Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel

Cecilia V. Rojas1, Alan Neely2, Gabriela Velasco-Loyden1, Verónica Palma3, and Manuel Kukuljan3

1 Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Casilla 138-11, Santiago; and 3 Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70005-7, Santiago, Chile; and 2 Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Mutations in the human skeletal muscle Na+ channel underlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na+ currents thought to depolarize the sarcolemma and thus inactivate normal Na+ channels. We expressed human wild-type or M1592V mutant alpha -subunits with the beta 1-subunit in Xenopus laevis oocytes and recorded Na+ currents using two-electrode and cut-open oocyte voltage-clamp techniques. The most prominent functional difference between M1592V mutant and wild-type channels is a 5- to 10-mV shift in the hyperpolarized direction of the steady-state activation curve. The shift in the activation curve for the mutant results in a larger overlap with the inactivation curve than that observed for wild-type channels. Accordingly, the current through M1592V channels displays a larger noninactivating component than does that through wild-type channels at membrane potentials near -40 mV. The functional properties of the M1592V mutant resemble those of the previously characterized HPP T704M mutant. Both clinically similar phenotypes arise from mutations located at a distance from the putative voltage sensor of the channel.

sodium current; ion channel; neuromuscular disease; gating; Xenopus oocytes

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

FAST ACTIVATION AND inactivation of voltage-gated Na+ channels underlie the rapid changes in Na+ permeability that account for the firing of short action potentials in nerve and muscle cells (12). The biophysical analysis of Na+ currents through native and recombinant channels, as well as data obtained from the study of voltage-gated K+ channels, has rendered a picture of the molecular mechanisms that link membrane potential to the gating of the Na+ permeation pathway (1, 2, 4, 6, 10, 11, 15, 26). The discovery that some inherited muscular diseases are caused by mutations in the gene that encodes the human skeletal muscle Na+ channel alpha -subunit has provided an additional basis both for a better knowledge of the structure-function relationship of Na+ channels and for the understanding of the pathophysiology of these syndromes at the molecular level (1, 2).

The first insights linking a group of hereditary muscular diseases to Na+ channel function came from electrophysiological recordings made from muscle biopsies of individuals affected by hyperkalemic periodic paralysis (HPP) (16). These recordings showed a persistent tetrodotoxin-sensitive Na+ current, which is thought to cause a depolarization of the sarcolemma that leads to the inactivation of Na+ channels and thus to the inability of the muscle to fire action potentials. More than a dozen naturally occurring mutations associated with HPP, paramyotonia congenita, and atypical myotonia have been found in the alpha -subunit of the skeletal muscle Na+ channel (1).

Eleven mutations responsible for paramyotonia congenita and atypical myotonia were shown to exhibit similar functional defects when expressed in heterologous systems (4, 10, 15, 17, 18, 26). The most striking alterations in the properties of these mutant channels are the shift in the steady-state inactivation toward depolarized membrane potentials, a decreased rate of inactivation, and a fast rate of recovery from fast inactivation.

To date, only four mutations associated with HPP have been described (23, 28, 29, 32). The functional characterization of the most common mutation, T704M (and its rat homologue rT698M), indicates a shift in the steady-state activation of the Na+ current toward membrane potentials negative to those of wild-type channels. No significant change in the rate of fast inactivation and virtually the same rate of recovery from inactivation as wild-type channels were found (7, 34). However, Cannon and Strittmatter (3), studying the rat homologue rT698M, did not report differences in the activation properties but found faster recovery from slow inactivation in the mutant than in the wild-type channels (7, 11).

