Muscle Sodium Research Articles

The Role of Individual Disulfide Bonds of μ-Conotoxin GIIIA in the Inhibition of NaV1.4.

μ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel NaV1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of NaV1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against NaV1.4 was assayed by whole cell patch-clamp on rNaV1.4, heterologously expressed in HEK293 cells. The corresponding IC50 values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA-1, 3.3 ± 0.2 μM for GIIIA-2, and 15.8 ± 0.8 μM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3–Cys15, Cys4–Cys20 and Cys10–Cys21, respectively). Other analogues were not active enough for IC50 measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of NaV1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10–Cys21 is the most important for the NaV1.4 potency.

Sex differences in sodium deposition in human muscle and skin

The aim of this work was to investigate possible sex differences in the patterns of sodium deposition between muscle and skin using sodium MRI. A total of 38 subjects were examined for comparisons: 20 males, aged 25–79years with a median age of 51; 18 females, aged 38–66years, median age 53. All subjects underwent sodium MRI scans of the calf muscles together with cross sections through four calibration standards containing known sodium contents (10mM, 20mM, 30mM, and 40mM). Tissue sodium concentrations (TSC) in muscle and skin were then calculated by comparing signal intensities between tissues and reference standards using a linear analysis. A Wilcoxon rank sum test was applied to the ΔTSC (=TSCmuscle−TSCskin) series of males and females to examine if they were significantly different. Finally, a multiple linear regression was utilized to account for the effects from two potential confounders, age and body mass index (BMI). We found that sodium content appears to be higher in skin than in muscle for men, however women tend to have higher muscle sodium than skin sodium. This sex-relevant sodium deposition is statistically significant (P=3.10×10−5) by the Wilcoxon rank sum test, and this difference in distribution seems to be more reliable with increasing age. In the multiple linear regression, gender still has a statistically significant effect (P<1.0×10−4) on the difference between sodium deposition in muscle and skin, while taking the effects of age and BMI into account.

A novel Ile1455Thr variant in the skeletal muscle sodium channel alpha-subunit in a patient with a severe adult-onset proximal myopathy with electrical myotonia and a patient with mild paramyotonia phenotype

In sodium channelopathies, a severe fixed myopathy caused by a dominant mutation is rare. We describe two unrelated patients with a novel variant, p.Ile1455Thr, with phenotypes of paramyotonia in one case and fixed proximal myopathy with latent myotonia in another. In-vitro whole cell patch-clamp studies show that the mutation slows inactivation and accelerates recovery, in line with other paramyotonia variants with destabilized fast inactivation as pathomechanism. Additionally, p.IleI1455 causes a loss-of-function by reduced membrane insertion, right-shift of activation, and slowed kinetics. Molecular dynamics simulations comparing wild type and mutant Nav1.4 showed that threonine substitution hindered D4S4 mobility in response to membrane depolarization, consistent with effects of the mutation on channel inactivation. The fixed myopathy is likely to be associated to gain-of-function leading to sodium accumulation, regional edema, T-tubular swelling and mitochondrial stress. A possible contribution of the loss-of-function features towards myotonia and myopathy is discussed.

A case of non-dystrophic myotonia with concomitant mutations in the SCN4A and CLCN1 genes

Non-dystrophic myotonias are caused by mutations of either the skeletal muscle chloride (CLCN1) or sodium channel (SCN4A) gene. They exhibit several distinct phenotypes, including myotonia congenita, paramyotonia congenita and sodium channel myotonia, and a genotype-phenotype correlation has been established. However, there are atypical cases that do not fit with the standard classification. We report a case of 27-year-old male who had non-dystrophic myotonia with periodic paralysis and two heterozygous mutations, E950K in CLCN1 and F1290L in SCN4A. His mother, who exhibited myotonia without paralytic attack, only harbored E950K, and no mutations were identified in his asymptomatic father. Therefore, the E950K mutation was presumed to be pathogenic, although it was reported as an extremely rare genetic variant. The proband experienced paralytic attacks that lasted for weeks and were less likely to be caused by CLCN1 mutation alone. Functional analysis of the F1290L mutant channel heterologously expressed in cultured cells revealed enhanced activation inducing membrane hyperexcitability. We therefore propose that the two mutations had additive effects on membrane excitability that resulted in more prominent myotonia in the proband. Our case stresses the value of performing genetic analysis of both CLCN1 and SCN4A genes for myotonic patients with an atypical phenotype.

