Brain, Vol. 122, No. 6, 1085-1092,
June 1999
© 1999 Oxford University Press
Mutant channels contribute <50% to Na+ current in paramyotonia congenita muscle
1 Departments of Applied and 2 General Physiology and 3 Neurology, University of Ulm, Germany and 4 Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, Tenn., USA
Correspondence to:
Dr Nenad Mitrovic, Department of Applied Physiology/Neurology, University of Ulm, D-89069 Ulm, Germany E-mail: nenad.mitrovic{at}medizin.uni-ulm.de
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An important question in the pathophysiology of dominantly inherited diseases, such as channelopathies, is the level of expression of the mutant protein. In our study, we address this issue by comparing the gating defects of two human muscle Na+ channel mutants (R1448C and R1448P) causing paramyotonia congenita in native muscle specimens from two patients with those of the same mutant recombinant channels expressed in human embryonic kidney (HEK-293) cells. Patch-clamp recordings of transfected HEK-293 cells revealed a pronounced slowing of the Na+ current decay, a left-shifted and decreased voltage dependence of steady-state inactivation, and an increased frequency of channel reopenings for mutant compared with wild-type channels. For R1448P channels, inactivation was almost six-fold and for R1448C it was three-fold slower than for wild-type channels. The same defects, though less pronounced, as expected for a disorder with dominant inheritance, were observed for muscle specimens from paramyotonia congenita patients carrying these mutations. Quantitative kinetic analysis of Na+ channel inactivation in the paramyotonic muscle specimens separating wild-type from mutant channels suggested that no more than 38% of the channels in the paramyotonia congenita muscle specimen were of the mutant type. Our data raise the possibility that variability in the ratio of mutant to wild-type Na+ channels in the muscle membrane has an impact on the clinical severity of the phenotype.
Na+ channel; inactivation; channelopathies; patch-clamp; human skeletal muscle
ISS/IPEAK = relative persistent Na+ current
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Introduction |
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TopAbstractIntroductionMaterial and methodsResults and discussionReferences |
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Paramyotonia congenita is a dominantly inherited muscle disorder characterized by paradoxical myotonia, which is muscle stiffness increasing with repeated activity, and weakness triggered by exposure to cold. Stiffness and weakness are caused by an unstable muscle fibre membrane potential. EMG recordings show long-lasting series of action potentials during muscle effort or electrical silence during periods of paralysis.
Normal excitability of muscle fibres requires a high resting potential and short-lasting action potentials. Neither requirement is fulfilled in paramyotonia congenita, as shown with microelectrode recordings on excised muscle from patients. Voltage-clamp studies of single fibres have revealed that inactivation of Na+ currents is incomplete (Lehmann-Horn et al., 1987
).
Recently, we applied the patch-clamp technique to blebs of muscle fibres from a paramyotonia congenita patient carrying the arginine-1448-proline (R1448P) mutation within the voltage sensor IV/S4 of the 
-subunit of the human skeletal muscle Na+ channel (Lerche et al., 1996). The major abnormal findings were that (i) the defective channels tend to reopen spontaneously after a `physiological' opening upon depolarization and (ii) the sum of responses from many channels showed a much slower Na+ current decay. These reopenings increase Na+ influx into the muscle fibres, causing either slight depolarization with hyperexcitability (myotonia) or substantial depolarization with inexcitability (paralysis) of the muscle.
About 20 different disease-causing mutations have been found in the gene encoding the -subunit of the adult skeletal muscle Na+ channel, six of them resulting in paramyotonia congenita [the others cause either hyperkalaemic periodic paralysis or potassium-aggravated myotonia, reviewed by Lehmann-Horn and Rüdel (Lehmann-Horn and Rüdel, 1996)]. The electrophysiological properties of an ion channel can be elegantly examined by expressing the recombinant channel DNA in cultivated cells. Advantages are that (i) the Na+ channel population is homogeneously wild type or mutant and (ii) muscle biopsy is not required. Direct comparison of wild-type and mutant channels in both natural and artificial milieus has not been performed. Here, we provide a detailed analysis of the R1448P mutation, which has not been expressed in human embryonic kidney (HEK-293) cells before and we compare two mutations causing paramyotonia congenita (R1448C and R1448P) in both expression systems—native muscle of paramyotonia congenita patients and HEK-293 cells. A quantitative analysis of Na+ channel inactivation kinetics allowed us to estimate that in the patient's muscle the mutant channels contribute only 20–38% of the Na+ current, less than expected for a disease with a dominant mode of inheritance.
