Mutations in the human skeletal muscle chloride channel gene (CLCN1) associated with dominant and recessive myotonia congenita

Myotonia, defined as delayed relaxation of muscle after contraction, is seen in a group of genetic disorders that includes autosomal dominant myotonia congenita (Thomsen's disease) and autosomal recessive myotonia congenita (Becker's disease). Both disorders are characterized electrophysiologically by increased excitability of muscle fibers, reflected in clinical myotonia. These diseases are similar except that transient weakness is seen in patients with Becker's, but not Thomsen's disease. Becker's and Thomsen's diseases are caused by mutations in the skeletal muscle voltage-gated chloride channel gene (CLCN1). Genetic screening of a panel of 18 consecutive myotonia congenita (MC) probands for mutation in CLCN1 revealed that a novel Gln-68-Stop nonsense mutation predicts premature truncation of the chloride channel protein. Four previously reported mutations, Arg-894-stop, Arg-338-Gln, Gly-230-Glu, and del 1437-1450, were also noted in our sample set. The Arg-338-Gln and Gly-230-Glu mutations were found in patients with different phenotypes from those of previous reports. Further study of the Arg-338-Gln and Gln-230-Glu alleles may shed light on variable modes of transmission (dominant versus recessive) in different families. Physiologic study of these mutations may lead to better understanding of the pathophysiology of myotonia in these patients and of voltage-gated chloride channel structure/function relationships in skeletal muscles.     

ClC-1 chloride channel mutations in myotonia congenita: variable penetrance of mutations shifting the voltage dependence

Mutations in the ClC-1 muscle chloride channel cause either recessive or dominant myotonia congenita. Using a systematic screening procedure, we have now identified four novel missense mutations in dominant (V286A, F307S) and recessive myotonia (V236L, G285E), and have analysed the effect of these and other recently described mutations (A313T, I556N) on channel properties in the Xenopus oocyte expression system. Mutations V286A, F307S and A313T displayed a 'classical' dominant phenotype: their voltage dependence was shifted towards positive potentials and displayed a dominant-negative effect by significantly imparting a voltage shift on mutant-wild-type heteromeric channels as found in heterozygous patients. In contrast, the recessive mutation V236L also shifted the voltage dependence to positive values, but co-expression with wild-type ClC-1 gave almost wild-type currents. I556N, a mutation found in patients with benign dominant myotonia, drastically shifts the voltage dependence, but only a slight shift is seen when co-expressed with wild-type ClC-1. Thus, the voltage dependence of mutant heteromeric channels is not always intermediate between those of the constituent homomeric channel subunits, a conclusion further supported by mixing different ClC-1 mutants. These complex interactions correlate clinically with various inheritance patterns, ranging from autosomal dominant with various degrees of penetrance to autosomal recessive.     

Myotonia and the muscle chloride channel: dominant mutations show variable penetrance and founder effect

The delayed relaxation or sustained contraction of skeletal muscle-myotonia-is frequently seen in myotonic dystrophy and sodium channelopathies (hyperkalemic periodic paralysis, paramyotonia congenita). Many cases of congenital myotonia without other clinical symptoms have been associated with mutations in the muscle chloride channel gene. Most cases reported to date show a recessive inheritance pattern, with loss of function of the corresponding protein. Six families have been reported with dominantly inherited myotonia and mutations of the chloride channel gene. Here we report clinical and molecular data on 38 family members from four new families with dominantly inherited myotonia congenita. Three families show a previously characterized G230E mutation, and we show that these three share a common affected ancestor despite living in different regions of the United States (linkage disequilibrium). One Italian family is shown to have a novel dominant mutation-I290M. This is the sixth mutation identified in Thomsen's myotonia. Genotype/phenotype correlations in these four families showed that both of the dominant mutations resulted in a mild clinical picture in 90% of the patients, and no symptoms in 10% of mutation-positive patients. The EMG was the clinical feature that most closely correlated with mutation data; however, 3 of 16 (19%) mutation-positive patients tested negative by electromyography at least once, and 1 (6%) tested negative despite multiple tests. Only about half (55%) of the mutation-positive patients tested positive for percussion myotonia. Most of the clinically symptomatic individuals stated that cold temperatures and stress substantially worsened their myotonia. Our data show that dominantly inherited Thomsen's myotonia is most often a very mild disorder that shows considerable clinical heterogeneity.     

Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis)

Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3' part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus.     