Recent advances in congenital myasthenic syndromes

AbstractCongenital myasthenic syndromes (CMS) are heterogeneous disorders caused by germline mutations in genes expressed at the neuromuscular junction. Mutations have been identified in 24 genes encoding acetylcholine receptor subunits (CHRNA1, CHRNB1, CHRND, CHRNE and CHRNG), skeletal muscle sodium channel (SCN4A), signaling molecules driving acetylcholine receptor subunits clustering (AGRN, LRP4, MUSK and DOK7), synaptic structural proteins (COLQ, LAMB2 and COL13A1), postsynaptic structural proteins (RAPSN and PLEC), presynaptic molecules (CHAT, SYT2), glycosylation enzymes (GFPT1, DPAGT1, ALG2, ALG14 and GMPPB), and other less characterized molecules (PREPL and SCL25A1). CMS are recessive disorders, except for slow channel CMS and synaptotagmin 2 (SYT2)‐CMS. Onsets are largely less than 2 years, but adult‐onset is not rare, especially in slow‐channel CMS and limb‐girdle type CMS caused by glycosylation defects and by DOK7 mutations. Clinical features include fatigable muscle weakness, amyotrophy and minor facial anomalies. Eye, facial and bulbar muscles are frequently affected, but sparing of these muscles is observed, especially in limb‐girdle type CMS. Serum creatine kinase levels are frequently elevated in slow‐channel CMS, glutamine‐fructose‐6‐phosphate aminotransferase 1 (GFPT1)‐CMS, and guanosine diphosphate mannose pyrophosphorylase B (GMPPB)‐CMS. Electrophysiological findings supporting compromised neuromuscular signal transmission are a prerequisite for diagnosing CMS. Most CMS patients are likely to be underdiagnosed, and recognition of CMS in undiagnosed muscle weakness and/or amyotrophy is critical for diagnosing CMS.

The frequently used intraperitoneal hyponatraemia model induces hypovolaemic hyponatraemia with possible model-dependent brain sodium loss.

What is the central question of this study? The brain response to acute hyponatraemia is usually studied in rodents by intraperitoneal instillation of hypotonic fluids (i.p. model). The i.p. model is described as 'dilutional' and 'syndrome of inappropriate ADH (SIADH)', but the mechanism has not been explored systematically and might affect the brain response. Therefore, in vivo brain and muscle response were studied in pigs. What is the main finding and its importance? The i.p. model induces hypovolaemic hyponatraemia attributable to sodium redistribution, not dilution. A large reduction in brain sodium is observed, probably because of the specific mechanism causing the hyponatraemia. This is not accounted for in current understanding of the brain response to acute hyponatraemia. Hyponatraemia is common clinically, and if it develops rapidly, brain oedema evolves, and severe morbidity and even death may occur. Experimentally, acute hyponatraemia is most frequently studied in small animal models, in which the hyponatraemia is produced by intraperitoneal instillation of hypotonic fluids (i.p. model). This hyponatraemia model is described as 'dilutional' or 'syndrome of inappropriate ADH (SIADH)', but seminal studies contradict this interpretation. To confront this issue, we developed an i.p. model in a large animal (the pig) and studied water and electrolyte responses in brain, muscle, plasma and urine. We hypothesized that hyponatraemia was induced by simple water dilution, with no change in organ sodium content. Moderate hypotonic hyponatraemia was induced by a single i.v. dose of desmopressin and intraperitoneal instillation of 2.5% glucose. All animals were anaesthetized and intensively monitored. In vivo brain and muscle water was determined by magnetic resonance imaging and related to the plasma sodium concentration. Muscle water content increased less than expected as a result of pure dilution, and muscle sodium content decreased significantly (by 28%). Sodium was redistributed to the peritoneal fluid, resulting in a significantly reduced plasma volume. This shows that the i.p. model induces hypovolaemic hyponatraemia and not dilutional/SIADH hyponatraemia. Brain oedema evolved, but brain sodium content decreased significantly (by 21%). To conclude, the i.p. model induces hypovolaemic hyponatraemia attributable to sodium redistribution and not water dilution. The large reduction in brain sodium is probably attributable to the specific mechanism that causes the hyponatraemia. This is not accounted for in the current understanding of the brain response to acute hyponatraemia.