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Material and methods |
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The arginine-1448-cysteine/proline mutations were created by site-directed mutagenesis using the Altered Sites system (Promega Corporation, Madison, Wis., USA) as described (Chahine et al., 1994
). Wild-type and mutant constructs were assembled in the mammalian expression vector pRC/CMV and transfected into human embryonic kidney cells (HEK-293) by the calcium phosphate precipitation method. Stable cell lines expressing either wild-type or mutant channels were obtained by antibiotic selection as described (Mitrovic et al., 1994).
Standard whole-cell recordings were performed using an EPC-7 amplifier (List, Darmstadt, Germany). The voltage error due to series resistance was <5 mV. Leakage and capacitative currents were subtracted automatically by a prepulse protocol (–P/4). All data were filtered with 3 kHz and digitized at 20 kHz using pClamp 6.02 (Axon Instruments, Foster City, Calif., USA). Analysis was based on pClamp, Excel 97 (Microsoft, Redmond, Mass., USA), Origin 5.0 (Microcal Software, Northampton, Mass., USA), Sigma Plot 5.0 (Jandel Scientific, San Rafael, Calif., USA) and our own software. The solutions were as follows (in mM, pH 7.4). Whole-cell recordings: pipette: 135 CsCl, 5 NaCl, 2 MgCl2, 5 EGTA and 10 HEPES; bath: 140 NaCl, 4 KCl, 2 CaCl2,1 MgCl2, 4 dextrose and 5 HEPES. Single channels were recorded in the cell-attached configuration using the following solutions: pipette: 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 TEACl and 10 HEPES; bath: 150 KCl, 5 NaCl, 2 CaCl2, 1 MgCl2 and 10 HEPES. Recordings from sarcolemmal blebs of native muscle were performed in the bleb-attached mode as described by Lerche and colleagues (Lerch et al., 1996). Biopsy was performed under local anaesthesia from the quadriceps muscle with informed consent of the patient, as described by Lerche and colleagues (Lerche et al., 1996). All procedures were in accordance with the Helsinki convention and were approved by the Ethical Committee of the University of Ulm. Experiments were performed at 21°–22°C if not stated otherwise. The temperature was controlled via a water-perfused Petri dish holder. For statistical evaluation, Student's t test was applied. All data are shown as mean ± standard error of the mean.
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Results and discussion |
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Voltage-gated Na+ channels are the basis for the generation and conduction of action potentials in nerve and muscle cells. They open briefly upon depolarization and then close to a fast inactivated state from which they reopen very rarely. Thus fast inactivation limits the duration of an action potential and initiates its repolarizing phase. The muscle membrane becomes inexcitable for a short period of time after an action potential; this is the so-called refractory period. This time is determined by the recovery of the Na+ channel from inactivation. Thus, recovery from inactivation limits the firing rate of nerve and muscle cells. These characteristics of Na+ channels can be determined electrophysiologically using the patch-clamp technique. By comparison of the mutant with the wild-type phenotype, gating defects can be elucidated in detail.
Whole-cell recordings of Na+ currents through R1448P channels in HEK-293 cells
The R1448P mutation results in the substitution of a neutral proline for a positively charged arginine in the S4 segment of domain IV of the Na+ channel -subunit, which is composed of four highly homologous domains containing six transmembrane segments each (for review, see Catterall, 1995). Positively charged residues in S4 segments constitute the Na+ channel voltage sensor and play a central role in Na+ channel gating (for review, see Horn, 1999). Families of Na+ currents for wild-type and R1448P channels are shown in Fig. 1A and B. Currents of the mutant channels decayed much more slowly than those of the wild type, indicating impaired channel inactivation. The current decay was best fitted with the sum of two exponentials, yielding two inactivation time constants (
h1 and h2). With respect to wild type, for R1448P the faster time constant h1 (relative weight >75%) was almost sixfold larger at a test potential of 0 mV (Fig. 3B).
