Overexcited or inactive: ion channels in muscle disease.
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Animals↗
Biomedical subjects
R Rüdel
Publications and source records attributed to R Rüdel.
Cerebrospinal fluid and serum from patients with inflammatory polyradiculoneuropathy have opposite effects on sodium channels.
The effects of cerebrospinal fluid (CSF) and serum from patients having Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) on voltage-dependent Na+ channels were compared. Bathing human myoballs in CSF substantially reduced their Na+ currents (by > 40% with 8 of 10 patients) elicited at 1 Hz under whole-cell recording conditions. This was because, at the resting potential, more Na+ channels were inactivated (left-shift of the h infinity curve). CSF from patients with other neurological diseases (OND) produces a similar, but smaller, effect. In contrast, serum samples from the same GBS and OND patients caused an increase of the Na+ currents by reducing the number of Na+ channels inactivated at the resting potential. This right-shift of the h infinity curve is in part explained by the effect of serum albumin. We confirm that the CSF of most GBS and CIDP patients contains factors inhibiting voltage-dependent Na+ currents. There is no indication that such factors are effective in the serum of these patients.
Cells, Cultured↗
Recombinant interleukin-2 acts like a class I antiarrhythmic drug on human cardiac sodium channels.
Human recombinant interleukin-2 (rIL-2) was bath-applied to isolated human cardiocytes while sodium currents were triggered and registered using the whole-cell recording technique. In the presence of the cytokine the sodium currents were reversibly blocked, 50% peak current reduction occurring at a concentration of 500 U/ml. The current-voltage relationship was not affected, but the steady-state inactivation curve was not affected, but the steady-state inactivation curve was shifted in the negative direction by 15 mV. When 35% of the sodium current was blocked the time constant of recovery from block at -135 mV was in the range of 63 +/- 27 ms. Use dependence was observed only at stimulation frequencies above 4 Hz. Addition of a polyclonal anti-IL-2 antibody to the extracellular solution prevented all of the above effects, while incubation of the cells with a function-blocking monoclonal anti-IL-2 receptor antibody had no influence on the described rIL-2 action. In contrast to rIL-2, recombinant tumor necrosis factor alpha (rTNF-alpha) did not affect the sodium currents. It is concluded that rIL-2 acts like a class I antiarrhythmic drug on human cardiac sodium channels. This might explain some of its proarrhythmic side effects when given intravenously in high doses.
Antibodies↗
An aspartic acid residue important for voltage-dependent gating of human muscle chloride channels.
A point mutation (D136G) predicting the substitution of glycine for aspartate in position 136 of the human muscle Cl- channel (hClC-1) causes recessive generalized myotonia. Heterologous expression of a recombinant D136G produces functional Cl- channels with profound alterations in voltage-dependent gating, without concomitant changes in pore properties. The mutant exhibits slowly activating current upon hyperpolarization, in contrast to wild-type channels, which display time-dependent current decay (deactivation) at negative membrane potentials. Steady-state activation of D136G depends upon the transmembrane Cl- gradient, reaching zero at voltages positive to the Cl- reversal potential in physiological Cl- distribution. This explains the reduced sarcolemmal Cl- conductance that causes myotonia. The functional disturbances exhibited by D136G may stem from a defect in the ClC-1 voltage sensor.
Action Potentials↗
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.
Humans↗
Local anaesthetic-like effect of interleukin-2 on muscular Na+ channels: no evidence for involvement of the IL-2 receptor.
The effects of interleukin-2 (IL-2) on muscular Na+ currents were studied in human myoballs. The transient Na+ inward currents, elicited by repetitive depolarizations at 1 Hz and recorded in the whole-cell mode, were inhibited by the cytokine, the half-maximum effect occurring at about 500 U/ml. The effects resembled those of local anaesthetics without use dependence, as the inactivation (h infinity) curve was shifted in a negative direction while the current/voltage curve was not affected. As with these local anaesthetics, depolarization at 1, 4 and 8 Hz in the presence of IL-2 did not produce any cumulative block. The interaction of IL-2 with the Na+ channels is very fast (within ms) and it is suggested that it occurs when the Na+ channels are in the state of fast inactivation. The recovery from inactivation was only slightly slowed by IL-2, in agreement with the absence of any use dependence. All effects were readily reversible on washout of the cytokine. The effects were seen both in tetrodotoxin-(TTX)-sensitive adult Na+ channels and in TTX-insensitive juvenile channels. In contrast to the whole-cell configuration, no inhibition was visible in the attached-patch configuration. Further, the preincubation with an anti-IL-2-receptor antibody did not prevent the inhibitory effect of IL-2 on the Na+ currents. It is concluded that the cytokine blocks the voltage-dependent muscular Na+ channels by keeping the channels in the state of fast inactivation. An IL-2 receptor and a second messenger system are not likely to be involved in this reaction.
