cells resulted in a loss of function of Na(v)1.5 channels. While voltage-clamp recordings of the WT channel showed a distinct acceleration of Na(v)1.5 activation and fast inactivation kinetics, the Na(v)1.5/W1191X mutant failed to generate any currents. Co-expression of the WT channel and the mutant channel resulted in a 50% reduction in I(Na). No effect on activation and inactivation were observed with this heterozygous expression. The W1191X mutation is associated with BS and resulted in the loss of function of the cardiac sodium channel.     

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.     

Modulation of the cardiac sodium channel Nav1.5 by fibroblast growth factor homologous factor 1B

We have previously shown that fibroblast growth factor homologous factor 1B (FHF1B), a cytosolic member of the fibroblast growth factor family, associates with the sensory neuron-specific channel Na(v)1.9 but not with the other sodium channels present in adult rat dorsal root ganglia neurons. We show in this study that FHF1B binds to the C terminus of the cardiac voltage-gated sodium channel Na(v)1.5 and modulates the properties of the channel. The N-terminal 41 amino acid residues of FHF1B are essential for binding to Na(v)1.5, and the conserved acidic rich domain (amino acids 1773-1832) in the C terminus of Na(v)1.5 is sufficient for association with this factor. Binding of the growth factor to recombinant wild type human Na(v)1.5 in human embryonic kidney 293 cells produces a significant hyperpolarizing shift in the voltage dependence of channel inactivation. An aspartic acid to glycine substitution at position 1790 of the channel, which underlies one of the LQT-3 phenotypes of cardiac arrythmias, abolishes the interaction of the Na(v)1.5 channel with FHF1B. This is the first report showing that interaction with a growth factor can modulate properties of a voltage-gated sodium channel.     

Engineering non-natural inhibitor sensitivity in protein tyrosine phosphatase H1

Protein tyrosine phosphatase H1, a member of the ubiquitous protein tyrosine phosphatase (PTP) superfamily of enzymes, is an important signaling molecule, mutant forms of which have been found in human colorectal cancers. Selective PTPH1 inhibitors would be valuable tools for investigating PTPH1's roles in cellular regulation. However, no PTPH1-specific inhibitors are known. To identify target-selective inhibitors of human PTPH1, we have redesigned a PTPH1/inhibitor interface. Structure-based protein design was used to identify two amino-acid residues, isoleucine 846 and methionine 883, that control PTPH1's sensitivity to oxalylaminoindole PTP inhibitors. Mutation of residues 846 and 883 to alanine and glycine, respectively, conferred novel inhibitor sensitivity onto PTPH1. From a small panel of putative inhibitors, compounds that potently and selectively target the inhibitor-sensitized PTPH1 mutants were identified.     

PTPH1 is a predominant protein-tyrosine phosphatase capable of interacting with and dephosphorylating the T cell receptor zeta subunit

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor zeta subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor zeta subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR zeta substrate (specific activity ranging from 0.23 to 40 pmol/min/microg). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-zeta. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR zeta ITAMs, with PTPH1 directly dephosphorylating zeta. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR zeta.     

A mutation in telethonin alters Nav1.5 function

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na(+) channels in excitable cells results in influx of Na(+) and cellular depolarization. The function of Na(v)1.5, an Na(+) channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming alpha-subunit of this channel is encoded by SCN5A. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCN5A are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na(+) channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Na(v)1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Na(v)1.5 kinetics and may play a role in intestinal pseudo-obstruction.     

Modulation of Nav1.5 channel function by an alternatively spliced sequence in the DII/DIII linker region

In the present study, we identified a novel splice variant of the human cardiac Na(+) channel Na(v)1.5 (Na(v)1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Na(v)1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Na(v)1.5d was not detected in the mouse heart, indicating that alternative splicing of Na(v)1.5 is species-dependent. In HEK293 cells, splice variant Na(v)1.5d generated voltage-dependent Na(+) currents that were markedly reduced compared with wild-type Na(v)1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Na(v)1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Na(v)1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Na(v)1.5.     