The second most common HPP mutation, M1592V, has only been made in the rat skeletal muscle Na+ channel alpha -subunit (rM1585V) (3). The functional characterization of the rM1585V mutant revealed no shift in activation, whereas it displayed larger noninactivating Na+ current than wild-type channels. It was proposed that this persistent current could arise from the increased probability that mutant Na+ channels activate in a slow mode of gating characterized by multiple reopenings. Here we undertook the biophysical characterization of the M1592V mutation in the human skeletal muscle Na+ channel. Our main interest was to assess whether the clinical HPP phenotype is associated with similar biophysical defects in the human skeletal muscle Na+ channel function and, if so, to set the basis for a further understanding of how distant residues located in "inconspicuous" domains of the alpha -subunit (Fig. 1) can affect the channel gating properties. We found a shift of the activation curve to less depolarized potentials in M1592V channels, allowing the expression of a noninactivating component of the Na+ current significantly larger than in wild-type channels. These results are compatible with the original observations of Lehmann-Horn et al. (16) and comparable to those reported for the HPP T704M mutant (7, 34). Remarkably, these mutated residues (T704 and M1592) are located in domains at a distance from the putative voltage sensor domains of the Na+ channel.


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Fig. 1.   Location of hyperkalemic periodic paralysis (HPP) mutations. Proposed topology of Na+ channel alpha -subunit is shown. Subunit consists of 4 homologous domains (I, II, III, and IV), each with 6 membrane-spanning segments. Mutation M1592V studied here (and its rat homologue M1585V) is located in 6th transmembrane segment of domain IV. T704M (and its rat homologue T689M), the most common HPP mutation, is located in 5th transmembrane segment of domain II.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Molecular cloning of the alpha - and beta 1-subunits. cDNAs of the alpha - and beta 1-subunits from the human skeletal muscle Na+ channel were cloned by RT-PCR from muscle biopsies. The design of PCR primers was based on the sequence for rat and human adult skeletal muscle Na+ channel alpha -subunit. The entire coding region of the wild-type alpha -subunit was assembled in the pBluescript vector (Stratagene, La Jolla, CA) from the RT-PCR-amplified fragments and sequenced (33). In this construct, the cDNA fragment encoding the first nine amino acids was replaced by the sequence that encodes the amino terminus of the human alpha -subunit reported by McClatchey et al. (22). The entire coding region of the beta 1-subunit was amplified from cDNA using PCR primers based on rat brain (14) and human brain (21) beta 1-subunit sequences and was cloned into pBluescript. The sequence of the amplified cDNA is identical to that reported for the human beta 1-subunit (20).

Mutagenesis. The coding region of the cDNA for the wild-type alpha -subunit was subcloned in pALTER vector (Promega, Madison, WI). Mutagenesis was performed by oligonucleotide-directed mutagenesis using the Altered Sites system (Promega). The M1592V mutation was confirmed by sequencing the cDNA fragment around base 4776. Two independent clones for both the wild-type and M1592V alpha -subunits were used in the functional expression studies.

In vitro transcription. For functional expression in Xenopus laevis oocytes, both alpha - and beta 1-subunit cDNAs were subcloned into pGEMHE. In this vector, the cloning site is flanked by 5' and 3' untranslated sequences from the X. laevis beta -globin gene (19). In vitro transcription and capping were carried out simultaneously by the T7 bacteriophage RNA polymerase, using the mMessage mMachine kit (Ambion, Austin, TX).

Microinjection of transcripts into X. laevis oocytes. Isolation and injection of stage V or VI oocytes were done according to standard procedures (8). After injection, oocytes were incubated for 1-5 days at 18°C in sterile ND-96 solution (in mM: 96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES; pH 7.6) supplemented with 5 mM sodium pyruvate and 50 µg/ml gentamicin. Oocytes were microinjected with 50 nl of a mixture of the transcripts for the alpha - and beta 1-subunits. The optimum ratio of alpha - and beta 1-subunit transcripts for injection was determined empirically by evaluating the kinetics of the decaying phase of macroscopic Na+ currents. Typically, a 6- to 10-fold molar excess of beta 1-subunit transcripts was injected.