Anesthetics, Local↗
Characterization of the sodium currents in isolated human cardiocytes.
The whole-cell recording technique was used to register Na+ currents from 403 atrial cardiocytes isolated from 80 human biopsies. With intracellular [Na+] ([Na+]i) raised to 70 mM, and at physiological extracellular [Na+] ([Na+]e, 145 mM) and room temperature, the Na+ currents were small enough for the error of the voltage clamp not to exceed 2 mV (series resistances 0.4-2 M omega). The threshold potential of the Na+ current was -64.0 +/- 7.7 mV. The peak amplitude was at -30.0 +/- 6.2 mV. The time course of fast inactivation was satisfactorily described with a single exponential. The time constant of inactivation was 2.0 ms at -55 mV and asymptotically approached 0.2 ms at positive membrane potentials. The steady-state inactivation curve was well fitted by a single Boltzmann distribution. Increasing the prepulse duration from 32 to 512 ms shifted the inflexion point of the curve from -61.7 +/- 6.4 to -72.2 +/- 2.6 mV. The time course of slow inactivation was also well described by a single exponential, the time constant ranging from 76.1 +/- 29.3 ms at -115 mV to 18.6 +/- 7.8 ms at -55 mV. Fitting the time course of recovery from inactivation required two time constants. At a recovery potential of -135 mV these were 1.6 +/- 0.2 and 8.6 +/- 2.9 ms and 15.9 +/- 9.4 and 53.2 +/- 33.3 ms at -95 mV. A 50% block of the Na+ currents was achieved by tetrodotoxin at 10 microM. It is concluded that the properties of human cardiac Na+ channels are similar to those of the juvenile Na+ channels of human skeletal muscle.
Heart Atria↗
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 microM 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 microM) 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylcholine↗
Role of the cytoskeleton in the regulation of Cl- channels in human embryonic skeletal muscle cells.
The effects of volume change and cytoskeleton manipulation on the Cl- channels in human embryonic skeletal muscle cells were studied. Trypsination, used for production of myoballs, changes the channel properties only a little. When the external osmolarity was reduced from 300 to 270 mosmol/l, the specific Cl- conductance, gCl, (at -80 mV) of myoballs increased from 5.1 +/- 1.9 to 30.4 +/- 12.2 microS/cm2 (SD; n = 6) within 15 min. Concomitantly, the kinetics of Cl- currents, elicited by clamping the membrane potential from a negative to positive values, changed from activation and subsequent slow inactivation to instantaneous activation with fast inactivation. G protein activation, protein kinase action or [Ca2+]i elevation seemed not to be involved in these effects. Similar changes were produced in the absence of a transmembrane osmotic gradient by 500 nM intracellular cytochalasin D (gCl = 34.3 +/- 10.3 microS/cm2; n = 6) or 12.5 microM colchicine (gCl = 15.4 +/- 1.4 microS/cm2; n = 5). When the external osmolarity was increased to 418 mosmol/l, 1 microM cytochalasin D did not affect gCl. In four of six cell-attached patches the open probability of the intermediate Cl- channel was increased after reduction of the bath osmolarity. In inside-out patches, the drugs increased the open probability of the channels. It is concluded that the Cl- channels are under control of the cytoskeleton.
Calcium↗
Proof of a non-functional muscle chloride channel in recessive myotonia congenita (Becker) by detection of a 4 base pair deletion.
Recessive myotonia congenita (Becker) is genetically linked to HUMCLC, the gene encoding the muscular chloride channel, localized on chromosome 7q35. Three point mutations have so far been reported in HUMCLC, one causing recessive Becker-type myotonia, the others causing the clinically similar Thomsen-type myotonia, which is inherited as a dominant trait. We report a homozygous patient having a 4 base pair deletion in HUMCLC that shifts the reading frame and causes early stop codons, thus destroying the gene's coding potential for several membrane-spanning domains. In addition, we report a patient homozygous for a novel point mutation located at the extracellular side of the first membrane-spanning domain that causes removal of a negative charge (aspartic acid-136-glycine). Both mutations lead to the recessive type of myotonia congenita. Since the patient having the deletion presents less severe clinical myotonia than the patient carrying the missense mutation, it seems that the absence or truncation of the channel protein may disturb muscle fibre function less than the substitution of a single amino acid.