Point-Mutations in Skeletal Muscle Voltage-Gated Sodium Channels Confer Resistance to Tetrodotoxin: But at a Cost?

The interaction between toxic newts (Taricha) and several resistant garter snakes (Thamnophis) provides a model system for the study of predator-prey coevolution (Brodie & Brodie 1999). Pacific newts secrete through their skin the potent neurotoxin, tetrodotoxin (TTX, Fuhrman et al., 1964), a compound that specifically binds to the outer pore of voltage-gated sodium channels (NaV proteins, Fozzard et al., 1994). TTX-ligation prevents sodium ion movement across the membrane and therefore abolishes excitability in skeletal muscle and peripheral nerves (Hille, 2001). Three species of garter snake that prey on sympatric newts have adapted to these toxic newts by circumventing the block through several independently evolved mutations within the pore loop of the skeletal muscle channel variant, NaV1.4 (Feldman et al., 2012). These mutations reduce the affinity of TTX to the pore and provide physiological resistance (Feldman et al., 2009). However, these same mutations are also expected to alter the biophysical properties of the channel, in particular the permeation and selectivity (Hille, 2001). We show that animals with greater TTX resistance displayed diminished skeletal muscle performance (phasic and tetanic contractions). We are currently testing the hypothesis that changes in skeletal muscle sodium channel current density, activation, inactivation, and recovery could potentially explain the tissue-level findings.

Probing Voltage-Dependent Structural Changes of the VSD in Mammalian Nav with LRET

Voltage-gated sodium channels (Nav) play an essential role in the generation and propagation of action potentials. Mammalian Nav alpha subunits are large single polypeptide chains organized in four different domains (DI-DIV). Each domain is composed of 6 transmembrane segments (S1-S6) in which S1-S4 constitute the voltage sensing domain (VSD) and S5 and S6 form the pore. Although Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. Recently, the crystal structures of several prokaryotic sodium channels have been solved, but they are homo-tetrameric proteins in contrast to the mammalian Navs and these studies only provide a single snapshot of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are needed. In this work we have used Lanthanide-based resonance energy transfer (LRET) to obtain structural information and conformational dynamics of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4). We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which binds a lanthanide (Tb3+) ion with high affinity, inserted in several strategic places at the top of the S4 segment in each domain. Two new conotoxin analogs were synthesized to function as LRET acceptors: KIIIA-Bodipy and GIIIA-Bodipy. We calculated multiple distances from each one of the domains to the pore region where the labeled toxin binds in both resting and slow inactivated states in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs, which remained functionally active. The results give a geometrical map of the S4 positions of each domain in the mammalian Nav channels and provide evidence for voltage-dependent structural changes. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Skeletal muscle sodium channelopathies.