Voltage-clamp recordings. Expressed currents were recorded from seven batches of oocytes. In three of these batches, currents were recorded from both wild-type and mutant channels. Two-electrode recordings were done as described by Stühmer (30), using an oocyte clamp OC725B amplifier (Warner Instruments, Hamden, CT). The solution in the bath was ND-96. Electrodes were made from borosilicate glass (Corning 7740) on a horizontal puller (P-87, Sutter Instruments, Novato, CA), and the tips were broken manually to obtain a resistance of 0.3-0.6 MOmega for the current electrode and 0.7-1 MOmega for the voltage electrode. Electrodes were filled with 3 M KCl. Voltage pulse protocols were applied using the pCLAMP software package (Axon Instruments, Foster City, CA). Na+ currents were also recorded using the cut-open oocyte voltage-clamp (COVC) technique with a CA-1 amplifier (Dagan, Minneapolis, MN). Oocytes were mounted in a three-compartment chamber (31). The oocyte membrane exposed to the bottom chamber was permeabilized by a brief treatment with 0.1% saponin. The voltage pipettes, filled with 3 M KCl, had tip resistances of 0.6-1.2 MOmega . The internal solution contained 120 mM N-methyl-D-glucamine, 10 mM EGTA, and 10 mM HEPES, adjusted to pH 7.0 with methanesulfonic acid. The solution in the external and in the guard compartment was ND-96. Leakage currents were subtracted online by a P/4 protocol. The maximal amplitude of the Na+ currents acquired for analysis ranged from 1 to 15 µA for two-electrode voltage-clamp (TEVC) recordings and from 0.1 to 0.5 µA for COVC recordings. Current signals acquired online were low-pass filtered at 2 kHz (8-pole Bessel filter) and digitized at 10 kHz.

To examine steady-state activation properties of Na+ currents, a series of depolarization pulses was applied from a holding potential of -110 mV (TEVC) or -90 mV (COVC). The activation curves were calculated with the equation G = I/(Vtest - Vrev), where G is the conductance, I is the measured peak current, Vtest is the test potential, and Vrev is the reversal potential calculated by extrapolation from I vs. Vtest plots to zero-current level. To determine half-maximal activation (V1/2) and the slope factor (k), the voltage dependence of activation was fitted with a Boltzmann distribution, G/Gmax = 1/{1 + exp[(V - V1/2)/k]}, where Gmax is maximum G. For steady-state fast inactivation analysis, 50-ms-long prepulses to different potentials were followed by a 40-ms test pulse to -10 mV. The holding potential was set at -140 mV. The steady-state inactivation curves were fitted with a Boltzmann distribution, I/Imax = 1/{1 + exp[(V - V1/2)/k]}, where Imax is maximum I and V is the prepulse potential. For the determination of the recovery from inactivation, two 10-ms pulses separated by an increasing time interval were given to -10 mV from the holding potential. The holding potential was set from -130 to -70 mV and the interval separating the depolarizing pulses ranged from 0.24 to 67 ms. The time constant of the recovery from inactivation (tau ) was calculated with the equation I/Imax = a - b · exp(-t/tau ), where a and b are constants and t is the time interval. To compare fast inactivation kinetics of wild-type and mutant currents, the rapid phase of the decay of macroscopic current traces was fitted to a single-exponential function I = a + b · exp(-t/tau h), where t is the time from the beginning of the test pulse and tau h is the time constant of the decay. Data were analyzed using pCLAMP and SigmaPlot software (Jandel Scientific, San Rafael, CA). Least squares fitting was done with the SigmaPlot program.

Data are presented as means ± SE. Statistical significance was determined by the Student's t-test for unpaired data, with a confidence limit of 95%.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

To study the electrophysiological consequences of the HPP-associated mutation M1592V, the A-to-G transversion was made by site-directed mutagenesis at position 4776 in the cDNA that encodes the Na+ channel alpha -subunit from human adult skeletal muscle. Functional expression was carried out in X. laevis oocytes injected with both alpha - and beta 1-subunit cRNAs. Transient inward currents evoked by depolarizing pulses displayed the typical pattern of activation and inactivation of voltage-gated Na+ currents. Figure 2, A and B, illustrates families of currents, recorded by the TEVC technique, that result from the expression of wild-type and M1592V channels. Normalized current-voltage (I-V) relationships are displayed in Fig. 2C. The maximal amplitude of the inward current for wild-type channels was elicited by depolarizing pulses to -12 mV, whereas M1592V-injected oocytes displayed inward currents with maximal amplitude at -15 mV. Time to peak at -15 mV was not statistically different (P = 0.5) between wild-type (1.62 ± 0.05 ms; n = 9) and mutant channels (1.56 ± 0.06 ms; n = 9). Normalized peak conductance-voltage (G-V) relationships are illustrated in Fig. 2D. Experimental data were fitted to a two-state Boltzmann distribution to determine the steady-state activation parameters. Half-maximal current activation was at -22.25 ± 0.15 mV (n = 14) for the wild-type channels and at -26.79 ± 0.13 mV (n = 14) for M1592V channels (P < 0.0001). The slope factor of the fitted function was 5.28 ± 0.13 mV/e-fold increase in conductance for the wild-type channels and 5.45 ± 0.49 mV/e-fold increase in conductance for the M1592V channels (P = 0.6).


