Figure 2 - available via license: CC BY
Content may be subject to copyright.
![Activation and inactivation curves of wild-type (WT) and p.V445M mutant channels in the CHO-K1 cells. (A) Sample sweeps of the activation (left panel) and inactivation (right panel) curves of the WT and p.V445M mutant channels with 0.1 mM Na v β4 peptide. For the current-voltage (I-V) plot, each patch cell configuration was held at −120 mV, and step test pulses were from −140 to +40 mV for 100 ms in each 5 mV escalation and plotted against test pulse voltage where each with 5 potential step. For the inactivation curve of Na + currents, the maximal currents at +10 mV were documented after 100 ms prepulses at various voltages. (B) The activation and fast steady-state inactivation curves of the WT and p.V445M mutant channels are fitted with a Boltzmann function according to the following formula: 1 / [1 + exp((V h − V) / k)], where V is the membrane potential, V h and k are −25.79 ± 1.5 mV and 6.67 ± 0.43 for the activation curve and −63.99 ± 2.17 mV and −8.93 ± 0.33 for the inactivation curve in the WT Na v 1.4 channel; −36.53 ± 1.4 mV and 6.61 ± 0.23 for the activation curve and −73.81 ± 1.2 mV and −9.11 ± 0.21 for the inactivation curve in the p.V445M mutant channel, respectively (n = 9 for each measurement).](publication/340533693/figure/fig1/AS:878408800157696@1586440386156/Activation-and-inactivation-curves-of-wild-type-WT-and-pV445M-mutant-channels-in-the.png)
Activation and inactivation curves of wild-type (WT) and p.V445M mutant channels in the CHO-K1 cells. (A) Sample sweeps of the activation (left panel) and inactivation (right panel) curves of the WT and p.V445M mutant channels with 0.1 mM Na v β4 peptide. For the current-voltage (I-V) plot, each patch cell configuration was held at −120 mV, and step test pulses were from −140 to +40 mV for 100 ms in each 5 mV escalation and plotted against test pulse voltage where each with 5 potential step. For the inactivation curve of Na + currents, the maximal currents at +10 mV were documented after 100 ms prepulses at various voltages. (B) The activation and fast steady-state inactivation curves of the WT and p.V445M mutant channels are fitted with a Boltzmann function according to the following formula: 1 / [1 + exp((V h − V) / k)], where V is the membrane potential, V h and k are −25.79 ± 1.5 mV and 6.67 ± 0.43 for the activation curve and −63.99 ± 2.17 mV and −8.93 ± 0.33 for the inactivation curve in the WT Na v 1.4 channel; −36.53 ± 1.4 mV and 6.61 ± 0.23 for the activation curve and −73.81 ± 1.2 mV and −9.11 ± 0.21 for the inactivation curve in the p.V445M mutant channel, respectively (n = 9 for each measurement).
Source publication
Myotonia congenita (MC) is a rare disorder characterized by stiffness and weakness of the limb and trunk muscles. Mutations in the SCN4A gene encoding the alpha-subunit of the voltage-gated sodium channel Nav1.4 have been reported to be responsible for sodium channel myotonia (SCM). The Nav1.4 channel is expressed in skeletal muscles, and its relat...
Contexts in source publication
Context 1
... compared the electrophysiological characteristics of the basic gating control of the WT and p.V445M mutant Na v 1.4 channels. Figure 2A shows the sweeps after the depolarization via either an escalating (from −140 to +40 mV, left panel of Figure 2A) or constant (+10 mV, right panel of Figure 2A) depolarizing current. The activation and inactivation curves of the WT and p.V445M mutant channels were fitted with the following Boltzmann function: ...
Context 2
... compared the electrophysiological characteristics of the basic gating control of the WT and p.V445M mutant Na v 1.4 channels. Figure 2A shows the sweeps after the depolarization via either an escalating (from −140 to +40 mV, left panel of Figure 2A) or constant (+10 mV, right panel of Figure 2A) depolarizing current. The activation and inactivation curves of the WT and p.V445M mutant channels were fitted with the following Boltzmann function: ...
