1 Instituto de Nutrición
y Tecnología de los Alimentos, 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
sodium current; ion channel; neuromuscular disease; gating; Xenopus oocytes
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 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 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
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
-subunits with the
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.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
-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).
-subunit of
the skeletal muscle Na+ channel
(1).
-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
-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.
View larger version (19K):
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Fig. 1.
Location of hyperkalemic periodic paralysis (HPP)
mutations. Proposed topology of
Na+ channel -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.
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METHODS |
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Molecular cloning of the - and
1-subunits.
cDNAs of the
- and
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
-subunit. The
entire coding region of the wild-type
-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
-subunit reported by
McClatchey et al. (22). The entire coding region of the
1-subunit was amplified from
cDNA using PCR primers based on rat brain (14) and human brain (21)
1-subunit sequences and was
cloned into pBluescript. The sequence of the amplified cDNA is
identical to that reported for the human
1-subunit (20).
Mutagenesis.
The coding region of the cDNA for the wild-type -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
-subunits were used in
the functional expression studies.
In vitro transcription.
For functional expression in Xenopus
laevis oocytes, both - and
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
-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 - and
1-subunits. The optimum ratio of
-
and
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
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 M for
the current electrode and 0.7-1 M
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 M
. 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.
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RESULTS |
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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 -subunit from human
adult skeletal muscle. Functional expression was carried out in
X. laevis oocytes injected with both
- and
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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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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Both wild-type and mutant channels exhibit fast inactivation as
described for - and
-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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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
(h) depicted in Fig.
4B shows that
h decreases similarly with
increasing depolarizing pulses for both wild-type and mutant channels.
A small difference in
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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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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DISCUSSION |
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We have studied the
M1592V mutation that underlies HPP
by functional expression of the human skeletal muscle
Na+ channel in X. laevis oocytes. The -subunit, either wild type or
mutant, was coinjected with the wild-type
1-subunit. It has been well
documented that Na+ channels
resulting from the coexpression of
- and
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
-subunits were
coinjected with the
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
-subunits devoid of
1-subunit is the source of this
noninactivating component, despite the severalfold molar excess of mRNA
coding for the
1-subunit over
the
-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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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
-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
-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 -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
-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 -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 -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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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