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R L Barchi

Publications and source records attributed to R L Barchi.

At least 37 records · Page 2Linked to original sources
PubMed1994

Sodium channel mutations in paramyotonia congenita exhibit similar biophysical phenotypes in vitro.

Mutations in the skeletal muscle voltage-gated Na+ channel alpha-subunit have been found in patients with two distinct hereditary disorders of sarcolemmal excitation: hyperkalemic periodic paralysis (HYPP) and paramyotonia congenita (PC). Six of these mutations have been functionally expressed in a heterologous cell line (tsA201 cells) using the recombinant human skeletal muscle Na+ channel alpha-subunit cDNA hSkM1. PC mutants from diverse locations in this subunit (T1313M, L1433R, R1448H, R1448C, A1156T) all exhibit a similar disturbance in channel inactivation characterized by reduced macroscopic rate, accelerated recovery, and altered voltage dependence. PC mutants had no significant abnormality in activation. In contrast, one HYPP mutation studied (T704M) has a normal inactivation rate but exhibits shifts in the midpoints of steady-state activation and inactivation along the voltage axis. These findings help to explain the phenotypic differences between HYPP and PC at the molecular and biophysical level and contribute to our understanding of Na+ channel structure and function.

Base Sequence↗
PubMed1994

Pharmacological modulation of human cardiac Na+ channels.

Pharmacological modulation of human sodium current was examined in Xenopus oocytes expressing human heart Na+ channels. Na+ currents activated near -50 mV with maximum current amplitudes observed at -20 mV. Steady-state inactivation was characterized by a V1/2 value of -57 +/- 0.5 mV and a slope factor (k) of 7.3 +/- 0.3 mV. Sodium currents were blocked by tetrodotoxin with an IC50 value of 1.8 microM. These properties are consistent with those of Na+ channels expressed in mammalian myocardial cells. We have investigated the effects of several pharmacological agents which, with the exception of lidocaine, have not been characterized against cRNA-derived Na+ channels expressed in Xenopus oocytes. Lidocaine, quinidine and flecainide blocked resting Na+ channels with IC50 values of 521 microM, 198 microM, and 41 microM, respectively. Use-dependent block was also observed for all three agents, but concentrations necessary to induce block were higher than expected for quinidine and flecainide. This may reflect differences arising due to expression in the Xenopus oocyte system or could be a true difference in the interaction between human cardiac Na+ channels and these drugs compared to other mammalian Na+ channels. Importantly, however, this result would not have been predicted based upon previous studies of mammalian cardiac Na+ channels. The effects of DPI 201-106, RWJ 24517, and BDF 9148 were also tested and all three agents slowed and/or removed Na+ current inactivation, reduced peak current amplitudes, and induced use-dependent block. These data suggest that the alpha-subunit is the site of interaction between cardiac Na+ channels and Class I antiarrhythmic drugs as well as inactivation modifiers such as DPI 201-106.

Animals↗
PubMed1994

Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation.

Mutations in the adult human skeletal muscle Na+ channel alpha subunit cause the disease paramyotonia congenita. Two paramyotonia congenita mutations, R1448H and R1448C, substitute histidine and cysteine for arginine in the S4 segment of domain 4. These mutations, expressed in a cell line, have only small effects on the activation of Na+ currents, but mutant channels inactivate more slowly with less voltage dependence than wild-type channels and exhibit an enhanced rate of recovery from inactivation. Increase of extracellular pH made the rate of inactivation of R1448H similar to that of R1448C, suggesting that this residue has an extracellular location and that its charge is important for normal inactivation. Analysis of single-channel data reveals that mutant channels inactivate normally from closed states, but poorly from the open state. The data suggest a critical role for the S4 helix of domain 4 in coupling between activation and inactivation.

Base Sequence↗
PubMed1994

Voltage-dependent regulation of modal gating in the rat SkM1 sodium channel expressed in Xenopus oocytes.