Context 3
... compared the electrophysiological characteristics of the basic gating control of the WT and p.V445M mutant Na v 1.4 channels. Figure 2A shows the sweeps after the depolarization via either an escalating (from −140 to +40 mV, left panel of Figure 2A) or constant (+10 mV, right panel of Figure 2A) depolarizing current. The activation and inactivation curves of the WT and p.V445M mutant channels were fitted with the following Boltzmann function: ...
Context 4
... V is the membrane potential and k is the slope factor. From the equation, V h and k were −25.79 ± 1.5 mV and 6.67 ± 0.43 for the activation curve and −63.99 ± 2.17 mV and −8.93 ± 0.33 for the fast steady-state inactivation curve in the WT channel, respectively and −36.53 ± 1.4 mV and 6.61 ± 0.23 for the activation curve and −73.81 ± 1.2 mV and −9.11 ± 0.21 for the inactivation curve in the p.V445M mutant channel, respectively (n = 9; p < 0.05; Figure 2B). Both the activation and fast steady-state inactivation curves in the p.V445M mutant channel were hyperpolarization-shifted compared with those in the WT channel. ...
Context 5
... the activation and fast steady-state inactivation curves in the p.V445M mutant channel were hyperpolarization-shifted compared with those in the WT channel. The slopes of both gating curves of the Na v 1.4 channels remained grossly unchanged ( Figure 2B). We further analyzed the window currents, which were defined by the area under the activation and fast steady-state of the inactivation curves. ...
Context 6
... activation curves of the transient Na + currents and those of the resurgent Na + currents of the WT and mutant channels were replotted. The activation curve of the transient sodium current showed a hyperpolarizing shift in the mutant channel ( Figure 5C), which is in line with our previous study ( Figure 2B). However, the activation curve of the resurgent Na + currents moved to a more depolarized membrane voltage range in the mutant channel compared with the WT channel ( Figure 5C). ...
Similar publications
In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker disease) experience debilitating bouts of transient weakness that remain poorly understood despite years of study. We performed intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechani...
Citations
... The main symptoms of these channelopathies, i.e. muscle stiffness and myotonia, episodic weakness and cold sensitivity, may variably combine to result in one of the three phenotypes; however, there are several reports of mutations causing mild, incomplete, or overlapping phenotypes, as well as unusual heat sensitivity or warm-up phenomenon, a feature typical of chloride channel (CLCN1 gene) myotonia [3][4][5][6][7][8][9][10] but increasingly seen in sodium channel myotonia. The electrophysiological behavior of mutated channels has been analyzed in detail for a number of mutations [11][12][13][14][15], disclosing impaired inactivation and/or enhanced activation as the main gating mechanisms causing the channel overactivity that determines hyperexcitability and muscle stiffness. The specific biophysical defect may also constitute a basis for a more selective therapeutic approach [16][17][18]. ...
Background:
The nondystrophic myotonias are rare muscle hyperexcitability disorders caused by gain-of-function mutations in the SCN4A gene or loss-of-function mutations in the CLCN1 gene. Clinically, they are characterized by myotonia, defined as delayed muscle relaxation after voluntary contraction, which leads to symptoms of muscle stiffness, pain, fatigue, and weakness. Diagnosis is based on history and examination findings, the presence of electrical myotonia on electromyography, and genetic confirmation.
Methods:
Next-generation sequencing including the CLCN1 and SCN4A genes was performed in patients with clinical neuromuscular disorders. Electromyography, Short Exercise Test, in vivo and in vitro electrophysiology, site-directed mutagenesis and heterologous expression were collected.
Results:
A heterozygous point mutation (c.1775C > T, p.Thr592Ile) of muscle voltage-gated sodium channel α subunit gene (SCN4A) has been identified in five female patients over three generations, in a family with non-dystrophic myotonia. The muscle stiffness and myotonia involve mainly the face and hands, but also affect walking and running, appearing early after birth and presenting a clear cold sensitivity. Very hot temperatures, menstruation and pregnancy also exacerbate the symptoms; muscle pain and a warm-up phenomenon are variable features. Neither paralytic attacks nor post-exercise weakness has been reported. Muscle hypertrophy with cramp-like pain and increased stiffness developed during pregnancy. The symptoms were controlled with both mexiletine and acetazolamide. The Short Exercise Test after muscle cooling revealed two different patterns, with moderate absolute changes of compound muscle action potential amplitude.