Nav1.5-dependent persistent Na+ influx activates CaMKII in rat ventricular myocytes and N1325S mice

Late Na(+) current (I(NaL)) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are both increased in the diseased heart. Recently, CaMKII was found to phosphorylate the Na(+) channel 1.5 (Na(v)1.5), resulting in enhanced I(NaL). Conversely, an increase of I(NaL) would be expected to cause elevation of intracellular Ca(2+) and activation of CaMKII. However, a relationship between enhancement of I(NaL) and activation of CaMKII has yet to be demonstrated. We investigated whether Na(+) influx via Na(v)1.5 leads to CaMKII activation and explored the functional significance of this pathway. In neonatal rat ventricular myocytes (NRVM), treatment with the I(NaL) activators anemone toxin II (ATX-II) or veratridine increased CaMKII autophosphorylation and increased phosphorylation of CaMKII substrates phospholamban and ryanodine receptor 2. Knockdown of Na(v)1.5 (but not Na(v)1.1 or Na(v)1.2) prevented ATX-II-induced CaMKII phosphorylation, providing evidence for a specific role of Na(v)1.5 in CaMKII activation. In support of this view, CaMKII activity was also increased in hearts of transgenic mice overexpressing a gain-of-function Na(v)1.5 mutant (N(1325)S). The effects of both ATX-II and the N(1325)S mutation were reversed by either I(NaL) inhibition (with ranolazine or tetrodotoxin) or CaMKII inhibition (with KN93 or autocamtide 2-related inhibitory peptide). Furthermore, ATX-II treatment also induced CaMKII-Na(v)1.5 coimmunoprecipitation. The same association between CaMKII and Na(v)1.5 was also found in N(1325)S mice, suggesting a direct protein-protein interaction. Pharmacological inhibitions of either CaMKII or I(NaL) also prevented ATX-II-induced cell death in NRVM and reduced the incidence of polymorphic ventricular tachycardia induced by ATX-II in rat perfused hearts. Taken together, these results suggest that a Na(v)1.5-dependent increase in Na(+) influx leads to activation of CaMKII, which in turn phosphorylates Na(v)1.5, further promoting Na(+) influx. Pharmacological inhibition of either CaMKII or Na(v)1.5 can ameliorate cardiac dysfunction caused by excessive Na(+) influx.     

Identification of the cell cycle regulator VCP (p97/CDC48) as a substrate of the band 4.1-related protein-tyrosine phosphatase PTPH1.

The human band 4.1-related protein-tyrosine phosphatase PTPH1 was introduced into NIH3T3 cells under the control of a tetracycline-repressible promoter. Ectopic expression of wild type PTPH1 dramatically inhibited cell growth, whereas a catalytically impaired mutant showed no effect. To identify the direct target of PTPH1 in the cell, we generated a substrate-trapping mutant, in which an invariant aspartate residue was changed to alanine (D811A in PTPH1). The PTPH1-D811A mutant trapped primarily a 97-kDa tyrosine-phosphorylated protein, which was determined to be VCP (also named p97 or yeast CDC48), from various cell lysates in vitro. However, when expressed in mammalian cells, the D811A mutant was observed to contain high levels of phosphotyrosine and did not trap substrates. Mutation of tyrosine 676 to phenylalanine (Y676F) in the PTPH1-D811A mutant led to a marked reduction in phosphotyrosine content. Furthermore, this double mutant specifically trapped VCP in vivo and recognized the C-terminal tyrosines of VCP, whose phosphorylation is important for cell cycle progression in yeast. Like wild type PTPH1, this double mutant also inhibited cell proliferation. Moreover, induction of wild type PTPH1 resulted in specific dephosphorylation of VCP without changing the overall phosphotyrosine profile of the cells. VCP has been implicated in control of a variety of membrane functions, including membrane fusions, and is a regulator of the cell cycle. Our results suggest that PTPH1 may exert its effects on cell growth through dephosphorylation of VCP, thus implicating tyrosine phosphorylation as an important regulator of VCP function.     

Colocalization of voltage-gated Na+ channels with the Na+/Ca2+ exchanger in rabbit cardiomyocytes during development