This is an update on skeletal muscle sodium channelopathies since knowledge in the field have dramatically increased in the past years. The relationship between two phenotypes and SCN4A has been confirmed with additional cases that remain extremely rare: severe neonatal episodic laryngospasm mimicking encephalopathy, which should be actively searched for since patients respond well to sodium channel blockers; congenital myasthenic syndromes, which have the particularity to be the first recessive Nav1.4 channelopathy. Deep DNA sequencing suggests the contribution of other ion channels in the clinical expressivity of sodium channelopathies, which may be one of the factors modulating the latter. The increased knowledge of channel molecular structure, the quantity of sodium channel blockers, and the availability of preclinical models would permit a most personalized choice of medication for patients suffering from these debilitating neuromuscular diseases. Advances in the understanding of the molecular structure of voltage-gated sodium channels, as well as availability of preclinical models, would lead to improved medical care of patients suffering from skeletal muscle, as well as other sodium channelopathies.

Is there more than one way to skin a newt? Convergent toxin resistance in snakes is not due to a common genetic mechanism.

Convergent evolution of tetrodotoxin (TTX) resistance, at both the phenotypic and genetic levels, characterizes coevolutionary arms races between amphibians and their snake predators around the world, and reveals remarkable predictability in the process of adaptation. Here we examine the repeatability of the evolution of TTX resistance in an undescribed predator-prey relationship between TTX-bearing Eastern Newts (Notophthalmus viridescens) and Eastern Hog-nosed Snakes (Heterodon platirhinos). We found that that local newts contain levels of TTX dangerous enough to dissuade most predators, and that Eastern Hog-nosed Snakes within newt range are highly resistant to TTX. In fact, these populations of Eastern Hog-nosed Snakes are so resistant to TTX that the potential for current reciprocal selection might be limited. Unlike all other cases of TTX resistance in vertebrates, H. platirhinos lacks the adaptive amino acid substitutions in the skeletal muscle sodium channel that reduce TTX binding, suggesting that physiological resistance in Eastern Hog-nosed Snakes is conferred by an alternate genetic mechanism. Thus, phenotypic convergence in this case is not due to parallel molecular evolution, indicating that there may be more than one way for this adaptation to arise, even among closely related species.

Episodic weakness and vacuolar myopathy in hypokalemic periodic paralysis

We report a 50-year-old woman who presented with a 20year history of gradually progressive lower extremity weakness, characterized by knee buckling with occasional falls and foot dragging. She also experienced difficulty in lifting her arms above her shoulders. The primary periodic paralyses are rare disorders caused by dysfunctional ion channels in skeletal muscle. The hypokalemic type is generally an autosomal dominant condition, due to missense mutations in the alpha subunits of the skeletal muscle L-type calcium channel genes, CACN1AS, or the skeletal muscle sodium channel gene, SCN4A. The affected patients typically present with episodic weakness. For our patient, the consumption of foods high in carbohydrates seemed to precipitate the episodes of weakness. Her family history was significant for six blood relatives, including three sons and three relatives on the paternal side, who had experienced similar symptoms. A biopsy of the left rectus femoralis muscle showed vacuolar myopathic changes in the scattered muscle fibers, accompanied by occasional degenerating and regenerating muscle fibers. There was no evidence of inflammation on the biopsy. The vacuoles were often associated with increased acid phosphatase staining. An electron microscopic examination showed that the vacuolar changes were due to T-tubule dilation, a characteristic of hypokalemic periodic paralysis. Other metabolic etiologies of vacuolar myopathy, such as acid phosphatase (lysosomal) associated acid maltase deficiency (a glycogen storage disease), need to be considered in the differential diagnosis.

SCN4A variants and Brugada syndrome: phenotypic and genotypic overlap between cardiac and skeletal muscle sodium channelopathies.