The TTX-sensitive rat skeletal muscle sodium channel (rSkM1) exhibits two modes of inactivation (fast vs slow) when the alpha subunit is expressed alone in Xenopus oocytes. In this study, two components are found in the voltage dependence of normalized current inactivation, one having a V1/2 in the expected voltage range (approximately -50 mV, I(N)) and the other with a more hyperpolarized V1/2 (approximately -130 mV, IH) at a holding potential of -90 mV. The I(N) component is associated with the gating mode having rapid inactivation and recovery from inactivation of the macroscopic current (N-mode), while IH corresponds to the slow inactivation and recovery mode (H-mode). These two components are interconvertible and their relative contribution to the total current varies with the holding potential: I(N) is favored by hyperpolarization. The interconversion between the two modes is voltage dependent and is well fit to a first-order two-state model with a voltage dependence of e-fold/8.6 mV and a V1/2 of -62 mV. When the rat sodium channel beta 1-subunit is coinjected with rSkM1, IH is essentially eliminated and the inactivation kinetics of macroscopic current becomes rapid. These two current components and their associated gating modes may represent two conformations of the alpha subunit, one of which can be stabilized either by hyperpolarization or by binding of the beta 1 subunit.

Animals↗
PubMed1994

Molecular cloning and functional analysis of the promoter of rat skeletal muscle voltage-sensitive sodium channel subtype 2 (rSkM2): evidence for muscle-specific nuclear protein binding to the core promoter.

rSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt -102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5' upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additional cis element (-645/-506). The positive and negative cis-elements together drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (-44/-29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (-44/-29) are competed by unlabeled -44/-29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides -40 to -38 and -34 to -32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-specific core promoter elements.

Animals↗
PubMed1993

Sodium channel mutations in paramyotonia congenita and hyperkalemic periodic paralysis.

Clinical and electrophysiological data have outlined a spectrum of similar yet distinct periodic paralyses, including potassium-sensitive (hyperkalemic periodic paralysis [HYPP]) and temperature-sensitive (paramyotonia congenita [PC]) forms. Recent work has revealed that these disorders result from allelic defects in the alpha-subunit of the adult, human skeletal muscle sodium channel. We report an additional mutation, a leucine-->arginine substitution in the S3 segment of domain 4 (L1433R), that results in the PC phenotype. Five other HYPP and PC families have been ascertained, and previously reported sodium channel mutations have been identified in each. Characterization of these mutations and phenotypic variations in such families will contribute to the understanding of sodium channel structure and function relationships, as well as channel malfunction in the periodic paralyses.

Adult↗
PubMed1993

Genomic organization of the human skeletal muscle sodium channel gene.

Voltage-dependent sodium channels are essential for normal membrane excitability and contractility in adult skeletal muscle. The gene encoding the principal sodium channel alpha-subunit isoform in human skeletal muscle (SCN4A) has recently been shown to harbor point mutations in certain hereditary forms of periodic paralysis. We have carried out an analysis of the detailed structure of this gene including delineation of intron-exon boundaries by genomic DNA cloning and sequence analysis. The complete coding region of SCN4A is found in 32.5 kb of genomic DNA and consists of 24 exons (54 to > 2.2 kb) and 23 introns (97 bp-4.85 kb). The exon organization of the gene shows no relationship to the predicted functional domains of the channel protein and splice junctions interrupt many of the transmembrane segments. The genomic organization of sodium channels may have been partially conserved during evolution as evidenced by the observation that 10 of the 24 splice junctions in SCN4A are positioned in homologous locations in a putative sodium channel gene in Drosophila (para). The information presented here should be extremely useful both for further identifying sodium channel mutations and for gaining a better understanding of sodium channel evolution.

Amino Acid Sequence↗
PubMed1993

Expression of the sodium channel beta 1 subunit in rat skeletal muscle is selectively associated with the tetrodotoxin-sensitive alpha subunit isoform.

Transcripts homologous to the rat brain sodium channel beta subunit (beta 1) are prominently expressed in both innervated and denervated adult skeletal muscle and in heart, but not in neonatal skeletal or cardiac muscle. Regulation of beta 1 mRNA expression closely parallels that of SkM1 alpha during development, after denervation in adult muscle, and in primary muscle culture, but does not follow SkM2 expression under any condition examined. In oocytes, beta 1 interacts functionally with SkM1 to modulate the abnormally slow inactivation kinetics observed with this alpha subunit expressed alone. We conclude that a common beta 1 subunit is expressed in skeletal muscle, heart, and brain and that in skeletal muscle, this subunit is specifically associated with the SkM1, rather than the SkM2, sodium channel isoform.