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Fig. 2.   Activation of wild-type and M1592V mutant Na+ currents recorded by 2-electrode voltage-clamp technique. Representative families of current traces recorded from oocytes expressing wild-type (A) and M1592V mutant (B) Na+ channels. Oocytes were injected with a mixture of alpha -subunit (mutant or wild type) and beta -subunit transcripts for human skeletal muscle Na+ channel. Displayed currents were elicited by test depolarizations from -60 to 3 mV in 3-mV steps. Holding potential was -110 mV. Maximal current amplitude at each voltage was normalized with respect to peak current for wild type (bullet ) or M1592V mutant (open circle ) and plotted against test potential (C). Na+ conductance was calculated as described in METHODS, normalized, and plotted against voltage (D). Data in D are means ± SE of 14 experiments. Dotted lines, best fits of Boltzmann distribution to data.

Because the COVC technique reportedly provides a faster and more accurate control of membrane potential, we used this approach to confirm the data obtained with the TEVC method. Figure 3, A and B, shows currents recorded from oocytes expressing wild-type and M1592V channels, respectively. The normalized I-V relationships are shown in Fig. 3C. The maximal amplitude of the inward current for wild-type channels was elicited by depolarizing pulses to -5 mV, and M1592V-injected oocytes displayed inward currents with a maximum at -15 mV. Time to peak for wild-type channels (1.15 ± 0.06 ms; n = 9) and for M1592V channels (1.09 ± 0.05 ms; n = 9) was statistically similar (P = 0.5). Steady-state activation parameters obtained from adjusting a Boltzmann distribution to the normalized G-V data (Fig. 3D) yielded V1/2 of -16.74 ± 0.22 mV (n = 17) for the wild-type channel and -26.74 ± 0.20 mV (n = 18) for the M1592V mutant (P < 0.0001). The slope factor was 6.49 ± 0.20 mV/e-fold increase in conductance for the wild type and 6.88 ± 0.18 mV/e-fold change for M1592V (P = 0.2).


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Fig. 3.   Activation of wild-type and M1592V mutant Na+ currents recorded by cut-open oocyte voltage-clamp (COVC) technique. Representative families of current traces recorded from oocytes expressing wild-type (A) and M1592V mutant (B) Na+ channels. Oocytes were injected with a mixture of alpha -subunit (mutant or wild type) and beta -subunit transcripts for human skeletal muscle Na+ channel. Displayed currents were elicited by test depolarizations from -70 to 25 mV in 5-mV steps. Holding potential was set at -110 mV. Maximal current amplitude at each voltage was normalized with respect to peak current for wild type (bullet ) or M1592V mutant (open circle ) and plotted against test potential (C). Na+ conductance was calculated as described in METHODS, normalized, and plotted against voltage (D). Data in D are means ± SE of 17 and 18 experiments for wild-type and mutant channels, respectively. Dotted lines, best fits of Boltzmann distribution to data.