High penetrance and pronounced variation in expressivity of GCH1 mutations in five families with dopa-responsive dystonia

A mathematical model is presented which examines the extent to which the intestinal epithelium is accessed by drug molecules. Morphological information from the literature for the jejunum, ileum, and colon of the rat and for human jejunum was incorporated. Perturbation theory was used to derive the limiting cases for total access to the entire epithelial surface, for transport by diffusion and by diffusion with convection, respectively. A parameter gamma = square root of (Ph2)/(Db) was identified to provide a measure of the ability of drug molecules to access the entire epithelial surface down to the crypt wells, where P is the cell permeability, D the aqueous diffusion coefficient, h the channel depth between the villi, and b is half the width of the idealized intervillous channel. When gamma < 1, diffusion is not a limitation and the entire surface is fully utilized for absorption of drug. This condition arises with drugs of low permeability and is more likely to be met with colonic than small intestinal epithelium. When gamma >/= 1, diffusion becomes a limitation and then not all of the epithelial surface is functionally accessible to drug molecules, a condition most likely to prevail with drugs of high permeability traversing the jejunum. Furthermore, water flux per se is predicted to have relatively little influence on enhancing surface accessibility. This simple, but quantitative approach showed that the ranking order of permeability jejunum >ileum> colon for low permeable drugs can at least in part be explained by the differences in surface amplification between these different epithelial regions. The analysis also indicates that for highly permeable drugs extreme caution should be exercised in extrapolating permeability measurements in vitro across various preparations and to events in vivo.     

Identification of functionally important regions of the muscular chloride channel CIC-1 by analysis of recessive and dominant myotonic mutations

Mutations in the muscular voltage-dependent Cl-channel, CIC-1, lead to recessive and dominant myotonia. Here we analyse the effects of one dominant (G200R) and three recessive (Y150C, Y261C, and M485V) mutations after functional expression in Xenopus oocytes. Glycine 200 is a highly conserved amino acid located in a conserved stretch in the putatively cytoplasmic loop between domains D2 and D3. Similar to several other dominant mutations the amino acid exchange G200R leads to a drastic shift by approximately 65 mV of the open probability curve to more positive voltages. As explored by co-expression studies, the shift is intermediate in heteromeric mutant/WT channels. Open channel properties such as single channel conductance, rectification or ion selectivity are not changed. Thus we identified a new region of the CIC-1 protein in which mutations can lead to drastic shifts of the voltage dependence. The recessive mutation M485V, which is located in a conserved region at the beginning of domain D10, leads to a drastic reduction of the single channel conductance from 1.5 pS for WT to approximately 0.3 pS. In addition, the mutant is strongly inwardly rectifying and deactivates incompletely at negative voltages. Ion-selectivity, however, is unchanged. These electrophysiological properties fully explain the recessive phenotype of the mutation and identify a new region of the protein that is involved in ion permeation and gating of the CIC-1 channel. The other two recessive mutations (Y150C and Y261C) had been found in a compound heterozygous patient. Surprisingly, expression of these mutants in oocytes yielded currents indistinguishable from WT CIC-1 when explored by two-electrode voltage clamp recording and patch clamping (either singly or both mutations co-expressed). Other mechanisms that are not faithfully represented by the Xenopus expression system must therefore be responsible for the myotonic symptoms associated with these mutations.     

Effects of electromagnetic radiation of various modes on heart activity (in experiments)

On spinal cord frogs and isolated interauricle to a partition of heart in vivo and in vitro influence the MICROWAVES of a radiation in continuous and modulated modes on function of heart (9.3 Hz is investigated; 0.348-0.16 and 0.016 mV/sm2, modulation from 1 up to 100 Hz). A possibility of influence of an electromagnetic exposure on heart frequency and rhythm is revealed. Pointing of heart in vitro by a neutral red resulted in large number of % of experience in a stop of irradiated heart.     