Animals↗
PubMed1993

Ion channels and disorders of excitation in skeletal muscle.

For skeletal muscle to contract normally, signals from the neuromuscular junction must be conveyed to the contractile proteins by action potentials that spread across the surface membrane and into the T-tubular system. Hyperexcitability in the surface membrane can lead to unwanted and persistent contraction, while reduced excitability can cause weakness or paralysis. Many of the human muscle diseases that are associated with abnormalities of membrane excitability have been linked to defects in the function of membrane ion channels. Using molecular biologic tools, these abnormal channels can be identified and their molecular defects defined. Advances in these areas now provide the basis for a rational, mechanistic approach to the classification and treatment of these disorders of membrane excitation.

Chlorides↗
PubMed1992

Chimeric study of sodium channels from rat skeletal and cardiac muscle.

Two isoforms of voltage-dependent Na channels, cloned from rat skeletal muscle, were expressed in Xenopus oocytes. The currents of rSkM1 and rSkM2 differ functionally in 4 properties: (i) tetrodotoxin (TTX) sensitivity, (ii) mu-conotoxin (mu-CTX) sensitivity, (iii) amplitude of single channel currents, and (iv) rate of inactivation. rSkM1 is sensitive to both TTX and mu-CTX. rSkM2 is resistant to both toxins. Currents of rSkM1 have a higher single channel conductance and a slower rate of inactivation than those of rSkM2. We constructed (i) chimeras by interchanging domain 1 (D1) between the two isoforms, (ii) block mutations of 22 amino acids in length that interchanged parts of the loop between transmembrane segments S5 and S6 in both D1 and D4, and (iii) point mutations in the SS2 region of this loop in D1. The TTX sensitivity could be switched between the two isoforms by the exchange of a single amino acid, tyrosine-401 in rSkM1 and cysteine-374 in rSkM2 in SS2 of D1. By contrast most chimeras and point mutants had an intermediate sensitivity to mu-CTX when compared with the wild-type channels. The point mutant rSkM1 (Y401C) had an intermediate single-channel conductance between those of the wild-type isoforms, whereas rSkM2 (C374Y) had a slightly lower conductance than rSkM2. The rate of inactivation was found to be determined by multiple regions of the protein, since chimeras in which D1 was swapped had intermediate rates of inactivation compared with the wild-type isoforms.

Amino Acid Sequence↗
PubMed1992

Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel.

The principal voltage-sensitive sodium channel from human heart has been cloned, sequenced, and functionally expressed. The cDNA, designated hH1, encodes a 2016-amino acid protein that is homologous to other members of the sodium channel multigene family and bears greater than 90% identity to the tetrodotoxin-insensitive sodium channel characteristic of rat heart and of immature and denervated rat skeletal muscle. Northern blot analysis demonstrates an approximately 9.0-kilobase transcript expressed in human atrial and ventricular cardiac muscle but not in adult skeletal muscle, brain, myometrium, liver, or spleen. When expressed in Xenopus oocytes, hH1 exhibits rapid activation and inactivation kinetics similar to native cardiac sodium channels. The single channel conductance of hH1 to sodium ions is about twice that of the homologous rat channel and hH1 is more resistant to block by tetrodotoxin (IC50 = 5.7 microM). hH1 is also resistant to mu-conotoxin but sensitive to block by therapeutic concentrations of lidocaine in a use-dependent manner.

Amino Acid Sequence↗
PubMed1992

Primary structure of the adult human skeletal muscle voltage-dependent sodium channel.

The gene encoding the principal voltage-dependent sodium channel expressed in adult human skeletal muscle (SCN4A) has recently been linked to the pathogenesis of human hyperkalemic periodic paralysis and paramyotonia congenita. We report the cloning and nucleotide sequence determination of the normal product of this gene. The 7,823 nucleotide complementary DNA, designated hSkM1, encodes a 1,836 amino acid protein that exhibits 92% identity with the tetrodotoxin-sensitive rat skeletal muscle sodium channel alpha subunit, but lower homology with either the human heart sodium channel or with other sodium channels from immature rat muscle or rat brain. Specific hSkM1 RNA transcripts are expressed in adult human skeletal muscle but not in heart, brain, or uterus. The SCN4A gene product, hSkM1, is the human homologue of rSkM1, the tetrodotoxin-sensitive sodium channel characteristic of adult rat skeletal muscle. This structural information should provide the necessary backdrop for identifying and evaluating mutations affecting the function of this channel in the periodic paralyses.