Both wild-type and mutant channels exhibit fast inactivation as described for alpha - and beta -subunits coexpressed in X. laevis oocytes (9, 13). However, both wild-type and mutant currents display a similar noninactivating component for test potentials above -20 mV, as illustrated in Fig. 4A. However, in our experiments, we detected a significant difference in the extent of whole current inactivation at the end of depolarizing pulses to -40 mV for wild-type and mutant channels. For TEVC recordings, the normalized persistent current at 8 ms was 0.041 ± 0.008 (n = 20) for wild-type channels and 0.092 ± 0.005 (n = 19) for M1592V mutant (P < 0.0001). For COVC recordings, the fractions of persistent current at 8 ms were 0.039 ± 0.003 (n = 12) and 0.101 ± 0.009 (n = 17) for wild-type and mutant channels (P < 0.0001), respectively. The overlap between steady-state inactivation and activation (see Fig. 7) leads to prediction of larger noninactivating currents at -40 mV for M1592V than for wild type.


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Fig. 4.   Kinetics of fast inactivation. Time course of current decay was nearly identical for wild-type and M1592V mutant channels. A: traces of normalized current elicited by depolarizing steps to -15 mV for 20 ms from a holding potential of -110 mV. B: time constants of current inactivation (tau h) for wild type (bullet ) and M1592V mutant (open circle ) were calculated by fitting fast decaying phase of current traces to single exponentials and plotted as a function of voltage. Currents were recorded with COVC technique.

The rapid decay phase of the current could be fitted to a monoexponential function. The plot of the voltage dependence of the time constants for the decay of the whole current (tau h) depicted in Fig. 4B shows that tau h decreases similarly with increasing depolarizing pulses for both wild-type and mutant channels. A small difference in tau h was seen at 10 mV (wild type, 0.39 ± 0.02 ms; M1592V mutant, 0.48 ± 0.03 ms; P < 0.05) and at 20 mV (wild type, 0.33 ± 0.02; M1592V mutant, 0.42 ± 0.02; P < 0.005).

In contrast to activation, steady-state inactivation parameters did not significantly differ for mutant and wild-type channels (Fig. 5). With the COVC technique, half-maximal current inactivation was obtained at -57.36 ± 0.11 mV (n = 15) and at -57.59 ± 0.21 mV (n = 14) for wild-type and mutant channels (P = 0.3), respectively. The slope factor was 7.02 ± 0.10 mV/e-fold decrease in the fraction of current for wild-type channels and 7.39 ± 0.18 mV/e-fold change for mutant channels (P = 0.1). Similarly, when the TEVC technique was used, no differences in the inactivation parameters calculated for wild-type and mutant channels were found (data not shown).


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Fig. 5.   Steady-state inactivation of wild-type and M1592V mutant channels. Steady-state inactivation for wild-type (bullet ) and M1592V mutant (open circle ) channels was measured by a double-pulse protocol, as described in METHODS. Currents recorded with COVC technique were normalized to largest current amplitude and plotted vs. prepulse potential. Data of 14 experiments are expressed as means ± SE. Dotted lines, best fits of data to Boltzmann distribution.

The rate of recovery from inactivation was examined by increasing the interval between two depolarizing pulses to -10 mV from potentials in the range between -140 and -70 mV. Figure 6, A and B, shows the results of experiments carried out by the COVC technique in which the holding and interpulse potentials were set at -70 mV. The time course of the current recovery for each tested voltage was fitted to a single exponential function, as illustrated in Fig. 6C for -70 mV. The time constant at each recovery potential was calculated from the exponential fit to the data and plotted as a function of the voltage (Fig. 6C). Statistical analysis of these data indicates that mutant channels recover slightly faster from inactivation than wild-type channels at voltages above -90 mV. The difference in the rate of recovery was more evident when currents were recorded by TEVC than by COVC. However, with both techniques, the relative difference in the rate of recovery between wild-type and mutant channels tended to decrease at hyperpolarized potentials. The ratios of the rate of recovery (M1592V rate/wild-type rate) calculated from data recorded with COVC were 1.38, 1.44, and 1.66, whereas those calculated from data recorded with the TEVC technique were 1.84, 2.71, and 2.81 for -90, -80, and -70 mV, respectively.