Melanocortin receptor 1 (MC1R) mutations and coat color in pigs

The melanocortin receptor 1 (MC1R) plays a central role in regulation of eumelanin (black/brown) and phaeomelanin (red/yellow) synthesis within the mammalian melanocyte and is encoded by the classical Extension (E) coat color locus. Sequence analysis of MC1R from seven porcine breeds revealed a total of four allelic variants corresponding to five different E alleles. The European wild boar possessed a unique MC1R allele that we believe is required for the expression of a wild-type coat color. Two different MC1R alleles were associated with the dominant black color in pigs. MC1R*2 was found in European Large Black and Chinese Meishan pigs and exhibited two missense mutations compared with the wild-type sequence. Comparative data strongly suggest that one of these, L99P, may form a constitutively active receptor. MC1R*3 was associated with the black color in the Hampshire breed and involved a single missense mutation D121N. This same MC1R variant was also associated with EP, which results in black spots on a white or red background. Two different missense mutations were identified in recessive red (e/e) animals. One of these, A240T, occurs at a highly conserved position, making it a strong candidate for disruption of receptor function.     

Novel mutations in aquaporin-2 gene in female siblings with nephrogenic diabetes insipidus: evidence of disrupted water channel function

Novel mutations of the aquaporin-2 (AQP2) gene have been detected in Japanese female siblings with autosomal-recessive nephrogenic diabetes insipidus. The patients were compound heterozygote for point mutations at nucleotide position 374 (C374T) and at position 523 (G523A) in exon 2 of the AQP2 gene, resulting in substitution of methionine for threonine at codon 125 (T125M) and arginine for glycine at codon 175 (G175R). The water permeability (Pf) of oocytes injected with wild-type complementary RNA increased 9.0-fold compared with the Pf of water-injected oocytes, whereas the increases in the Pf of oocytes injected with T125M and G175R complementary RNA were only 1.7-fold and 1.5-fold, respectively. Immunoblot and immunocytochemistry indicated that the plasma membrane expressions of T125M and G175R AQP2 proteins were comparable to that of the wild-type, suggesting that although neither the T125M nor G175R mutation had a significant effect on plasma membrane expression, they both distorted the structure and function of the aqueous pore of AQP2. These results provide evidence that the nephrogenic diabetes insipidus in patients with T125M and G175R mutations is attributable not to the misrouting of AQP2, but to the disrupted water channel function.     

Adrenal hypoplasia congenita with hypogonadotropic hypogonadism: evidence that DAX-1 mutations lead to combined hypothalmic and pituitary defects in gonadotropin production

Adrenal hypoplasia congenita (AHC) is an X-linked disorder that typically presents with adrenal insufficiency during infancy. Hypogonadotropic hypogonadism (HHG) has been identified as a component of this disorder in affected individuals who survive into childhood. Recently, AHC was shown to be caused by mutations in DAX-1, a protein that is structurally similar in its carboxyterminal region to orphan nuclear receptors. We studied two kindreds with clinical features of AHC and HHG. DAX-1 mutations were identified in both families. In the JW kindred, a single base deletion at nucleotide 1219 was accompanied by an additional base substitution that resulted in a frameshift mutation at codon 329 followed by premature termination. In the MH kindred, a GGAT duplication at codon 418 caused a frameshift that also resulted in truncation of DAX-1. Baseline luteinizing hormone (LIT), follicle-stimulating hormone (FSH), and free-alpha-subunit (FAS) levels were determined during 24 h of frequent (q10 min) venous sampling. In patient MH, baseline LH levels were low, but FAS levels were within the normal range. In contrast, in patient JW, the mean LH and FSH were within the normal range during baseline sampling, but LH secretion was erratic rather than showing typical pulses. FAS was apulsatile for much of the day, but a surge was seen over a 3-4-h period. Pulsatile gonadotropin releasing hormone (GnRH) (25 ng/kg) was administered every 2 h for 7 d to assess pituitary responsiveness to exogenous GnRH. MH did not exhibit a gonadotropin response to pulsatile GnRH. JW exhibited a normal response to the first pulse of GnRH, but there was no increase in FAS. In contrast to the priming effect of GnRH in GnRH-deficient patients with Kallmann syndrome, GnRH pulses caused minimal secretory responses of LH and no FAS responses in patient JW. The initial LH response in patient JW implies a deficiency in hypothalamic GnRH. On the other hand, the failure to respond to pulsatile GnRH is consistent with a pituitary defect in gonadotropin production. These two cases exemplify the phenotypic heterogeneity of AHC/HHG, and suggest that DAX-1 mutations impair gonadotropin production by acting at both the hypothalamic and pituitary levels.     