Conclusions:
The p.Thr592Ile mutation in the SCN4A gene identified in this Sardinian family was responsible of clinical phenotype of myotonia.
... SCN4A encodes the α-subunit of the skeletal voltage-gated sodium channel, Nav1.4. This channel, expressed in skeletal muscle, in uences muscle excitability 39 . Mutations in SCN4A lead to nondystrophic myotonia and/or periodic paralysis. ...
Objective: Investigating the Role of Epicardial Adipose Tissue as a Catalyst for Reversal and Reconstruction of Atrial Myocardial Cells in the Context of Dialogue with Atrial Myocardial Cells. Implications for Breakthroughs in Preventing Paroxysmal Atrial Fibrillation Progression.
Methods: We obtained three datasets (GSE41177, GSE31821, and GSE135455) associated with atrial fibrillation (AF) from the Gene Expression Omnibus (GEO) database, which were subsequently merged for comprehensive analysis. Differentially expressed genes (DEGs) were identified using the "limma" package in the R software. Candidate AF genes were selected through machine learning techniques, including the LASSO regression algorithm and SVM-RFE algorithm. The diagnostic efficacy of these genes was evaluated using Receiver Operating Characteristic (ROC) curves. Additionally, CIBERSORT was employed to investigate the proportions of infiltrating immune cells in each sample, while the Pearson method was applied to examine the correlation between genes and immune cells. Further validation of the DEGs were performed by PCR in atrial fibrillation rats.
Results: A total of 310 Differentially Expressed Genes (DEGs) were identified in atrial cardiomyocytes with epicardial adipose tissue. Using the LASSO regression and SVM-RFE algorithms, ID1, SCN4A, COL4A5, COLEC11, and SNAI2 were pinpointed as key genes associated with Atrial Fibrillation (AF). In both the training and validation datasets, these genes exhibited robust effectiveness. The immune infiltration analysis revealed that, in comparison to sinus rhythm (SR), atrial samples from patients with AF exhibited higher levels of neutrophils, while T cells follicular helper were relatively lower. Correlation analysis highlighted significant associations between ID1, SCN4A, COL4A5, COLEC11, SNAI2, and infiltrating immune cells. The outcomes of the RT- qPCR analysis in our investigation were consistent with the findings of bioinformatics analysis.
Conclusions: In summary, this study posits that ID1, SCN4A, COL4A5, COLEC11, and SNAI2 emerge as pivotal genes in Atrial Fibrillation (AF), exhibiting correlation with infiltrating immune cells. Furthermore, it underscores the indispensable roles played by infiltrating immune cells in the context of AF.
... On the other hand, PMC arises due to a mutation in the sodium channel gene (SCN4A), impairing rapid sodium channel inactivation during repolarization. The influx of numerous Na + ions through Nav1.4 elevates the equilibrium potential of Na + and the magnitude of myocyte action potential depolarization [22,23]. Consequently, potential amplitudes rise, with these dual mechanisms synergistically generating giant-amplitude potentials. ...
BACKGROUND
Paramyotonia congenita (PMC) stands as a rare sodium channelopaty of skeletal muscle, initially identified by Eulenburg. The identification of PMC often relies on electromyography (EMG), a diagnostic technique. The child’s needle EMG unveiled trains of myotonic discharges with notably giant amplitudes, alongside irregular wave trains of myotonic discharges. This distinctive observation had not surfaced in earlier studies.
CASE SUMMARY
We report the case of a 3-year-old female child with PMC, who exhibited laryngeal stridor, muffled speech, myotonia from birth. Cold, exposure to cool water, crying, and physical activity exacerbated the myotonia, which was relieved in warmth, yet never normalized. Percussion myotonia was observable in bilateral biceps. Myotonia symptoms remained unchanged after potassium-rich food consumption like bananas. Hyperkalemic periodic paralysis was excluded. Cranial magnetic resonance imaging yielded normal results. Blood potassium remained within normal range, while creatine kinase showed slight elevation. Exome-wide genetic testing pinpointed a heterozygous mutation on chromosome SCN4A: c.3917G>A (p.G1306E). After a six-month mexiletine regimen, symptoms alleviated.