Reverse-mode activity of the Na(+)/Ca(2+) exchanger (NCX) has been previously shown to play a prominent role in excitation-contraction coupling in the neonatal rabbit heart, where we have proposed that a restricted subsarcolemmal domain allows a Na(+) current to cause an elevation in the Na(+) concentration sufficiently large to bring Ca(2+) into the myocyte through reverse-mode NCX. In the present study, we tested the hypothesis that there is an overlapping expression and distribution of voltage-gated Na(+) (Na(v)) channel isoforms and the NCX in the neonatal heart. For this purpose, Western blot analysis, immunocytochemistry, confocal microscopy, and image analyses were used. Here, we report the robust expression of skeletal Na(v)1.4 and cardiac Na(v)1.5 in neonatal myocytes. Both isoforms colocalized with the NCX, and Na(v)1.5-NCX colocalization was not statistically different from Na(v)1.4-NCX colocalization in the neonatal group. Western blot analysis also showed that Na(v)1.4 expression decreased by sixfold in the adult (P < 0.01) and Na(v)1.1 expression decreased by ninefold (P < 0.01), whereas Na(v)1.5 expression did not change. Although Na(v)1.4 underwent large changes in expression levels, the Na(v)1.4-NCX colocalization relationship did not change with age. In contrast, Na(v)1.5-NCX colocalization decreased ～50% with development. Distance analysis indicated that the decrease in Na(v)1.5-NCX colocalization occurs due to a statistically significant increase in separation distances between Na(v)1.5 and NCX objects. Taken together, the robust expression of both Na(v)1.4 and Na(v)1.5 isoforms and their colocalization with the NCX in the neonatal heart provides structural support for Na(+) current-induced Ca(2+) entry through reverse-mode NCX. In contrast, this mechanism is likely less efficient in the adult heart because the expression of Na(v)1.4 and NCX is lower and the separation distance between Na(v)1.5 and NCX is larger.     

Sodium current function in adult and aged canine atrial cells

The incidence of atrial fibrillation increases with age, but it is unknown whether there are changes in the intrinsic function of Na+ currents in cells of the aged atria. Thus, we studied right (RA) and left (LA) atrial cells from two groups of dogs, adult and aged (>8 yr), to determine the change in Na+ currents with age. In this study all dogs were in normal sinus rhythm. Whole cell voltage clamp techniques were used to compare the Na+ currents in the two cell groups. Immunocytochemical studies were completed for the Na+ channel protein Na(v)1.5 to determine whether there was structural remodeling of this protein with age. In cells from aged animals, we found that Na+ currents are similar to those we measured in adult atria. However, Na+ current (I(Na)) density of the aged atria differed depending on the atrial chamber with LA cell currents being larger than RA cell currents. Thus with age, the difference in I(Na) density between atrial chambers remains. I(Na) kinetic differences between aged and adult cells included a significant acceleration into the inactivated state and an enhanced use-dependent decrease in peak current in aged RA cells. Finally, there is no structural remodeling of the cardiac Na+ channel protein Na(v)1.5 in the aged atrial cell. In conclusion, with age there is no change in I(Na) density, but there are subtle kinetic differences contributing to slight enhancement of use dependence. There is no structural remodeling of the fast Na+ current protein with age.     

The intracellular domain of the beta 2 subunit modulates the gating of cardiac Na v 1.5 channels

We have previously shown that the transmembrane segment plus either the extracellular or intracellular domain of the beta1 subunit are required to modify cardiac Na(v)1.5 channels. In this study, we coexpressed the intracellular domain of the beta2 subunit in a beta1/beta2 chimera with Na(v)1.5 channels in Xenopus oocytes and obtained an atypical recovery behavior of Na(v)1.5 channels not reported before for other Na(+) channels: Recovery times of up to 20 ms at -120 mV produced a similar fast recovery as observed for Na(v)1.5/beta1 channels, but the current amplitude decreased again at longer recovery times and reached a steady-state level after 1-2 s with current amplitudes of only 43 +/- 2% of the value at 20 ms. Current reduction was accompanied by slowed inactivation and by a shift of steady-state activation toward depolarized potentials by 9 mV. All effects were reversible and they were not seen when deleting the beta2 intracellular domain. These results describe the first functional effects of a beta2 subunit region on Na(v)1.5 channels and suggest a novel closed state in cardiac Na(+) channels accessible at hyperpolarized potentials.     

Irreversible block of cardiac mutant Na+ channels by batrachotoxin

Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.     

RPTPmu and protein tyrosine phosphorylation regulate K(+) channel mRNA expression in adult cardiac myocytes

Previously, we reported that cell-cell contact regulates K(+) channel mRNA expression in cultured adult rat cardiac myocytes. Here we show that exposing cardiac myocytes to tyrosine kinase inhibitors (genistein, tyrphostin A25), but not inactive analogs, prevents downregulation of Kv1.5 mRNA and upregulation of Kv4.2 mRNA normally observed when they are cultured under low-density conditions. Furthermore, cardiac myocytes cocultured with cells that endogenously (Mv 1 Lu) or heterologously (Chinese hamster ovary cells) express the receptor-type protein tyrosine phosphatase mu (RPTPmu) display Kv1.5 mRNA levels paralleling that which was observed in myocytes cultured under high-density conditions and in intact tissue. In contrast, myocytes cocultured with control cells failed to produce this response. Finally, it is shown that Kv4.2 mRNA expression is unaffected by RPTPmu. These findings reveal that multiple tyrosine phosphorylation-dependent mechanisms control cardiac myocyte K(+) channel genes. Furthermore, we conclude that RPTPmu specifically regulates cardiac myocyte Kv1.5 mRNA expression. Thus this receptor protein tyrosine phosphatase may be important in responses to pathological conditions associated with the loss of cell-cell interactions in the heart.     