Novel compound heterozygous mutations of growth hormone (GH) receptor gene in a patient with GH insensitivity syndrome

A girl with severe growth retardation had the clinical features of Laron syndrome. Her serum insulin-like growth factor-I level was completely unresponsive to exogenous GH administration. The serum GH-binding protein (GHBP) level was below the detectable limit in the patient, but it was normal in her parents and brother. Her parents and brother were normal in their height. Amplification with PCR and direct sequencing of her GH receptor gene revealed compound heterozygous mutations. The allele from her mother contained a transversion of G to T in exon 7 that could introduce a stop codon in place of a glutamic acid at amino acid 224. Another mutation was found in the allele in her father and also identified in her brother. It was a C deletion at position 981 in exon 10 that could introduce a frame shift, thereby causing the production of 20 novel amino acids (310-329) instead of the wild-type sequence, the premature termination at codon 330, and the subsequent deletion of the C terminal portion of the intracellular domain. RT-PCR of her father's lymphocytes and sequencing of its complementary DNA revealed that only the wild-type GH receptor messenger RNA was expressed in his lymphocytes, though the mechanism remains unclear. These results suggest that neither of the mutant alleles could generate a functional GH receptor, which would be consistent with the patient's severe growth retardation and undetectable serum GHBP.     

Novel GLRA1 missense mutation (P250T) in dominant hyperekplexia defines an intracellular determinant of glycine receptor channel gating

Missense mutations as well as a null allele of the human glycine receptor alpha1 subunit gene GLRA1 result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, Mendelian Inheritance in Man (MIM) #149400]. In a pedigree showing dominant transmission of hyperekplexia, we identified a novel point mutation C1128A of GLRA1. This mutation encodes an amino acid substitution (P250T) in the cytoplasmic loop linking transmembrane regions M1 and M2 of the mature alpha1 polypeptide. After recombinant expression, homomeric alpha1(P250T) subunit channels showed a strong reduction of maximum whole-cell chloride currents and an altered desensitization, consistent with a prolonged recovery from desensitization. Apparent glycine binding was less affected, yielding an approximately fivefold increase in Ki values. Topological analysis predicts that the substitution of proline 250 leads to the loss of an angular polypeptide structure, thereby destabilizing open channel conformations. Thus, the novel GLRA1 mutant allele P250T defines an intracellular determinant of glycine receptor channel gating.     

Kinds and locations of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase gene of human T-lymphocytes by 1-nitrosopyrene, including those caused by V(D)J recombinase

The detection of an increase in the frequency of mutants in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of circulating T-cells has been proposed as a method to evaluate the biological effects of human exposure to environmental mutagens. We exposed adult human T-cells in vitro to 1-nitrosopyrene (1-NOP), a partially reduced metabolite of 1-nitropyrene, a ubiquitous environmental carcinogen. In populations of T-cells from two unrelated donors, a dose of 1-NOP that reduced survival to 40% of the untreated cells increased the HPRT mutant frequency 6 to 7 times over the background frequency of 5 x 10(-6). The coding region of 35 independent mutants was amplified by polymerase chain reaction and sequenced. Single base substitutions were found in 63% of the mutants (22 of 35). These were distributed randomly throughout the gene. Most of the substitutions (82%) involved G-C base pairs, mainly G.C-->A.T transitions and G.C-->T.A transversions. Fifteen mutants were lacking one or more exons; 9 of the 15 were lacking exons 2 and 3. Examination showed that at least four of the latter had resulted from V(D)J recombinase acting illegitimately to recombine sites located in introns 1 and 3 of the HPRT gene. T-cells from a second unrelated donor were exposed to 1-NOP and 38 additional independent mutants were analyzed. The results indicated that such mutations occurred at a frequency of 2.4 x 10(-6) compared to a background frequency of less than 0.3 x 10(-6). This recombinase, which plays an important role in leukemogenesis, is normally present in developing, but not mature, B- and T-cells such as those used here as target cells for 1-NOP. The present study is the first report showing that exposure to an environmental carcinogen can cause mutations induced by the action of this enzyme.     