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Fig. 6.   Kinetics of recovery from inactivation. Time course of recovery from inactivation was examined using a 2-pulse protocol. Both depolarizing pulses to -10 mV lasted 10 ms. Currents were recorded for wild-type (A) and M1592V mutant (B) channels at increasing interpulse intervals, with -70 mV as holding and interpulse potential. Current amplitudes elicited during 2nd pulse were normalized with respect to amplitude during 1st pulse and plotted against interpulse interval for wild-type (bullet ) and mutant (open circle ) channels (C). Time constant for recovery is plotted as a function of recovery voltage (D). Dotted lines, best fits to single-exponential equations.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

We have studied the M1592V mutation that underlies HPP by functional expression of the human skeletal muscle Na+ channel in X. laevis oocytes. The alpha -subunit, either wild type or mutant, was coinjected with the wild-type beta 1-subunit. It has been well documented that Na+ channels resulting from the coexpression of alpha - and beta 1-subunits in oocytes display gating properties that are similar to those of native channels (14, 27). Accordingly, we observed the typical pattern of inward Na+ currents evoked by depolarizing pulses when wild-type or mutant alpha -subunits were coinjected with the beta 1-subunits in X. laevis oocytes.

Human skeletal muscle Na+ channels bearing the M1592V mutation display a relevant functional difference with respect to wild-type channels: a shift of the steady-state activation toward less depolarized membrane potentials. This leftward shift in the V1/2 of steady-state activation for M1592V channels was first detected with the TEVC method. When using this technique, V1/2 for M1592V was found to occur at ~5 mV negative to the wild type. We also used the COVC technique, which provides a faster and more accurate control of potential across a segment of the oocyte membrane and found a consistent shift of 10 mV toward hyperpolarized potentials in the V1/2 for the M1592V mutant. The difference in the magnitude of the shift in V1/2 obtained with TEVC and COVC may arise from spatial and time resolution differences that are intrinsic to these methods of clamp control. The slope parameter for the G-V relationship was similar for wild-type and mutant channels, independent of the current recording technique.

Analysis of the steady-state inactivation shows that M1592V channels inactivate in a voltage range and with a voltage dependence indistinguishable from wild-type channels. Therefore, M1592V does not affect the onset of fast inactivation of the human skeletal muscle Na+ channel. Examination of the kinetics of the recovery from the inactivation shows that M1592V channels recover from the inactivated state at a slightly faster rate than the wild-type channels, at voltages above -90 mV.

Currents expressed by the mutant and the wild-type channels displayed similar fast inactivation. Thus currents arising from the expression of M1592V mutants inactivated with a time constant similar to the one measured from oocytes expressing wild-type channels. However, a variable fraction of macroscopic Na+ currents did not inactivate at the end of 8- to 30-ms-long depolarizing pulses above -20 mV. We cannot rule out that the expression of alpha -subunits devoid of beta 1-subunit is the source of this noninactivating component, despite the severalfold molar excess of mRNA coding for the beta 1-subunit over the alpha -subunit messenger injected in each case. It should be noted that at potentials near -40 mV we detected a significant difference between the wild-type and mutant channels in the amplitude of the noninactivating current. At this membrane potential, which corresponds to the region of overlap between steady-state activation and inactivation (Fig. 7), the M1592V mutant displayed twice the persistent current of wild-type channels.


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Fig. 7.   Overlapping of steady-state activation and inactivation. Data from Figs. 3D and 5 show steady-state inactivation for wild-type (black-triangle) and M1592V mutant (triangle ) channels and steady-state activation for wild-type (bullet ) and M1592V mutant (open circle ) channels. Currents were recorded with COVC technique.

Mutations equivalent to the HPP M1592V have been constructed in Na+ channels other than the human skeletal muscle channel. The M1770V mutation was made in the rat brain type IIA Na+ channel (25). M1770 is equivalent to M1592 in the rat brain type IIA alpha -subunit isoform. This mutation did not cause significant effects in the inactivation of the Na+ current expressed in X. laevis oocytes (25). However, substitution of other neighboring amino acid residues in the 6th segment of domain IV had dramatic functional consequences for the brain type IIA Na+ channel (24, 25). The V1774A mutant exhibited the most prominent alterations, with a negative shift in the voltage dependence of activation and incomplete inactivation, although the parameters for steady-state inactivation were similar to those shown by wild-type channels. To assess the functional consequences of the M1592V mutation, Cannon and Strittmatter (3) made the M-to-V substitution in the alpha -subunit from rat skeletal muscle (rM1585V) and used HEK-293t cells for functional expression. The rM1598V mutation caused an enlarged steady-state current (7.5% for mutant vs. 1.4% for wild type at -10 mV) that was explained by an increased frequency of the slow mode of gating in the mutant channel. The authors did not report differences in the voltage dependence of activation or inactivation.