CONCLUSION
In this case revealed the two types of myotonic discharges, and had not been documented in other studies. We underscore two distinctive features: Giant-amplitude potentials and irregular waves.
... Depolarization of the Nav1.4 channel produces an action potential for muscle fiber contraction, and regular fast/slow inactivation process for refraining from myotonia (gain-of-function) or paralysis (loss-of-function) [33]. Functional analyses of mutated Nav1.4 channels revealed hyperpolarizing shift of the activation curve (p.V445M, p.I693T, p.T1313M, p.L1433R, and p.R1448H), leading to hyperexcitability with repetitive firing of muscle fiber membrane [34][35][36][37]; slowing of fast inactivation and acceleration of recovery from inactivation (p.G1306V, p.T1313M, p.L1433R, and p.R1448H) [33,34,36]; or prolonged recovery of slow inactivation (p.V445M and p.I693T) [35,37]. Otherwise, partial lossof-function effect, typically uncovered from paralysis-related SCN4A variants, was demonstrated from p.R225W, albeit shared by two NDM patients from current study and a previous report [38,39]. ...
... Depolarization of the Nav1.4 channel produces an action potential for muscle fiber contraction, and regular fast/slow inactivation process for refraining from myotonia (gain-of-function) or paralysis (loss-of-function) [33]. Functional analyses of mutated Nav1.4 channels revealed hyperpolarizing shift of the activation curve (p.V445M, p.I693T, p.T1313M, p.L1433R, and p.R1448H), leading to hyperexcitability with repetitive firing of muscle fiber membrane [34][35][36][37]; slowing of fast inactivation and acceleration of recovery from inactivation (p.G1306V, p.T1313M, p.L1433R, and p.R1448H) [33,34,36]; or prolonged recovery of slow inactivation (p.V445M and p.I693T) [35,37]. Otherwise, partial lossof-function effect, typically uncovered from paralysis-related SCN4A variants, was demonstrated from p.R225W, albeit shared by two NDM patients from current study and a previous report [38,39]. ...
Non-dystrophic myotonias (NDM) are rare skeletal muscle channelopathies, mainly linked to two voltage-gated ion channel genes, CLCN1 and SCN4A. The aim of this study is to identify the clinical and genetic features of patients with NDM in Japan. We collected a Japanese nationwide case series of patients with clinical diagnosis of NDM (1999–2021). Among 71 out of 88 pedigrees, using Sanger and next-generation sequencing targeting both CLCN1 and SCN4A genes, variants classified as pathogenic/likely pathogenic/unknown significance were detected from CLCN1 (31 probands), SCN4A (36 probands), or both genes (4 probands), and 11 of them were novel. Pedigrees carrying mono-allelic CLCN1 variants were more commonly seen than that with bi-allelic/double variants (24:7). Compared to patients with CLCN1 variants, patients harboring SCN4A variants showed younger onset age (5.64 ± 4.70 years vs. 9.23 ± 5.21 years), fewer warm-up phenomenon, but more paramyotonia, hyperCKemia, transient muscle weakness, and cold-induced myotonia. Haplotype analysis verified founder effects of the hot spot variants in both CLCN1 (p.T539A) and SCN4A (p.T1313M). This study reveals variants in CLCN1 and SCN4A from 80.7% of our case series, extending genetic spectrum of NDM, and would further our understanding of clinical similarity/diversity between CLCN1- and SCN4A-related NDM, as well as the genetic racial differences.
... Although the Na v β4 subunit contains 228 amino acids, only a short peptide from subunit β4 with approximately 14 residues was used in electrophysiological recording (KKLITFILKKTREK, the cytoplasmic tail of the entire β4 subunit) [27]. This peptide was dissolved in distilled water to make a 10 mM stock solution and then diluted into an intracellular solution to a final concentration of 0.1 mM [28,29]. ...
... Resurgent Na + currents can be observed in voltage-gated Na + channels with an internal Navβ4 peptide during the repolarization process [29,[31][32][33]. The traces of resurgent Na + currents at the repolarization (0 to −120 mV) stage in the mutant channels (p.V781I and p.A1737T) and WT are shown in Figure 4A. ...