Activation of BK(Ca) channel via endothelin ET(A) receptors in porcine coronary artery smooth muscle cells

It has been demonstrated previously that endothelin-1 stimulates the Ca2+-activated K+ (BK(Ca)) channel activity in porcine coronary artery smooth muscle cells. The purpose of the present study was to delineate the endothelin receptor subtype involved in this action. In receptor binding studies, [125I]endothelin-1 was shown to bind to the homogenate of porcine primary coronary artery smooth muscle cells in a single class of binding sites with K(D) and Bmax values of 73 pM and 99 fmol/mg protein, respectively. Furthermore, endothelin-1 and endothelin-3 displaced the binding of [125I]endothelin-1 to these cells with respective IC50 values of 70 and 17000 pM, a 240-fold difference in potency. The effects of endothelin-3 on the activity of the BK(Ca) channel in porcine coronary artery smooth muscle cells were examined using the cell-attached patch-clamp technique. Similar to endothelin-1, endothelin-3 also exhibited a bell-shaped concentration-response curve. A maximal increase of 95% in channel open-state probability (Po) was induced by 100 nM endothelin-3 as compared with the 320% increase in Po by 1 nM endothelin-1. Thus, endothelin-1 was about 100-fold more potent and 3.4-fold more efficacious than endothelin-3 in this action. Both the receptor binding and the electrophysiological results suggest that the effects of endothelins on the BK(Ca) channel are mediated through the endothelin ET(A) receptor subtype.     

Localization of type-1 porin channel (VDAC) in the sarcoplasmatic reticulum

Eucaryotic porin channels or voltage-dependent anion channels (VDACs) are expressed in the outer mitochondrial membranes and in the plasmalemma of mammalian cells. Subfractions of sarcoplasmatic reticulum (SR) obtained from rabbit skeletal muscle display type-1 porin channels in transverse tubuli (TT) when analysed by immunoblot analysis with type-1 porin specific monoclonal antibodies. These data are in agreement with our recent proposal suggesting the presence of porin channels in non-mitochondrial eucaryotic membranes.     

Occlusion and ion exchange in the molten (lithium chloride+potassium chloride+alkaline-earth chloride) salt+zeolite 4A system with alkaline-earth chlorides of calcium and strontium and in the molten (lithium chloride+potassium chloride+actinide chloride) salt+zeolite 4A system with the actinide chloride of uranium

The interaction between molten salts of the type LiCl-KCl-MeCl _n (Me=Ca, Sr, U; = to 0.45; and x _KCl/x _LiCl=0.69) and zeolite 4A have been studied at 823 K. The main interactions between these salts and zeolite are molten salt occlusion to form salt-loaded zeolite and ion exchange between the molten salt and salt-loaded zeolite. An irreversible chemical reaction has been observed in the LiCl-KCl-UCl₃+zeolite system. The extent of occlusion is a function of the concentration of MeCl _n in the zeolite and is equal to 10±1 Cl⁻ per zeolite unit cell, (AlSiO₄)₁₂, at infinite MeCl _n dilution. The ion-exchange mole-fraction equilibrium constants (separation factors) with respect to Li are decreasing functions of the concentration of SrCl₂ and UCl₃, but an increasing function of the concentration of CaCl₂ in the zeolite. At infinite MeCl _n dilution, they are equal to 0.9, 11.9, and 13 for CaCl₂, SrCl₂, and UCl₃, respectively. The standard ion-exchange chemical potentials are equal to −50.0, −84, and −101.1 kJ·mol⁻¹ for Ca²⁺, Sr²⁺, and U³⁺, respectively.     

Structural and functional characterization of human potassium channel subunit beta 1 (KCNA1B)

Voltage-activated Shaker-related potassium channels (kv1) consist of alpha and beta subunits. We have analysed the structure of the human KCNA1B (hKv beta 1) gene. KCNA1B is > 250 kb in size and encodes at least three Kv beta 1 splice variants. The Kv beta 1 open reading frame is divided into 14 exons. In contrast, genes coding for family members of KCNA (Kv 1 alpha) subunits are markedly smaller and have intronless open reading frames. The expression of Kv 1 alpha and Kv beta mRNA was compared in Northern blots of poly(A+) RNA isolated from various human brain tissues. The results suggest an intricate and cell-specific regulation of Kv 1 alpha and Kv beta mRNA synthesis such that distinct combinations of alpha and beta subunits would occur in different nuclei of the brain. The splice variants hKv beta 1.1 and hKv beta 1.2 were functionally characterized in coexpression studies with hKv 1.5 alpha subunits in 293 cells. It is shown that the confer rapid inactivation on hKv 1.5 channels with different potencies. This may be due to differences in their amino terminal sequences and/or inactivating domains. It is also shown that the amino terminal Kv beta 1.1 and Kv 1.4 alpha inactivating domains compete with each other, probably for the binding to the same receptor site(s) on Kv 1 alpha-subunits.