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The persistent Na+ current (ISS) represents the proportion of non-inactivating channels. We have shown previously that ISS may cause a sustained membrane depolarization in paramyotonia congenita muscle fibres, resulting in the clinical symptom of paralysis (Lehmann-Horn et al., 1987
; Lerche et al., 1996). Therefore, we determined a persistent current at the end of a depolarizing test pulse and normalized it to the peak current (IPEAK). It was increased 1.5-fold in R1448P channels compared with wild-type channels (Fig. 1C
).
For R1448P channels, the steady-state activation curve describing the voltage dependence of channel opening was shifted by –8 mV (Fig. 1D). This shift might represent a real alteration of the activation process, but could also reflect the influence of the slowed inactivation on our procedure for determining the activation curve: all currents were normalized to the largest peak current, which was delayed in R1448P. The steady-state inactivation curve, representing the proportion of non-inactivated Na+ channels, was shifted by –7 mV and its slope was markedly reduced (Fig. 1D). These changes have two important implications with regard to the weakness in paramyotonia congenita: (i) at the resting potential, the number of Na+ channels available for an action potential is markedly reduced (at –80 mV only 60% of mutant channels versus 95% of wild-type channels), and (ii) at less negative potentials the increased overlap of the activation and inactivation curves creates a larger permanent `window' current. The first point directly reduces excitability; the second furthers depolarization, i.e. it also reduces excitability.
Recovery from inactivation was significantly accelerated and its voltage dependence decreased for R1448P (Fig. 1E). The faster recovery from inactivation, which was also found for several other paramyotonia congenita or potassium-aggravated myotonia-causing mutations (Chahine et al., 1994; Yang et al., 1994; Mitrovic et al., 1994), promotes the development of myotonia by shortening the refractory period after an action potential.
The tail current decay describes Na+ channel deactivation, the closing of open channels upon membrane repolarization. For some mutations causing potassium-aggravated myotonia (Mitrovic et al., 1995) but also for the R1448P mutation (Featherstone et al., 1998), it has been proposed that slowed deactivation increases the Na+ inward current and may therefore contribute to membrane depolarization and myotonia. In our study, the deactivation time constants were identical for both channel types in the voltage range where no inactivation occurred (Fig. 1F), suggesting normal deactivation for R1448P.
In summary, R1448P affects inactivation and recovery from inactivation with minor effects on activation. Recent studies have shown that S4 segments in Na+ channels are specialized in their function. Whereas S4s in the first three domains play a more important role in activation, the S4 segment of the fourth domain seems to serve a double duty, but a more important one in inactivation (Stühmer et al., 1989; Chahine et al., 1994; Chen et al., 1996; Yang et al., 1996; Mitrovic et al., 1998).
The electrophysiological phenotype of R1448C channels has been described in detail before (Chahine et al., 1994). We would like to summarize the most important gating differences from wild-type and R1448P channels found in our study, which is important in the light of the comparison of native channels with those expressed in HEK cells, since these experiments were performed with both the R1448P and the R1448C mutation. R1448C channels were expressed, measured and analysed in the same way as described above for wild-type and R1448P channels. The fast inactivation time constant, h1, was 1.46 ± 0.08 ms at 0 mV, thus only threefold higher than that for the wild type, whereas R1448P channels showed a 5.8-fold increase in h1. The negative shift of steady-state inactivation was more pronounced for R1448C than for R1448P channels (V0.5 = –79 ± 2 mV for R1448C), while the steepness was not decreased as much for R1448C (k = 11.4 ± 0.3 mV). Steady-state activation was slightly shifted towards more negative potentials, and recovery from inactivation was accelerated for R1448C compared with wild type, as has been described previously (Chahine et al., 1994).
Single-channel studies and the effects of temperature
The slowing of inactivation and the increased persistent current could be due to an increased frequency of reopenings, to prolonged openings and/or later first openings of the mutant channels. To discriminate between these possibilities, we recorded single-channel currents using test pulses from a holding potential of –120 mV to –30 mV. First latencies were similar (0.6 ± 0.1 versus 0.7 ± 0.1 ms in wild type, n = 3–4) and mean open times were slightly prolonged for R1448P compared with wild-type channels. The mean open time histograms were best fitted with the sum of two exponentials. The respective time constants for R1448P and wild type were 1 = 0.26 ± 0.02 and 0.19 ± 0.01 ms (P < 0.01), 2 = 0.97 ± 0.06 and 0.83 ± 0.10 ms (P > 0.05); relative weights of 2 were 50 ± 3 and 25 ± 4% (P < 0.001) (n = 8–13). Normalized late Na+ currents calculated from reopenings were significantly increased in R1448P (Fig. 2A), corresponding to the larger persistent current in macroscopic recordings.
