MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1

The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families—muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins—are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5′ end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.     

Low single channel conductance of the major skeletal muscle chloride channel, ClC-1.

We expressed the skeletal muscle chloride channel, ClC-1, in HEK293 cells and investigated it with the patch-clamp technique. Macroscopic properties are similar to those obtained after expression in Xenopus oocytes, except that faster gating kinetics are observed in mammalian cells. Nonstationary noise analysis revealed that both rat and human ClC-1 have a low single channel conductance of about 1 pS. This finding may explain the lack of single-channel data for chloride channels from skeletal muscle despite its high macroscopic chloride conductance.     

Multimeric structure of ClC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen).

Voltage-gated ClC chloride channels play important roles in cell volume regulation, control of muscle excitability, and probably transepithelial transport. ClC channels can be functionally expressed without other subunits, but it is unknown whether they function as monomers. We now exploit the properties of human mutations in the muscle chloride channel, ClC-1, to explore its multimeric structure. This is based on analysis of the dominant negative effects of ClC-1 mutations causing myotonia congenita (MC, Thomsen's disease), including a newly identified mutation (P480L) in Thomsen's own family. In a co-expression assay, Thomsen's mutation dramatically inhibits normal ClC-1 function. A mutation found in Canadian MC families (G230E) has a less pronounced dominant negative effect, which can be explained by functional WT/G230E heterooligomeric channels with altered kinetics and selectivity. Analysis of both mutants shows independently that ClC-1 functions as a homooligomer with most likely four subunits.     

Quantification of chloride channel 2 (CLCN2) gene isoforms in normal versus lesion- and epilepsy-associated brain tissue

The chloride channel 2 (CLCN2) gene codes for a protein organized in N- and C-terminal regions with regulatory functions and a transmembrane region which forms the ring of the pore. Mutations in the gene have previously been described in patients with idiopathic familial epilepsy. In this study we looked for new isoforms of CLCN2 and we estimated expression levels by real time PCR in brain tissue containing epileptic foci. Samples used in this study were first analyzed and selected to exclude mutations in the coding region of the gene. Four isoforms (skipping exons 3, 16, 22 and 6/7) were identified and quantified by Real Time PCR and compared with total expression of the gene. Expression of the region common to all CLCN2 isoforms was 50% less in epilepsy-associated brain tissue than in controls. The ratio of the various isoforms was slightly greater in epileptic than control tissue. The greatest difference was recorded in the temporal lobe for the isoform with skipped exon 22. Analysis of these isoforms in brain tissue containing epileptic foci suggests that CLCN2 could be implicated in epilepsy, even in the absence of mutations.     

Temperature-sensitive mutations in the III-IV cytoplasmic loop region of the skeletal muscle sodium channel gene in paramyotonia congenita.

Paramyotonia congenita (PMC), a dominant disorder featuring cold-induced myotonia (muscle stiffness), has recently been genetically linked to a candidate gene, the skeletal muscle sodium channel gene SCN4A. We have now established that SCN4A is the disease gene in PMC by identifying two different single-base coding sequence alterations in PMC families. Both mutations affect highly conserved residues in the III-IV cytoplasmic loop, a portion of the sodium channel thought to pivot in response to membrane depolarization, thereby blocking and inactivating the channel. Abnormal function of this cytoplasmic loop therefore appears to produce the Na+ current abnormality and the unique temperature-sensitive clinical phenotype in this disorder.     

The gene for the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel (Cchl1a3) maps to mouse chromosome 1.

Cchl1a3 encodes the dihydropyridine-sensitive calcium channel alpha 1 subunit isoform predominantly expressed in skeletal muscle. mdg (muscular dysgenesis) has previously been implicated as a mutant allele of this gene. Hybridization of a rat brain cDNA probe for Cchl1a3 to Southern blots of DNAs from a panel of Chinese hamster x mouse somatic cell hybrids suggested that this gene maps to mouse Chromosome 1. Analysis of the progeny of an inbred strain cross-positioned Cchl1a3 1.3 cM proximal to the Pep-3 locus on Chr 1.     