... According to our previous studies, the Nav1.4 channel may have two different open states which govern the genesis of transient and resurgent Na + currents [29,33]. To evaluate the activation kinetics of both transient and resurgent currents, the cells were subjected to an incremental depolarization current (from −100 to +180 mV) for 50 ms, followed by repolarization at −60 mV for 150 ms. ...
Paramyotonia congenita (PMC) is a rare skeletal muscle disorder characterized by muscle stiffness upon repetitive exercise and cold exposure. PMC was reported to be caused by dominant mutations in the SCN4A gene encoding the α subunit of the Nav1.4 channel. Recently, we identified two missense mutations of the SCN4A gene, p.V781I and p.A1737T, in two PMC families. To evaluate the changes in electrophysiological properties caused by the mutations, both mutant and wild-type (WT) SCN4A genes were expressed in CHO-K1 and HEK-293T cells. Then, whole-cell patch-clamp recording was employed to study the altered gating of mutant channels. The activation curve of transient current showed a hyperpolarizing shift in both mutant Nav1.4 channels as compared to the WT channel, whereas there was a depolarizing shift in the fast inactivation curve. These changes confer to an increase in window current in the mutant channels. Further investigations demonstrated that the mutated channel proteins generate significantly larger resurgent currents as compared to the WT channel and take longer to attain the peak of resurgent current than the WT channel. In conclusion, the current study demonstrates that p.V781I and p.A1737T mutations in the Nav1.4 channel increase both the sustained and the resurgent Na+ current, leading to membrane hyperexcitability with a lower firing threshold, which may influence the clinical phenotype.
... The cells were grown and incubated under the culture condition as in our previous study [37,38]. Briefly, CHO-K1 cells were incubated in the F12K medium (Thermo Fisher Scientific, Waltham, MA, USA) with 5% CO 2 at 37 • C, supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 0.2% Normocin (InvivoGen, San Diego, CA, USA). ...
... Homology modeling and docking interaction of the Na v 1.4 channel with cannabidiol. (A) The homology model was built based on the X-ray crystal structures of human Na v 1.4 channels (PDB: 6AGF) using the software Discovery Studio 2020[37,38]. Four subunits of the Na v 1.4 channels homology modeling is shown. ...
Cannabidiol (CBD), one of the cannabinoids from the cannabis plant, can relieve the myotonia resulting from sodium channelopathy, which manifests as repetitive discharges of muscle membrane. We investigated the binding kinetics of CBD to Nav1.4 channels on the muscle membrane. The binding affinity of CBD to the channel was evaluated using whole-cell recording. The CDOCKER program was employed to model CBD docking onto the Nav1.4 channel to determine its binding sites. Our results revealed no differential inhibition of sodium current by CBD when the channels were in activation or fast inactivation status. However, differential inhibition was observed with a dose-dependent manner after a prolonged period of depolarization, leaving the channel in a slow-inactivated state. Moreover, CBD binds selectively to the slow-inactivated state with a significantly faster binding kinetics (>64,000 M−1 s−1) and a higher affinity (Kd of fast inactivation vs. slow-inactivation: >117.42 μM vs. 51.48 μM), compared to the fast inactivation state. Five proposed CBD binding sites in a bundle crossing region of the Nav1.4 channels pore was identified as Val793, Leu794, Phe797, and Cys759 in domain I/S6, and Ile1279 in domain II/S6. Our findings imply that CBD favorably binds to the Nav1.4 channel in its slow-inactivated state.
... The homology modeling procedure was performed using methods similar to those previously used [18][19][20]. A homology model of the WT Na v 1.4 channel encoded by the SCN4A gene was built from X-ray crystal structure data corresponding to the human voltage-gated Na v 1.4 channel (human Na v 1.4; PDB code: 6AGF) [21]. ...
... The amino acid sequence of the WT human Na v 1.4 channel was obtained from the UniProt database (P35499). The aligned sequences of the WT Na v 1.4, p.T1313M, and p.R1488H mutant channels were processed using Discovery Studio 2018 (Dassault Systèmes, Vélizy-Villacoublay, France) to generate the secondary structures and assign the relative positions [18][19][20]. ...