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Since the symptoms of paramyotonia congenita are aggravated by cold, we evaluated the effects of temperature on the kinetics of Na+ channel inactivation and on late Na+ currents in the range from 15° to 30°C. Slower current decay and increased persistent current were seen over the whole temperature range for R1448P channels (Fig. 2A and B
). The temperature sensitivity was determined as the activation energy Ea from Arrhenius plots for h1. The value of Ea was not increased for R1448P channels (Fig. 2B). These data suggest that it is not an increased temperature dependence of mutant channels that underlies the sensitivity to cold in paramyotonia congenita, as has also been found for several other paramyotonia congenita mutations (Chahine et al., 1994; Yang et al., 1994; Hayward et al., 1996; Fleischhauer et al., 1998). We therefore propose that a certain threshold of inactivation failure has to be exceeded in a cold environment to induce myotonia and/or paralysis.
Pathophysiology of paramyotonia congenita
One of the key symptoms in paramyotonia congenita is paradoxical myotonia, muscle stiffness increasing with continued activity, which is in contrast to the `warm-up' phenomenon seen with the chloride channel myotonias Thomsen and Becker. Paramyotonia congenita-causing mutations result predominantly in a slowing of fast inactivation (Chahine et al., 1994; Yang et al., 1994; Hayward et al., 1996), which is most pronounced in R1448P. The slowed current decay provides a plausible explanation for paradoxical myotonia since in this case continued exercise would result in the accumulation of intracellular Na+ and muscle depolarization.
Previous studies on paramyotonia congenita muscle fibres have shown that paralysis is caused by increased Na+ influx depolarizing the muscle fibres to –40 mV and rendering them inexcitable (Lehmann-Horn et al., 1987; Lerche et al., 1996). With regard to our electrophysiological results, we propose that a combination of an increased persistent current, exceeding a certain threshold in the cold, and the left-shift of steady-state inactivation cause weakness and paralysis in paramyotonia congenita.
Comparison of currents through mutant channels in HEK-293 cells and in paramyotonia congenita muscle
Inactivation was more slowed in HEK cells than in the paramyotonia congenita muscle (Fig. 3) because the former contain 100% mutant channels, whereas the muscle should contain 50% according to dominant inheritance. To pursue our main aim, i.e. to calculate the percentage of mutant channels in muscle from patients carrying the R1448P or R1448C mutations, we analysed and compared the decay of the Na+ currents in HEK cells and in muscle. This approach was justified because the properties of wild-type channels were very similar in the two expression systems. In both systems, the current decay of wild-type channels was well fitted to a second-order exponential function with a major fast time constant h1 (relative weight > 90%) only slightly prolonged in muscle (0.48 ± 0.01 ms at 0 mV in HEK cells and 0.57 ± 0.03 ms at –15 mV in muscle). We compared a test potential of 0 mV in HEK cells with –15 mV in muscle since the I–V curve was shifted by –15 mV and peaked at these potentials [the steady-state activation and inactivation curves were shifted in the hyperpolarizing direction by –15 and –30 mV, respectively, most probably owing to giga-seal formation in cell-attached patches (Fahlke and Rüdel, 1992)].