SCN5A mutations involving the α-subunit of the cardiac voltage-gated muscle sodium channel (NaV1.5) result in different cardiac channelopathies with an autosomal-dominant inheritance such as Brugada syndrome. On the other hand, mutations in SCN4A encoding the α-subunit of the skeletal voltage-gated sodium channel (NaV1.4) cause non-dystrophic myotonia and/or periodic paralysis. In this study, we investigated whether cardiac arrhythmias or channelopathies such as Brugada syndrome can be part of the clinical phenotype associated with SCN4A variants and whether patients with Brugada syndrome present with non-dystrophic myotonia or periodic paralysis and related gene mutations. We therefore screened seven families with different SCN4A variants and non-dystrophic myotonia phenotypes for Brugada syndrome and performed a neurological, neurophysiological and genetic work-up in 107 Brugada families. In the families with an SCN4A-associated non-dystrophic myotonia, three patients had a clinical diagnosis of Brugada syndrome, whereas we found a remarkably high prevalence of myotonic features involving different genes in the families with Brugada syndrome. One Brugada family carried an SCN4A variant that is predicted to probably affect function, one family suffered from a not genetically confirmed non-dystrophic myotonia, one family was diagnosed with myotonic dystrophy (DMPK gene) and one family had a Thomsen disease myotonia congenita (CLCN1 variant that affects function). Our findings and data suggest a possible involvement of SCN4A variants in the pathophysiological mechanism underlying the development of a spontaneous or drug-induced type 1 electrocardiographic pattern and the occurrence of malignant arrhythmias in some patients with Brugada syndrome.

Louis Ptáček receives the 2015 ASCI/Stanley J. Korsmeyer Award. Interview by Sarah Jackson.

Channelopathies encompass a diverse set of diseases that are caused by mutations in genes encoding ion channel subunits or their regulators. The first recognition of a channelopathy came from Louis Ptacek (Figure 1), whose lab cloned a mutated muscle sodium channel gene found in a family with hyperkalemic periodic paralysis. His subsequent work extends far beyond channelopathies, as he has continued studying a number of inherited, familial disorders. Ptacek’s research has identified a wide range of genes that are mutated in patients with migraine, epilepsy, cardiac arrhythmias, neurodegeneration, and circadian rhythm disorders. This year, the ASCI honors Ptacek’s outstanding work using human genetics to provide fundamental insights into the molecular mechanisms that drive disease pathophysiology. The JCI recently spoke with Ptacek about his path to success as a physician-scientist.

Magnetic resonance–determined sodium removal from tissue stores in hemodialysis patients

We have previously reported sodium is stored in skin and muscle. The amounts stored in hemodialysis (HD) patients are unknown. We determined whether 23Na magnetic resonance imaging (sodium-MRI) allows assessment of tissue sodium and its removal in 24 HD patients, and 27 age-matched healthy controls. We also studied 20 HD patients before and shortly after HD with a batch dialysis system with direct measurement of sodium in dialysate and ultrafiltrate. Age was associated with higher tissue sodium content in controls. This increase was paralleled by an age-dependent decrease of circulating levels of vascular endothelial growth factor-C (VEGF-C). Older (over 60 years) HD patients showed increased sodium and water in skin and muscle, and lower VEGF-C levels than age-matched controls. After HD, patients with low VEGF-C levels had significantly higher skin sodium content than patients with high VEGF-C levels (low VEGF-C: 2.3 ng/ml and skin sodium: 24.3 mmol/L; high VEGF-C: 4.1ng/ml and skin sodium: 18.2mmol/L). Thus, sodium-MRI quantitatively detects sodium stored in skin and muscle in humans and allows studying sodium storage reduction in ESRD patients. Age and VEGF-C-related local tissue-specific clearance mechanisms may determine the efficacy of tissue sodium removal with HD. Prospective trials on the relationship between tissue sodium content and hard endpoints could provide new insights into sodium homeostasis, and clarify whether increased sodium storage is a cardiovascular risk factor.