Molecular identity of the late sodium current in adult dog cardiomyocytes identified by Nav1.5 antisense inhibition

Late Na(+) current (I(NaL)) is a major component of the action potential plateau in human and canine myocardium. Since I(NaL) is increased in heart failure and ischemia, it represents a novel potential target for cardioprotection. However, the molecular identity of I(NaL) remains unclear. We tested the hypothesis that the cardiac Na(+) channel isoform (Na(v)1.5) is a major contributor to I(NaL) in adult dog ventricular cardiomyocytes (VCs). Cultured VCs were exposed to an antisense morpholino-based oligonucleotide (Na(v)1.5 asOligo) targeting the region around the start codon of Na(v)1.5 mRNA or a control nonsense oligonucleotide (nsOligo). Densities of both transient Na(+) current (I(NaT)) and I(NaL) (both in pA/pF) were monitored by whole cell patch clamp. In HEK293 cells expressing Na(v)1.5 or Na(v)1.2, Na(v)1.5 asOligo specifically silenced functional expression of Na(v)1.5 (up to 60% of the initial I(NaT)) but not Na(v)1.2. In both nsOligo-treated controls and untreated VCs, I(NaT) and I(NaL) remained unchanged for up to 5 days. However, both I(NaT) and I(NaL) decreased exponentially with similar time courses (tau = 46 and 56 h, respectively) after VCs were treated with Na(v)1.5 asOligo without changes in 1) decay kinetics, 2) steady-state activation and inactivation, and 3) the ratio of I(NaL) to I(NaT). Four days after exposure to Na(v)1.5 asOligo, I(NaT) and I(NaL) amounted to 68 +/- 6% (mean +/- SE; n = 20, P < 0.01) and 60 +/- 7% (n = 11, P < 0.018) of those in VCs treated by nsOligo, respectively. We conclude that in adult dog heart Na(v)1.5 sodium channels have a "functional half-life" of approximately 35 h (0.69tau) and make a major contribution to I(NaL).     

Molecular basis of isoform-specific micro-conotoxin block of cardiac,skeletal muscle,and brain Na+ channels

mu-Conotoxins (mu-CTXs) block skeletal muscle Na(+) channels with an affinity 1-2 orders of magnitude higher than cardiac and brain Na(+) channels. Although a number of conserved pore residues are recognized as critical determinants of mu-CTX block, the molecular basis of isoform-specific toxin sensitivity remains unresolved. Sequence comparison of the domain II (DII) S5-S6 loops of rat skeletal muscle (mu1, Na(v)1.4), human heart (hh1, Na(v)1.5), and rat brain (rb1, Na(v)1.1) Na(+) channels reveals substantial divergence in their N-terminal S5-P linkers even though the P-S6 and C-terminal P segments are almost identical. We used Na(v)1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Na(v)1.1 and Na(v)1.5. The Na(v)1.4-->Na(v)1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Na(v)1.4 numbering) reduced block of Na(v)1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more "cardiac-like." Conversely, an Na(v)1.5--> Na(v)1.4 chimeric construct in which the Na(v)1.4 DII S5-P linker replaces the analogous segment in Na(v)1.5 showed enhanced mu-CTX block. However, these variant determinants are conserved between Na(v)1.1 and Na(v)1.4 and thus cannot explain their different sensitivities to mu-CTX. Comparison of their sequences reveals two variants at Na(v)1.4 positions 729 and 732: Ser and Asn in Na(v)1.4 compared with Thr and Lys in Na(v)1.1, respectively. The double mutation S729T/N732K rendered Na(v)1.4 more "brain-like" (30-fold downward arrow in block), and the converse mutation T925S/K928N in Na(v)1.1 reproduced the high affinity blocking phenotype of Na(v)1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in mu-CTX binding, thus shaping isoform-specific toxin sensitivity.