Previously, we compared Na+ channels in muscle from a paramyotonia congenita patient carrying the R1448P mutation and from normal controls by fitting the current decay to a second-order exponential function (Lerche et al., 1996; Fig. 3A). The largest difference between paramyotonia congenita and control was a 1.6-fold increase in h1 (0.57 ± 0.03 and 0.93 ± 0.05 ms; n = 7–15). In contrast, in HEK cells the most pronounced difference was a 5.8-fold increase in h1 for R1448P compared with wild-type channels (Fig. 3B; h1 at 0 mV, 0.48 ± 0.01 and 2.77 ± 0.11 ms; n = 10–16). This result already suggests that the percentage of mutant channels in the paramyotonia congenita muscle specimen is less than 50%. Compared with the drastic changes in the major fast inactivation time constant, differences in h2 and its relative weight were small, or at least that their contribution to the slowing of the current decay was not large (values in native control versus paramyotonia congenita muscle at –15 mV: h2, 6.2 ± 0.4 versus 7.2 ± 1.0 ms; A2, 6.8 ± 0.9 versus 23.3 ± 1.9%; values in HEK cells for wild type versus R1448P channels at 0 mV: h2, 8.1 ± 1.3 versus 12.2 ± 2.1 ms; A2, 3.3 ± 0.5 versus 18.5 ± 2.9%).
In order to separate the different time constants deriving from mutant and wild-type channels in paramyotonia congenita muscle, we fitted multiple exponentials to the current decay at –15 mV. Theoretically, there should be four different time constants of inactivation, i.e. h1 and h2 for both wild-type and mutant channels. However, it was not possible to separate the two slow time constants because their amplitudes were too small and their difference was only marginal (see above). Three time constants could be well separated. We concluded that the shortest time constant represented the fast inactivation component of the wild-type channels: the intermediate time constant represented the fast inactivation component of the mutant channels and the slowest time constant represented the slow inactivation components of both channel types: I(t)/IPEAK = AWT exp(–t/h1WT) + AMT exp(–t/h1MT) + A2 exp(–t/h2) + ISS/IPEAK (where WT = wild type and MT = mutant). For accurate separation of the wild-type and mutant time constants and determination of all relative amplitudes, we kept h1WT fixed at the average value found for control muscle (0.57 ms) and h2 at the value obtained from a monoexponential fit of the slowly decaying part of the current (15–45 ms after onset of the depolarization). The values of h1MT and all amplitudes were left as free parameters. With these settings, h1MT for R1448P channels was 2.25 ± 0.17 ms with a relative amplitude A1MT/(A1WT + A1MT) of 38 ± 5% (n = 5). When we repeated the fit using the fixed value of h1MT = 2.77 ms obtained from R1448P channels expressed in HEK cells, the result was similar (35 ± 3% mutant channels). When the currents of control muscle were fitted in the same way, A1MT/(A1WT + A1MT) was negligibly small (4 ± 2%, n = 8, h2 = 3.09 ± 0.68 ms).
For a different biopsied muscle specimen from a patient with a moderate form of paramyotonia congenita carrying the R1448C mutation (Becker, 1970), we analysed the current decay using the same fitting procedure as described above. The relative proportion of R1448C channels A1MT/(A1WT + A1MT) was determined to be even smaller than for R1448P (only 19.5 ± 1.6%), with a time constant for the mutant channels of h1MT = 1.71 ± 10.18 ms (n = 5). The relative amplitudes for all patches recorded from both paramyotonia congenita muscles and normal controls are summarized in Fig. 4. Using this third-order exponential fit, the third time constant was similar for control and both paramyotonia congenita muscles, and generally had a small amplitude [h2, 12.4 ± 2.7 (wild type) versus 11.3 ± 1.8 (R1448P) and 12.0 ± 1.8 (R1448C) ms; A2, 6.3 ± 1.7 versus 6.3 ± 4.0 and 3.0 ± 1.3%]. We therefore conclude that this slow time constant did not significantly disturb our analysis.