Phenotypic Variability in Childhood of Skeletal Muscle Sodium Channelopathies

BackgroundMutations of the SCN4A gene cause several skeletal muscle channelopathies and overlapping forms of these disorders. However, the variability of the clinical presentation in childhood is confusing and not fully understood among pediatric neurologists. PatientsWe found three different mutations (p.V445M, p.I693L, and a novel mutation, p.V1149L) in SCN4A but not in the CLCN1 gene. The patient with p.V445M showed the clinical phenotype of sodium channel myotonia, but her clear symptoms did not appear until 11 years of age. Her younger sister and mother, who have the same mutation, displayed marked intrafamilial phenotypic heterogeneity from mild to severe painful myotonia with persistent weakness. The patient with p.I693L exhibited various symptoms that evolved with age, including apneic episodes, tonic muscular contractions during sleep, fluctuating severe episodic myotonia, and finally episodic paralyses. The patient with the novel p.V1149L mutation exhibited episodic paralyses starting at 3 years of age, and myotonic discharges were detected at 11 years of age for the first time. ConclusionThe present cohort reveals the complexity, variability, and overlapping nature of the clinical features of skeletal muscle sodium channelopathies. These are basically treatable disorders, so it is essential to consider genetic testing before the full development of a patient's condition.

Conditional knockout of smooth muscle sodium calcium exchanger type-1 lowers blood pressure and attenuates Angiotensin II-salt hypertension.

The functions of smooth muscle sodium calcium exchanger (NCX) in the vasculature are controversial and poorly understood. To determine the possible roles of NCX in the vascular phenotype and function, we developed a novel mouse model (SM‐NCX1 KO) in which the smooth muscle‐specific NCX type‐1 (NCX1) was conditionally knocked out using tamoxifen‐inducible Cre‐loxP recombination technique. SM‐NCX1 KO mice exhibit significantly lower blood pressure and attenuated angiotensin II (Ang II)‐salt‐induced hypertension (measured by radio telemetry and intra‐arterial catheterization). Isolated, pressurized mesenteric small resistance arteries from SM‐NCX1 KO mice, compared to control arteries, were characterized by the following: (1) ~90% reduced NCX1 protein expression; (2) impaired functional responses to (i) acute NCX inhibition by SEA0400 or SN‐6, (ii) NCX activation by low [Na+]o, and (iii) Na+ pump inhibition by ouabain; (3) attenuated myogenic reactivity; and (4) attenuated vasoconstrictor response to phenylephrine but not Ang II. These results provided direct evidence that arterial NCX1 normally mediates net Ca2+ influx that helps maintain basal vascular tone in small resistance arteries and blood pressure under physiological conditions. Importantly, NCX1 contributes to blood pressure elevation in Ang II‐salt hypertension, possibly by regulating α‐adrenergic receptor activation.

Autosomal Recessive Inheritance of Congenital Myasthenic Syndrome is Associated with Skeletal Muscle Sodium Channel Mutations

The SCN4A gene, that encodes the pore-forming α-subunit of the skeletal muscle voltage-gated sodium channel, has long been associated with dominantly-inherited myotonia, and periodic paralysis mutations. One single severe case of congenital myasthenic syndrome (CMS) due to a dominantly-inherited SCN4A mutation has been reported. CMS is a clinically and genetically heterogeneous group of rare disorders characterized by muscle weakness and fatigability. They are due to impairment of neuromuscular transmission and result from mutations in genes encoding for proteins critical for the neuromuscular junction. We have identified a novel homozygous SCN4A mutation (p.R1454W) in a patient with a rare form of recessively-inherited CMS. The p.R1454W missense mutation is located on the segment S4 of domain IV of the Nav1.4 channel. Expression of p.R1454W mutant Nav1.4 in the human embryonic kidney 293 cells induced dramatic changes in the biophysical properties of the mutated channel. These include fast and slow inactivation,kinetics of fast inactivation, and recovery from inactivation. A slower current decay combined with a shift in channel availability at resting potentials can ultimately lead to membrane inexcitability and muscle weakness. Our data confirm that this CMS phenotype belongs to the group of sodium channel disorders and question the clinical overlapping between periodic paralyses and CMS. We suggest that while Nav1.4 mutations exerting a dominant-negative effect cause periodic paralysis, a CMS phenotype may result from recessively-inherited mutations that decrease the Nav1.4 availability for muscle action potential genesis at the neuromuscular junction and its propagation along the sarcolemma.