Adult↗
PubMed1992

Localization of epitopes for antibodies that differentially label sodium sodium channels in skeletal muscle surface and T-tubular membranes.

We previously characterized two monoclonal antibodies, A/B2 and L/D3, that bind to the amino-terminus of the sodium channel but produce distinct immunocytochemical patterns in innervated adult skeletal muscle. Because these findings suggested the presence of several channel isoforms, we sought to define the epitopes for each antibody. Five peptides encompassing the amino-terminal 126 residues of the adult skeletal muscle sodium channel were synthesized and tested by radioimmunoassay against each antibody. Both monoclonals bound only to a peptide comprising residues 1-30 (I1-30). A nested set of peptides within this region was then synthesized and used to compete for antibody binding to I1-30. L/D3 binding was quantitatively inhibited by oligopeptides 1-30, 7-30, 13-30, and 19-30 but not 25-30, while binding of A/B2 was blocked only by the intact I1-30 peptide. This data implies that the epitope for L/D3 lies within residues 19-25 while the epitope for A/B2 is contained within residues 1-6. These tentative epitope localizations were confirmed using both proteolytic cleavage of I1-30 and immunoreactivity of a peptide corresponding to residues 1-12 with A/B2 but not L/D3. Therefore, epitopes for each monoclonal antibody are present in the SkM-1 sequence and are in close proximity in the amino-terminus of the protein. Their characteristic immunocytochemical labeling patterns may reflect differing accessibility of the epitopes in various membrane environments.

Amino Acid Sequence↗
PubMed1992

Lidocaine block of human heart sodium channels expressed in Xenopus oocytes.

The tertiary amine lidocaine is used clinically for preventing cardiac arrhythmias, and has been widely studied on mammalian tissue. Xenopus oocytes were used as an expression system to study the effect of lidocaine on a sodium (Na) channel, derived from a full-length human heart (hH1) cDNA clone. The concentration dependence of the lidocaine block of hH1 Na current was consistent with a binding stoichiometry of 1:1. At low frequency stimulation, and at holding potentials < or = 100 mV, the IC50 was 226 microM, comparable to values found in mammalian cardiac cells. Lidocaine also shifted the steady-state inactivation of hH1 Na current to hyperpolarized potentials in a dose-dependent manner. Our experiments suggest that lidocaine block is state dependent, with high affinity for an inactivated state (KI = 11 microM) and low affinity for the resting state (KR = 3.9 mM). The quaternary amine derivative of lidocaine, QX-314, had no effect on Na current at an extracellular concentration of 1 mM.

Animals↗
PubMed1992

Mutations in an S4 segment of the adult skeletal muscle sodium channel cause paramyotonia congenita.

The periodic paralyses are a group of autosomal dominant muscle diseases sharing a common feature of episodic paralysis. In one form, paramyotonia congenita (PC), the paralysis usually occurs with muscle cooling. Electrophysiologic studies of muscle from PC patients have revealed temperature-dependent alterations in sodium channel (NaCh) function. This observation led to demonstration of genetic linkage of a skeletal muscle NaCh gene to a PC disease allele. We now report the use of the single-strand conformation polymorphism technique to define alleles specific to PC patients from three families. Sequencing of these alleles defined base pair changes within the same codon, which resulted in two distinct amino acid substitutions for a highly conserved arginine residue in the S4 helix of domain 4 in the adult skeletal muscle NaCh. These data establish the chromosome 17q NaCh locus as the PC gene and represent two mutations causing the distinctive, temperature-sensitive PC phenotype.

Alleles↗
PubMed1992

Sodium channel gene defects in the periodic paralyses.

Abnormal Na+ currents that produce membrane depolarization have been associated with the episodes of muscle weakness that are the hallmark of the periodic paralyses. There is now strong evidence that various point mutations in the gene encoding the adult skeletal muscle voltage-dependent Na+ channel produce these abnormal currents, and are responsible for the expression of the disease phenotype.

Humans↗