The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia

Single-channel recordings of the currents mediated by the muscle Cl- channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with approximately 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations.     

Purification and characterization of the major aminopeptidase from human skeletal muscle.

The major aminopeptidase from human quadriceps muscle was purified (as judged by polyacrylamide-gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The enzyme showed maximum activity at pH 7.3, in the presence of 1 mM-2-mercaptoethanol and 0.5 mM-Ca2+ ions; activation of the enzyme occurred in the presence of several other bivalent cations. Inhibition of activity was obtained in the presence of metal-ion-chelating agents and inhibitors of aminopeptidases and thiol proteinases. The molecular weight of the enzyme was 102 000 (by gel filtration). The enzyme hydrolysed several amino acyl-7-amido-4-methylcoumarin derivatives; highest activity was obtained with alanyl-7-amido-4-methylcoumarin. The enzyme also degraded a series of dipeptides, alanine oligopeptides and some naturally occurring peptides. Of particular interest was the high activity of the enzyme towards the enkephalins.     

Refined localisation of the voltage-gated chloride channel, CLCN3, to 4q33

Mutations in ion channels have been shown to be responsible for a variety of neurological and muscular diseases. The voltage-gated chloride channel CLCN3 was recently mapped to chromosomal region 4q32. We are analysing a young female patient with Wolf-Hirschhorn syndrome and chorea associated with an inversion-deletion of chromosome 4 [46XX,inv(4)del(4)(qter→q33:: p15.32→q33]. Considering that chorea in this patient might be due to the disruption of a gene at either of the 4p15.32 or 4q33 breakpoints, CLCN3 was considered as a candidate gene. We showed by FISH analysis with a CLCN3 YAC that the gene was not broken by the inv-del event, and was therefore an unlikely candidate. Using high resolution techniques, we refined the localisation of CLCN3 to 4q33. Received: 15 June 1997 / Accepted: 5 November 1997     

Clinical and molecular diagnosis of a Costa Rican family with autosomal recessive myotonia congenita (Becker disease) carrying a new mutation in the CLCN1 gene

Myotonia congenita is a muscular disease characterized by myotonia, hypertrophy, and stiffness. It is inherited as either autosomal dominant or recessive known as Thomsen and Becker diseases, respectively. Here we confirm the clinical diagnosis of a family diagnosed with a myotonic condition many years ago and report a new mutation in the CLCN1 gene. The clinical diagnosis was established using ocular, cardiac, neurological and electrophysiological tests and the molecular diagnosis was done by PCR, SSCP and sequencing of the CLCN1 gene. The proband and the other affected individuals exhibited proximal and distal muscle weakness but no hypertrophy or muscular pain was found. The myotatic reflexes were lessened and sensibility was normal. Electrical and clinical myotonia was found only in the sufferers. Slit lamp and electrocardiogram tests were normal. Two affected probands presented diminution of the sensitive conduction velocities and prolonged sensory distal latencies. The clinical spectrum for this family is in agreement with a clinical diagnosis of Becker myotonia. This was confirmed by molecular diagnosis where a new disease-causing mutation (Q412P) was found in the family and absent in 200 unaffected chromosomes. No latent myotonia was found in this family; therefore the ability to cause this subclinical sign might be intrinsic to each mutation. Implications of the structure-function-genotype relationship for this and other mutations are discussed. Adequate clinical diagnosis of a neuromuscular disorder would allow focusing the molecular studies toward the confirmation of the initial diagnosis, leading to a proper clinical management, genetic counseling and improving in the quality of life of the patients and relatives.     