... For the current-voltage (I-V) plot, patch cell configurations were held at −120 mV and subjected to different test pulses increasing in 5-mV increments from −160 to +40 mV Biomedicines 2021, 9, 51 4 of 19 for approximately 100 ms [18][19][20]. The peak amplitude of inward Na + currents was plotted against test membrane potentials to give a I-V plot, including a progressive line between 0 and +40 mV. ...
Paramyotonia congenita (PMC) is a rare hereditary skeletal muscle disorder. The major symptom, muscle stiffness, is frequently induced by cold exposure and repetitive exercise. Mutations in human SCN4A gene, which encodes the α-subunit of Nav1.4 channel, are responsible for PMC. Mutation screening of SCN4A gene from two PMC families identified two missense mutations, p.T1313M and p.R1448H. To elucidate the electrophysiological abnormalities caused by the mutations, the p.T1313M, p.R1448H, and wild-type (WT) SCN4A genes were transient expressed on Chinese hamster ovary (CHO-K1) cells. The detailed study on the gating defects of the mutant channels using the whole-cell patch clamping technique was performed. The mutant Nav1.4 channels impaired the basic gating properties with increasing sustained and window currents during membrane depolarization and facilitated the genesis of resurgent currents during repolarization. The mutations caused a hyperpolarization shift in the fast inactivation and slightly enhanced the slow inactivation with an increase in half-maximal inactivation voltage. No differences were found in the decay kinetics of the tail current between mutant and WT channels. In addition to generating the larger resurgent sodium current, the time to peak in the mutant channels was longer than that in the WT channels. In conclusion, our results demonstrated that the mutations p.T1313M and p.R1448H in Nav1.4 channels can enhance fast inactivation, slow inactivation, and resurgent current, revealing that subtle changes in gating processes can influence the clinical phenotype.
NaV1.4 is a voltage-gated sodium channel subtype that is predominantly expressed in skeletal muscle cells. It is essential for producing action potentials and stimulating muscle contraction, and mutations in NaV1.4 can cause various muscle disorders. The discovery of the cryo-EM structure of NaV1.4 in complex with β1 has opened new possibilities for designing drugs and toxins that target NaV1.4. In this review, we summarize the current understanding of channelopathies, the binding sites and functions of chemicals including medicine and toxins that interact with NaV1.4. These substances could be considered novel candidate compounds or tools to develop more potent and selective drugs targeting NaV1.4. Therefore, studying NaV1.4 pharmacology is both theoretically and practically meaningful.
Plasminogen activator inhibitor-1 (PAI-1) is a specific and rapid-acting inhibitor of endogenous plasminogen activators (uPA and tPA). The global PAI-1 knockout mice (PAI-1KO) develop age-dependent cardiac-selective fibrosis, and young global PAI-1KO mice exhibit augmented susceptibility to developing cardiac fibrosis in response to hypertension. Here, we tested the hypothesis that cardiomyocyte PAI-1 is necessary to provide cardioprotective effects in a left ventricular pressure overload-induced murine model of cardiac hypertrophy and fibrosis using cardiomyocyte-specific PAI-1 knockout (cmPAI-1KO) mice. The results revealed that cmPAI-1KO mice display significantly worse cardiac fibrosis than controls. To investigate the molecular mechanisms responsible for these effects, genome-wide cardiac transcriptome analysis was performed. Loss of cardiomyocyte PAI-1 led to differential expression of 978 genes compared to controls in response to left ventricular pressure overload. Pathway enrichment analysis identified the inflammatory response, cell substrate adhesion, regulation of cytokine production, leukocyte migration, extracellular matrix organization, and cytokine-mediated signaling pathways as being significantly upregulated in cmPAI-1KO hearts. Conversely, specific epigenetic repressors, cation transmembrane transport, muscle system processes, and nitric oxide signaling were significantly downregulated in cmPAI-1KO hearts compared to control hearts in response to left ventricular pressure overload. Collectively, the present study provides strong evidence of the impact of cardiomyocyte PAI-1 in regulation of the transcriptome network involved in the cardiac stress response. In response to stress, the deregulatory impact of cardiomyocyte PAI-1 loss on the cardiac transcriptome may be the underlying cause of cardiac-selective accelerated fibrogenesis in global PAI-1-deficient mice.