Amino Acid Sequence↗
Filtration of cerebrospinal fluid in acute inflammatory demyelinating polyneuropathy (Guillain-Barré syndrome).
INTRODUCTION: Acute Guillain-Barré syndrome (GBS) is a disease where cell-mediated and humoural immune reactions play a key role. Breakdown of the blood-nerve barrier, inflammation of the nerve roots and conduction block are leading symptoms. As it is likely that pathological humoural or cellular factors are concentrated in the subarachnoid space and since factors in the cerebrospinal fluid (CSF) of GBS patients were shown to block sodium channels, a direct therapeutic intervention in the intrathecal compartment seemed worthwhile. METHODS: For this purpose, we developed a technique to purify CSF from pathological factors. Filtration was achieved by withdrawal of CSF (through a lumbally inserted 18 G catheter) and reinfusion via specially developed filters. RESULTS: Twenty severely affected GBS patients were treated with 4 to 38 CSF filtrations. This eliminated cells (> 99%) and reduced the protein content. Clinical improvement was achieved probably by improvement of nerve conduction following a reduction of sodium channel blocking factors. The median time to one grade improvement was 19 days, to reach grade 2 (independent walking) was 42 days. Ventilated patients were weaned from the ventilator after 16 days (median). Patients for which CSF filtration was the first kind of treatment improved faster than patients that had not responded to other treatments, such as plasma exchange or intravenous immunoglobulins. DISCUSSION AND CONCLUSION: Severe side effects were not observed, except sometimes transient headache during the withdrawal phase. This therapy seams to shorten treatment time (in comparison to times reported in studies with and without GBS-specific treatment by plasma exchange or immunoglobulins) and to reduce the degree of remaining neurological deficits. An open randomized controlled study is currently being conducted to compare CSF filtration with plasma exchange.
Acute Disease↗
Factors in the cerebrospinal fluid of multiple sclerosis patients interfering with voltage-dependent sodium channels.
The effect of cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) on voltage-dependent Na+ channels in human myoballs was studied. The transient Na+ currents, elicited by whole-cell depolarization from -85 to -20 mV, were decreased to 75-25% the control value in the presence of CSF from all 7 MS patients investigated. The effect was complete in about 5 s and was fully reversible on admission of standard external fluid. Such decrease was not or only to a minor extent observed with 10 out of 11 control CSFs from patients without inflammatory neurological disease. The origin of the factors interfering with the Na+ channels is unknown. It is suggested that, in addition to demyelination, impaired Na+ channel function might cause the symptoms in MS.
Cells, Cultured↗
Specific modifications of the membrane fatty acid composition of human myotubes and their effects on the muscular sodium channels.
The fatty acid (FA) composition of human myotube primary cultures was varied by modifications of the contents of FA in the culture medium. An incubation time of 18 h with a defined FA mixture resulted in the most effective alteration of the original FA pattern of the cells. The increases reached for the relative amounts of palmitic acid (16:0), linoleic acid (18:2) or arachidonic acid (20:4) were 3-5-fold. More than 50% of the extra FA were incorporated in the phospholipid fraction, the remaining share in the triglyceride fraction. Shorter incubation times resulted in less FA incorporation, longer incubation times raised the uptake of FA into the triacylglycerol fraction. For a study of the influence of the membrane modification on the function of the sodium channels, the myotubes were converted into myoballs. The sodium channel properties were then determined using the whole-cell clamp technique. The modified cultures showed no significant alterations in the time constants of activation and inactivation, in the voltage dependence of inactivation (h infinity curves) or in the average amplitudes of the sodium currents.
Arachidonic Acid↗
Genotype-phenotype correlations in human skeletal muscle sodium channel diseases.