Characteristics of two types of chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

A comparison is made of two types of chloride-selective channel in skeletal muscle sarcoplasmic reticulum (SR) vesicles incorporated into lipid bilayers. The I/V relationships of both channels, in 250/50 mM Cl- (cis/trans), were linear between -20 and +60 mV (cis potential,) reversed near Ecl and had slope conductances of approximately 250 pS for the big chloride (BCl) channel and approximately 70 pS for the novel, small chloride (SCl) channel. The protein composition of vesicles indicated that both channels originated from longitudinal SR and terminal cisternae. BCl and SCl channels responded differently to cis SO4(2-) (30-70 mM), 4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid (8-80 microM) and to bilayer potential. The BCl channel open probability was high at all potentials, whereas SCl channels exhibited time-dependent activation and inactivation at negative potentials and deactivation at positive potentials. The duration and frequency of SCl channel openings were minimal at positive potentials and maximal at -40 mV, and were stationary during periods of activity. A substate analysis was performed using the Hidden Markov Model (S. H. Chung, J. B. Moore, L. Xia, L. S. Premkumar, and P. W. Gage, 1990, Phil. Trans. R. Soc. Lond. B., 329:265-285) and the algorithm EVPROC (evaluated here). SCl channels exhibited transitions between 5 and 7 conductance levels. BCl channels had 7-13 predominant levels plus many more short-lived substates. SCl channels have not been described in previous reports of Cl- channels in skeletal muscle SR.     

Identification of three cysteines as targets for the Zn2+ blockade of the human skeletal muscle chloride channel

Currents through the human skeletal muscle chloride channel hClC-1 can be blocked by external application of 1 mM Zn2+ or the histidine-reactive compound diethyl pyrocarbonate (DEPC). The current block by Zn2+ strongly depends on the external pH (pKa near 6.9), whereas the block by DEPC is rather independent of the pH in the range of 5.5 to 8.5. To identify the target sites of these reagents, we constructed a total of twelve cysteine- and/or histidine-replacement mutants, transfected tsA201 cells with them, and investigated the resulting whole-cell chloride currents. The majority of the mutants exhibited a similar sensitivity toward Zn2+ or DEPC as wild type (WT) channels. Block by 1 mM Zn2+ was nearly absent only with the mutant C546A. Four mutants (C242A, C254A, H180A, and H451A) were slightly less sensitive to Zn2+ than WT. Tests with double, triple, and quadruple mutants yielded that, in addition to C546, C242 and C254 are also most likely participating in Zn2+-binding.     

Structural characterization of the human fast skeletal muscle troponin I gene (TNNI2)

Three troponin I genes have been identified in vertebrates that encode the isoforms expressed in adult cardiac muscle (TNNI3), slow skeletal muscle (TNNI1) and fast skeletal muscle (TNNI2), respectively. While the organization and regulation of human cardiac and slow skeletal muscle genes have been investigated in detail, the fast skeletal troponin I gene has to date only been examined in birds. Here, we describe the structure and complete sequence of the human fast skeletal muscle troponin I gene (TNNI2) and identify putative regulatory elements within both the 5' flanking region and the first intron. In particular, a region containing MEF-2, E-box, CCAC and CAGG elements was identified in intron 1 that closely resembles the fast internal regulatory element (FIRE) of the quail intronic enhancer. We have previously shown that the fast skeletal muscle troponin I gene is located at 11p15.5 and noted potential close linkage with the fast skeletal muscle troponin T gene (TNNT3). Here, we have isolated two independent human PAC genomic clones that contain either TNNI2 or TNNT3 and demonstrate by interphase FISH mapping that they are less than 100 kb apart in the genome. The results demonstrate that the human TNNI2 gene is closely related to its avian counterparts with conserved elements within both the putative promoter and first intron. Our data further confirm close physical linkage of TNNI2 and TNNI3 on 11p15.5.     

Sequence and genomic structure of the human adult skeletal muscle sodium channel alpha subunit gene on 17q.

The amino acid sequence of the sodium channel alpha subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the alpha subunit from rat adult skeletal muscle and 70% identity to the alpha subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene.