Reinhardt Rüdel

Increased calcium entry into dystrophin‐deficient muscle fibres of MDX and ADR‐MDX mice is reduced by ion channel blockers

1 Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic‐dystrophic double mutant ADR‐MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2 Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura‐2, were slightly higher in MDX (63 ± 20 nm; means ±s.d.; n= 454 analysed fibres) and ADR‐MDX (65 ± 12 nm; n= 87) fibres than in controls (51 ± 20 nm; n= 265). 3 The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50% of MDX fibres (n= 68), but in only 20% of controls (n= 54), the decay time constants were > 35 ms. 4 Bath application of Mn2+ resulted in a progressive quench of fura‐2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 ± 1.9% min−1; n= 275) than in controls (2.03 ± 1.4% min−1; n= 204). The quench rate in ADR‐MDX fibres (2.49 ± 1.4% min−1; n= 87) was closer to that of controls. 5 The Mn2+ influx into MDX fibres was reduced to 10% by Gd3+, to 19% by La3+ and to 47% by Ni2+ (all at 50 μm). Bath application of 50 μm amiloride inhibited the Mn2+ influx to 37%. 6 We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR‐MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability.

A common mutation (ε1267delG) in congenital myasthenic patients of Gypsy ethnic origin

Objective: Mutation analysis of the acetylcholine receptor (AChR) e subunit gene in patients with sporadic or autosomal recessive congenital myasthenic syndromes (CMS). Background: The nicotinic AChR of skeletal muscle is a neurotransmitter-gated ion channel that mediates synaptic transmission at the vertebrate neuromuscular junction. Mutations in its gene may cause congenital myasthenic syndromes. A recently described mutation in exon 12 of the AChR e subunit (e1267delG) disrupts the cytoplasmic loop and the fourth transmembrane region (M4) of the AChR e subunit. Methods: Forty-three CMS patients from 35 nonrelated families were clinically classified as sporadic cases of CMS (group III according to European Neuromuscular Centre consensus) and were analyzed for e1267delG by PCR amplification and sequence analysis. Results: The authors report the complete genomic sequence and organization of the gene coding for the e subunit of the human AChR (accession number AF105999). Homozygous e1267delG was identified in 13 CMS patients from 11 independent families. All e1267delG families were of Gypsy or southeastern European origin. Genotype analysis indicated that they derive from a common ancestor (founder) causing CMS in the southeastern European Gypsy population. Phenotype analysis revealed a uniform pattern of clinical features including bilateral ptosis and mild to moderate fatigable weakness of ocular, facial, bulbar, and limb muscles. Conclusions: The mutation e1267delG might be frequent in European congenital myasthenic syndrome patients of Gypsy ethnic origin. In general, patients (e1267delG) were characterized by the onset of symptoms in early infancy, the presence of ophthalmoparesis, positive response to anticholinesterase treatment, and the benign natural course of the disease.

Anti-GM1 antibodies can block neuronal voltage-gated sodium channels

Anti‐GM1 antibodies, frequently found in the serum of patients with Guillain‐Barré syndrome (GBS), have been suggested to interfere with axonal function. We report that IgG anti‐GM1 antibodies, raised in rabbits, can reversibly block the voltage‐gated Na+ channels of nerve cells, thus causing a reduction of the excitatory Na+ current. The block was, however, only substantial when the antibodies were applied together with rabbit complement factors. A solution containing anti‐GM1 sera (dilution 1:100) and complement (1:50) reduced the Na+ current to 0.5 ± 0.2 times control (mean value ± SD). Applications of the antibody by itself, complement by itself, or anti‐GM2 or anti‐GM4 antibodies (1:100) plus complement had little effect. The complexes of anti‐GM1 antibodies and complement factors block the ion‐conducting pore of the channel directly. In addition, they increase the fraction of channels that are inactivated at the resting potential and alter channel function by changing the membrane surface charge. The described effects may be responsible for conduction slowing and reversible conduction failure in some GBS patients.

Molecular pathophysiology of voltage-gated ion channels

In addition to the clarification of the pathology of a whole group of hereditary diseases, the study of the consequences of these mutations at the levels of the whole system (patient), organ and cells (excised muscle specimens), and of the channel proteins has taught us that our current opinions on channel structure-function relations are far from being comprehensive. For instance it had been assumed that as in the potassium channel, also in the sodium and calcium channel proteins, the S4 unit is mainly responsible for channel activation. This notion has to be corrected as mutations in S4 of repeat IV were found to affect mainly channel inactivation. Moreover mutations affecting other channel domains, such as interlinkers or other intramembraneous subunits cause virtually the same alterations, not only when tested with the limited probe of the patch clamp but also on the level of the patient. The lack of mutations in other parts of the genes, in particular those coding for sodium or calcium channels, may indicate that proper function of the corresponding protein domains is essential for life. Thus the knowledge derived from the experiments of Nature, as these diseases may be looked upon by the cell biologist, provides a valuable addition to the results from site-directed mutagenesis. For a final understanding of the pathology of the diseases, for example, triggering effects of cold or potassium, it seems that the regression from the proteins back to the cellular or even systemic levels is unavoidable.

Intracellular Ca2+ concentrations are not elevated in resting cultured muscle from Duchenne (DMD) patients and in MDX mouse muscle fibres

The free intracellular calcium concentration, [Ca2+]i, was studied in single myotubes using the fluorescent Ca2+ indicator fura-2. Myotubes cultured from satellite cells of small muscle specimens from Duchenne muscular dystrophy (DMD) patients were compared with human control myotubes and with myotubes cultured from MDX and control mouse muscle satellite cells. The resting [Ca2+]i levels in DMD and control myotubes were not significantly different, i. e. 104 ±26 nM (mean ± SD, n=190 cells from eight DMD patients) compared with 97±25 nM (175/seven controls) and were not significantly lower than the corresponding murine values (154±33 nM, n=135 MDX myotubes; 159±34 nM, n=135 controls). All myotubes reacted to 10 μM acetylcholine or 40 mM KCl with fast transient increases of [Ca2+]i. After application of a hyposmotic (130 mOsm) solution, [Ca2+]i was increased 1.5- to 3-fold within 2–3 min, the DMD myotubes tending to stronger reactions (significantly higher [Ca2+]i in 2 out of 6 cases). The response was usually transient, [Ca2+]i decreasing to the initial level within 10 min. Gadolinium (50 μM) reduced the response by 50%–70%, indicating that the osmotic shock increased Ca2+ influx. During exposure to high (15 mM) [Ca2+]e, [Ca2+]i of DMD and control cells was 1.5- to 2-fold higher. Adult muscle fibres from MDX mice and controls showed identical Ca2+ resting levels (n=45 fibres from three mice in each case), but did not respond to decreased external osmolarity with a change in [Ca2+]i. The results indicate that lack of dystrophin in muscle fibres does not necessarily lead to increased [Ca2+]i. It is suggested that increased [Ca2+]i is probably a secondary consequence of fibre damage.

An endogenous pentapeptide acting as a sodium channel blocker in inflammatory autoimmune disorders of the central nervous system.

Reversible blockade of sodium channels by endogenous substances has been claimed to account for the fast exacerbations and relapses commonly seen in demyelinating autoimmune diseases. Evidence has been provided that in the cerebrospinal fluid of patients with multiple sclerosis or Guillain-Barré syndrome, a sodium-channel-blocking factor exists that has properties of local anesthetic agents. This factor could contribute to the nerve conduction block and paresis seen in these disorders. We describe here a previously unknown endogenous substance in human cerebrospinal fluid with distinct channel-blocking properties even at very low (0.00001 M) concentrations. The pentapeptide with the sequence Gln-Tyr-Asn-Ala-Asp exerted its blocking action by shifting the steady-state inactivation curve of the sodium channels to more-negative potentials, as most local anesthetics do. In the cerebrospinal fluid of healthy individuals, its concentration was about 3 μM, whereas in patients with multiple sclerosis and Guillain-Barré syndrome, it increased 300–1,400%. At these concentrations, the peptides blocking efficacy was higher than that of 50 μM lidocaine. At a concentration of 10 μM, lidocaine is able to ‘unmask’ subclinical lesions in multiple sclerosis; thus, the endogenous pentapeptide may well contribute to the fast changes of symptoms. Furthermore, it may become valuable as a marker of disease activity.