BACKGROUND: Over the past 3 years, the genetics of the myotonic diseases have been substantially elaborated. Three genetically different groups of myotonic disease can be discerned: (1) the chloride channel myotonias, (2) the adynamia-paramyotonia complex, and (3) myotonic dystrophy. METHODS AND RESULTS: Electrophysiology has suggested and molecular biology has proven that the diseases belonging to the adynamia-paramyotonia complex, ie, paramyotonia congenita, hyperkalemic and normokalemic periodic paralysis, and some rare forms of myotonic disease, are caused by point mutations in the gene encoding the alpha subunit of the sodium channel in adult human skeletal muscle, located on chromosome 17q23. Thirteen different mutations have been described by various groups in the United States and Germany. The various mutations causing a particular form of the complex are not located in the gene in a predictable or easily understandable regular manner. CONCLUSIONS: Further study of the genotype-phenotype correlations should not only increase our understanding of the variability of signs in this group of diseases, it could also provide us with a deeper insight in the function of the various regions of the sodium channel protein.
Electrophysiology↗
Chloride channels with reduced single-channel conductance in recessive myotonia congenita.
Myoballs were cultured from muscle biopsies of patients having Becker-type recessive generalized myotonia (RGM). Single-channel recordings showed that the conductance of the "intermediate" Cl- channel, the Cl- channel having the highest membrane density and accounting for most of the Cl- conductance in myoballs, was reduced to about 50%. All other channel parameters tested, such as gating properties, ion selectivity, and regulatory pathways, were not different from controls. Two other Cl- channel types were only rarely observed and did not seem different from controls. Also, the macroscopic Cl- conductance measured with tight-seal whole-cell recording was smaller than controls, particularly in the positive membrane potential domain. These results might explain myotonia in RGM on the single-channel level.
Cell Membrane↗
Non-dystrophic myotonias and periodic paralyses. A European Neuromuscular Center Workshop held 4-6 October 1992, Ulm, Germany.
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Chloride Channels↗
Culture conditions for the production of porcine myotubes and myoballs.
The existence of a method for culturing porcine muscle cells would greatly facilitate the development of new breeding criteria for stress resistance and growth regulation in pig breeding. Also, many effects of nutritional or pharmacological components that influence animal performance could be studied first in muscle cell preparations. Therefore, we developed a specific procedure to culture porcine skeletal muscle cells from well-established methods for murine and human muscle cell culturing. Best results were obtained by isolating satellite cells from muscle tissue removed postmortem after normal slaughter procedure, using enzymatic dissociation. The satellite cells were allowed to proliferate for 3 to 5 d in a culture medium composed of 83% Ham's F-12 medium, 15% fetal calf serum (FCS), and 2% chick embryo extract (CEE). Well before reaching confluence, the cells were transferred to collagen-coated dishes filled with Dulbecco's modified Eagle's medium containing 5% horse serum (HS) for the differentiation to multinucleated myotubes. Also, .5% FCS can be used instead of HS. Besides the fusion to myotubes, the presence of voltage-sensitive Na+ channels is regarded as a specific feature of the muscle phenotype of the cells. To perform electrophysiological experiments of good quality, myotubes were converted into freely floating "myoballs." Voltage-clamp experiments in the whole-cell mode showed transient inward currents that had kinetics and voltage dependences very similar to those of the Na+ currents in human myoballs. The porcine Na+ currents were almost completely blocked by 1 mumol/L of tetrodotoxin, indicative of the presence of the adult form of the Na+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Giga-seal formation alters properties of sodium channels of human myoballs.
The influence of giga-seal formation on the properties of the Na+ channels within the covered membrane patch was investigated with a whole-cell pipette and a patch pipette applied to the same cell. Current kinetics, current/voltage relation and channel densities were determined in three combinations: (i) voltage-clamping and current recording with the whole-cell pipette, (ii) voltage-clamping with the whole-cell pipette and current recording with the patch pipette and, (iii) voltage-clamping and current recording with the patch pipette. The Hodgkin-Huxley (1952) parameters tau m and tau h were smaller for the patch currents than for the whole cell, and the h infinity curve was shifted in the negative direction. The channel density was of the order of 10 times smaller. All effects were independent of the extracellular Ca2+ concentration. The capacitive current generated in the patch by the whole-cell Na+ current and its effect on the transmembrane voltage of the patch were evaluated. The kinetic parameters of the Na+ channels in the patch did not depend on whether the voltage was clamped with the whole-cell pipette or the patch pipette. Thus, the results are not due to spurious voltage.
Electric Conductivity↗