The results we report here are comparable to those arising from the study of the HPP T704M mutation (7, 34). Cummins et al. (7) made the mutation corresponding to T704M in the alpha -subunit from rat skeletal muscle (rT698M). In their study, the most significant effect of the rT698M mutation was a 12-mV leftward shift in the voltage dependence of activation of the mutant channel; they also found a small noninactivating current component. In contrast, Cannon and Strittmatter (3) reported a larger persistent current arising from the disruption of fast inactivation as the main consequence of the rT698M mutation. Yang et al. (34) made the T704M mutation in the human skeletal muscle isoform of the Na+ channel alpha -subunit. Their results agree with those of Cummins et al. (7) with respect to the shift in the hyperpolarizing direction of the midpoint of steady-state activation (9 mV) and to the small persistent current. In addition, they report a shift of the steady-state inactivation curve in the depolarizing direction (13 mV). No significant changes were found in the inactivation kinetics or in the rate of recovery from inactivation with respect to wild-type channels. According to Cummins et al. (7) and Yang et al. (34), the shift of both activation and inactivation curves along the voltage axis results in an increased overlap between steady-state activation and steady-state inactivation. Therefore, a larger fraction of mutant channels, compared with wild-type channels, would be available to open in the voltage range of -75 to -40 mV. The window Na+ current would slightly depolarize the membrane of muscle cells and inactivate Na+ channels in HPP-affected individuals. All of these findings are in agreement with the observed persistent Na+ current at negative potentials in muscle fibers from HPP-affected individuals (16).

The fact that the substitution of a single amino acid has different functional consequences when expressed in human skeletal muscle, rat skeletal muscle, or rat brain type IIA alpha -subunit might be explained by subtle differences in the structure-function relationship among these isoforms of the Na+ channel. Accordingly, the functional analysis of mutations that underlie human neuromuscular disorders made in Na+ channel isoforms other than the human skeletal muscle may yield misleading information. Furthermore, potential divergences in posttranslational modifications in different expression systems or the technique used for current recordings may be partly responsible for some of the discrepancies found in the literature.

With consideration of the above statements, our results support the view that M1592V HPP arises from a functional defect that resembles the previously described T704M mutation. The persistent Na+ current observed in individuals affected by HPP seems to be caused by an increased overlap of activation and inactivation curves of channels carrying the M1592V or the T704M mutations. Thus similar clinical phenotypes are likely to arise from common functional defects in the Na+ channel. The fact that these two point mutations that lie far away in the primary structure as well as in the proposed tertiary structure of the alpha -subunit (and which are apparently unrelated to the voltage sensor) modify the activation gating provides some clues to further explore the molecular basis of the voltage-dependent gating of this class of ion channels.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Ricardo Bull and to Dr. Miguel Allende for helpful comments on the manuscript. We thank Fernando Vergara for assistance with the oocyte microinjection. The pGEMHE vector was kindly provided by Dr. Emily R. Liman (Howard Hughes Medical Institute, Harvard Medical School).

    FOOTNOTES

This study was supported by Fondo Nacional de Ciencia y Tecnología Grant 1930082 (to C. Rojas) and a Visiting Professor Fellowship from Fundación Andes (to A. Neely).

C. Rojas was affiliated with Centro de Estudios Científicos de Santiago during early stages of this work.

Present address of G. Velasco-Loyden: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, CP 04510, Mexico DF, Mexico.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. V. Rojas, INTA, Universidad de Chile, Casilla 138-11, Santiago, Chile.

Received 24 April 1998; accepted in final form 7 October 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Barchi, R. L. The molecular pathology of the skeletal muscle sodium channel. Annu. Rev. Physiol. 57: 355-358, 1995[Medline].

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