Improved therapy of myotonia with the lidocaine derivative tocainide

SummaryThe antimyotonic effect of the antiarrhythmic drug tocainide was tested in 14 patients. With 1200 to 1600 mg/d all patients reported good improvement. This was substantiated by objective tests. Only four patients noticed minor side effects.ZusammenfassungDie antimyotone Wirkung des Antiarrhythmikums Tocainid wurde an 14 myotonen Patienten untersucht. Bei Tagesdosen von 1200–1600 mg berichteten alle Patienten über gute Unterdrückung der Myotonie; dies wurde durch objektive Tests bestätigt. Nur 4 Patienten berichteten geringfügige Nebeneffekte.

The dominant chloride channel mutant G200R causing fluctuating myotonia : Clinical findings, electrophysiology, and channel pathology

Clinical, electrophysiological, and molecular findings are reported for a family with dominant myotonia congenita in which all affected members have experienced long‐term fluctuations of the symptom of myotonia. In some patients myotonia is combined with myalgia. The myotonia‐causing mutation in this family is in the gene encoding the muscular chloride channel, hClC‐1, predicting the amino acid exchange G200R. We have constructed recombinant DNA vectors for expression of the mutant protein in tsA201 cells and investigation of the properties of the mutant channel. The most prominent alteration was a +100‐mV shift of the midpoint of the activation curve. Therefore, within the physiological range the open probability of the mutant channel is markedly smaller than in wild‐type. This shift is likely to be responsible for the myotonia in the patients. The fluctuating symptoms of this chloride channelopathy are discussed with respect to short‐term fluctuations of myotonia in the sodium channelopathy of potassium‐aggravated myotonia.

Regulation of the human skeletal muscle chloride channel hClC‐1 by protein kinase C

1 The regulation of a recombinant human muscle chloride channel, hClC‐1, by protein kinase C (PKC) was investigated in human embryonic kidney (HEK 293) cells. 2 External application of 4β‐phorbol esters (4β‐PMA) reduced the instantaneous whole‐cell current amplitude over the entire voltage range tested. This effect was abolished when the cells were intracellularly perfused with a specific protein kinase C inhibitor, chelerythine. Inactive 4α‐phorbolesters did not affect the chloride currents. We conclude that the effect of 4β‐phorbol esters is mediated by protein kinase C (PKC). 3 Activation of PKC resulted in changes in macroscopic current kinetics. The time course of current deactivation determined in the presence and absence of 4β‐phorbol esters could be fitted with the sum of two exponentials and a constant value. In the presence of phorbol esters, the fast time constants and the minimum value of the fraction of non‐deactivating current were increased, whereas the voltage dependence of all fractional current amplitudes remained unchanged. PKC‐induced phosphorylation had only small effects on the voltage dependence of the relative open probability and the maximum absolute open probability was unaffected by treatment with 4β‐PMA, as shown by non‐stationary noise analysis. 4 The kinetic changes indicate that phosphorylation alters functional properties of active channels. Since the absolute open probability is not reduced, the observed macroscopic current reduction implies alterations of the ion permeation process. 5 Phosphorylation by PKC appears to affect ion transfer and gating processes. It is postulated that the phosphorylation site may be located at the cytoplasmic vestibule face of the pore.

Hereditary nondystrophic myotonias and periodic paralyses.

The hereditary disorders of muscle excitability are now recognized to be caused by defects in the genes encoding muscle ion channels. This led to a new classification of this disease group. The pathophysiology of these disorders has been elucidated on the molecular level to an extent that exceeds the understanding of the disease mechanisms of most other neuromuscular diseases. The seemingly minor variants of the symptom of myotonia were found to be caused by the remarkable difference that either chloride or sodium channel function is impaired. Even more surprising, the basic defects for hyper- and hypokalemic periodic paralysis, often clinically very difficult to distinguish, turned out to be in the sodium and calcium channels, respectively; these channels are considered to have very different functions in muscle physiology. Three new types of myotonic disease, that is, myotonia, fluctuans, myotonia permanens and proximal myotonic myopathy were discovered. An explanation has been provided as to why myotonia congenita may be transmitted as a dominant or recessive trait.