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For the R1448P mutation, the drastic difference in
h1 ensured a good separation of wild-type and mutant fast inactivation time constants in the paramyotonia congenita muscle specimen. We could clearly separate three time constants of inactivation, whereas in control muscle the relative weight of the second time constant designated to detect mutant channels (h1MT) was negligible. For the R1448C mutation, the result of 20% mutant channels is less reliable because of the smaller inactivation defect (only 3-fold increase in h1), hence the difficulty in clearly separating the time constants from wild-type and mutant channels. The steady-state inactivation curve for the R1448C mutation measured in HEK cells was shifted by –18 mV compared with wild type (V0.5 = –61.5 ± 1.2 and –79.2 ± 2.4 mV; k = 6.1 ± 0.1 and 11.4 ± 0.3 mV; n = 8), and in the paramyotonia congenita muscle containing this mutation this shift was –11.5 mV (V0.5 = –93.7 ± 0.4 and –105.2 ± 2.2 mV; k = 8.4 ± 0.3 and 9.3 ± 0.3 mV; n = 5–12). However, it was not possible to separate both mutant and wild-type channels by fitting the inactivation curves to the sum of two Boltzmann functions in paramyotonia congenita muscle from both patients. A reason for an underestimation of the percentage of mutant channels could be that a higher proportion of mutant channels is inactivated at the prepulse potential of –140 mV. We therefore tested prepulses of up to –160 mV, and could not find a difference in the current decay (results not shown). A prepulse value of –160 mV should be sufficient to let all channels recover from fast inactivation within 300 ms, which was the duration of the prepulse. Since slow inactivation was not significantly different for wild-type and R1448C channels (N. Mitrovic, unpublished results), the holding potential of –100 mV should not have influenced our analysis.
Altogether, we think that our method reliably estimated the percentage of mutant channels in paramyotonia congenita muscle containing the R1448P mutation due to the large difference in h1, which enabled a clear separation of mutant and wild-type channels. The 20% share calculated for R1448C channels could be an underestimate since the difference in h1 was only threefold. Nevertheless, in both cases the proportion of mutant channels was clearly smaller than 50%, as expected from the dominant mode of inheritance.
Using a kinetic analysis of Na+ currents, we were able to estimate the percentage of functional mutant protein in diseased muscle, a parameter so far unknown in the pathophysiology of channelopathies and other dominantly inherited diseases. In contrast to mRNA measurements, western blots, antibody staining and other molecular biological and biochemical approaches, which determine the level of mutant RNA or protein without regard to its function, the electrophysiogical evaluation allowed us to determine the ratio of the mutant protein that is pathophysiologically relevant. The reduced percentage of mutant channels may have several reasons. The mutated gene could be less expressed or the translation could be reduced. Also, glycolysation of the protein could be impaired or its incorporation in the membrane could be impeded. In any case, a share of the mutant channels smaller than 50% should reduce the severity of the clinical phenotype. For another Na+ channelopathy, the equine periodic paralysis in Quarter horses, the severity of the clinical phenotype has been shown to correspond to mRNA levels in native muscle probes (Zhou et al., 1994).
The phenotype of our patient carrying the R1448P mutation is severe (Wang et al., 1995; Lerche et al., 1996), while the phenotype of the other patient is moderate [kinship Q, patient V/26 in Becker (Becker, 1970); Haass et al., 1981]. This was also verified by recent clinical examinations in Ulm. Both patients showed paradoxical myotonia at room temperature, and muscle stiffness upon cooling in ice-cold water followed by weakness lasting up to an hour. The myotonic features were much more pronounced for the patient carrying the R1448P mutation. The gait of this patient was never normal. She walked on her toes and complained about pain in her calves (Wang et al., 1995; Lerche et al., 1996). In the case of the patient carrying the R1448C mutation, the less severe myotonic phenotype could be explained by both the smaller inactivation defect and a reduced number of mutant channels. However, we would like to mention that the R1448C mutation may also cause a severe phenotype in other patients. A possible explanation for this discrepancy may be a higher percentage of mutant channels in these patients.
Although the R1448C patient experiences less severe myotonic symptoms, he suffers from episodes of weakness in a warm environment, similar to patients with hyperkalaemic periodic paralysis, which has not been reported for the R1448P patient. This is in accordance with our hypothesis that weakness is favoured by a hyperpolarizing shift of the steady-state inactivation curve (Lerche et al., 1996; Wagner et al., 1997), which is clearly more pronounced for R1448C than for R1448P channels.
In summary, our data raise the possibility that the clinical phenotype of paramyotonia congenita depends not only on the degree of abnormality of the current conducted by the mutant channels but also on their fraction in the membrane.
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We wish to thank Drs R. Horn and A. C. Ludolph for helpful discussion, Dr R. Böhlen for collaboration and U. Pika-Hartlaub for performing expert cell culture. This study was supported by the Deutsche Forschungsgemeinschaft (Le481/3), the Muscular Dystrophy Association and the National Institutes of Health (NS32387).
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Received June 25, 1998. Revised January 11, 1999. Accepted February 1, 1999.
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