Resting State of S4 Identified for Each Domain of Nav1.2 using Omega Current Technique

During gating transitions the voltage sensor S4 slides through the narrow so-called gating pore. The positively charged amino acids of S4, arginine (R) or lysine (K) sense the transmembranal electric field and promote S4 in or out. If one or more long R or K is replaced by the short neutral glutamine (Q), a leak (omega-) pore is created through which a leak current, the omega current will flow when S4 is at the appropriate position. The occupancy of this leaky position can then be electrically monitored. Rat brain sodium channels Nav1.2 were studied at high expression in X. laevis oocytes with two-electrode voltage clamping at strong hyperpolarization to force S4 into the resting state. Mutant channels with single gaps R1Q, R2Q or R3Q as well as with double gaps RRn,n+1QQ were tested for the presence of omega leaks. We found unambiguous omega currents for double gaps in domain DI (RR12QQ), DII (RR12QQ), DIII (RR23QQ) and DIV (RR12QQ), indicating the resting position of S4 in each domain. These findings are in contrast to skeletal muscle sodium channels Nav1.4 where single gap omega leaks are reported for DII and DIII. However, our single gap mutants in Nav1.2 produced only very small leak currents similar to artifacts sometimes also occurring at wild-type channels at very strong hyperpolarizing pulses and at least ten times smaller than those with double gaps. Based on this study, we currently use our double gap channel constructs as a tool to selectively investigate which S4 of the four domains I-IV are immobilized by inactivation.

Dynamics of sodium and potassium in plasma, erythrocytes, and muscles of freshwater species under the effect of long-term combined stress

In the initial period of acute stress, the concentration of Na in the plasma of roach and bream decreases by 30 and 70% in several hours, thereby causing a difference in the osmotic concentration of cellular and extracellular liquids of an organism. In response to this, defensive systems are activated. They have extracted Na and K ions from the cell against their electrochemical gradient. As a result, the content of Na and K in erythrocytes and muscles of fish has decreased to a minimum. During the acclimatization of fish to new conditions, the ion concentration in plasma, erythrocytes, and muscles has stabilized at low levels, reflecting the physiological state of the organism. It is assumed that the counteraction of hyponatremia and hypoosmolality has been achieved due to the intensification of the Na+-K+-2Cl− cotransport system.

Roles of basic amino acid residues in the activity of μ-conotoxin GIIIA and GIIIB, peptide blockers of muscle sodium channels.

To study in detail the roles of basic amino acid residues in the activity of μ-conotoxin GIIIA (μ-GIIIA) and GIIIB (μ-GIIIB), specific blockers of muscle sodium channels, seven analogs of μ-GIIIA, and two analogs of μ-GIIIB were synthesized. μ-GIIIA analogs were synthesized by replacing systematically the three Arg residues (Arg1, Arg13, and Arg19) with one, two, and three Lys residues. μ-GIIIB analogs were synthesized by replacing simultaneously all four Lys residues (Lys9, Lys11, Lys16, and Lys19) with Arg residues and further replacement of acidic Asp residues with neutral Ala residues. Circular dichroism spectra of the synthesized analogs suggested that the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the side chain guanidino group of Arg13 of μ-GIIIA was important for the activity, whereas that of Arg19 had little role for biological activity. Although [Arg9,11,16,19]μ-GIIIB showed higher activity than native μ-GIIIB, highly basic [Ala2,12, Arg9,11,16,19]μ-GIIIB showed lower activity, suggesting that there was an appropriate molecular basicity for the maximum activity.