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The Journal of Neuroscience, June 1, 1998, 18(11):4063-4075
Connexin32 Mutations Associated with X-Linked
Charcot-Marie-Tooth Disease Show Two Distinct Behaviors: Loss of
Function and Altered Gating Properties
Catherine
Ressot1, 2,
Danielle
Gomès1,
André
Dautigny2,
Danielle
Pham-Dinh2, and
Roberto
Bruzzone1
1 Unité de Neurovirologie et
Régénération du Système Nerveux, Institut
Pasteur, F-75724 Paris Cedex 15, France, and 2 Laboratoire
de Neurogénétique Moléculaire, Unité de
Recherche Associée 1488, Centre National de la Recherche
Scientifique, Institut des Neurosciences, Université de Paris VI,
F-75252 Paris Cedex 05, France
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ABSTRACT |
The X-linked form of Charcot-Marie-Tooth disease (CMTX) is
associated with mutations in the gene encoding connexin32 (Cx32), which
is expressed in Schwann cells. We have compared the functional properties of 11 Cx32 mutations with those of the wild-type protein by
testing their ability to form intercellular channels in the paired
oocyte expression system. Although seven mutations were functionally
incompetent, four others were able to generate intercellular currents
of the same order of magnitude as those induced by wild-type Cx32
(Cx32wt). In homotypic oocyte pairs, CMTX mutations retaining functional activity induced the development of junctional currents that
exhibited changes in the sensitivity and kinetics of voltage dependence
with respect to that of Cx32wt. The four mutations were also capable of
interacting in heterotypic configuration with the wild-type protein,
and in one case the result was a marked rectification of junctional
currents in response to voltage steps of opposite polarity. In
addition, the functional CMTX mutations displayed the same selective
pattern of compatibility as Cx32wt, interacting with Cx26, Cx46, and
Cx50 but failing to do so with Cx40. Although the functional mutations
exhibited sensitivity to cytoplasmic acidification, which induced a
 80% decrease in junctional currents, both the rate and extent of
channel closure were enhanced markedly for two of them. Together, these
results indicate that the functional consequences of CMTX mutations of Cx32 are of two drastically distinct kinds. The presence of a functional group of mutations suggests that a selective deficit of Cx32
channels may be sufficient to impair the homeostasis of Schwann cells
and lead to the development of CMTX.
Key words:
gap junction; channel; myelin; Schwann cell; neuropathy; peripheral nervous system
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INTRODUCTION |
Connexins comprise a multigene
family of proteins that form the intercellular channels clustered at
gap junctions, thereby allowing adjacent cells to share ions, small
metabolites, and second messengers (Goodenough et al., 1996 ; Gros and
Jongsma, 1996; Kumar and Gilula, 1996; Yamasaki and Naus, 1996;
Bruzzone and Ressot, 1997). Intercellular channels span the plasma
membranes of two neighboring cells and result from the association of
two half-channels, or connexons, contributed separately by each of the
two participating cells. Each connexon, in turn, is a hexameric assembly of connexin subunits (for review, see Sosinsky, 1996; Yeager
and Nicholson, 1996). The specific role of connexins in different
tissues has been highlighted by the demonstration that two genetic
disorders are linked to mutations of connexin genes. Thus, connexin26
(Cx26) mutations are found in hereditary nonsyndromic deafness
(Denoyelle et al., 1997; Kelsell et al., 1997; Zelante et al., 1997),
whereas patients with the X-linked form of Charcot-Marie-Tooth disease (CMTX) have mutations in the gene encoding Cx32 (Bergoffen et
al., 1993).
Charcot-Marie-Tooth disease, the most common genetic disorder of the
peripheral nervous system, is characterized by distal muscle weakness
and amyotrophy, decreased or absent tendon reflexes, and pes cavus
deformity (Vance, 1991; Harding, 1995; Suter and Snipes, 1995). In
keeping with the proposed role as a candidate gene for CMTX, Cx32 is
expressed at high levels in myelinating Schwann cells and is regulated
in parallel to other myelin genes (Bergoffen et al., 1993; Scherer et
al., 1995; Chandross et al., 1996b; Satake et al., 1997).
Immunocytochemical studies have indicated that Cx32 is localized mainly
in noncompacted domains of myelin, such as paranodal loops and
Schmidt-Lanterman incisures (Bergoffen et al., 1993; Miyazaki et al.,
1995; Scherer et al., 1995; Spray and Dermietzel, 1995; Chandross et
al., 1996b; Nelles et al., 1996). This distribution is incompatible
with the formation of orthodox intercellular channels between adjacent
cells but, instead, has led to the hypothesis that Cx32 forms reflexive
intracellular channels and provides a radial pathway traversing the
myelin sheath and connecting the main body to the adaxonal portion of
Schwann cells (Bergoffen et al., 1993; Paul, 1995; Bone et al.,
1997).
The initial analysis of CMTX mutations has demonstrated that some
interfere with the channel-forming ability of Cx32 (Bruzzone et al.,
1994b; Omori et al., 1996), thus suggesting that loss-of-function of
Cx32 in Schwann cells and blockade of the pathway it provides across
the myelin turns represent the molecular basis of the disease. This
hypothesis has been strengthened by the demonstration that mice lacking
Cx32 develop a late-onset peripheral neuropathy with features similar
to those of CMTX (Anzini et al., 1997). One CMTX mutation, however,
appears to retain functional activity (Rabadan-Diehl et al., 1994;
Omori et al., 1996), although its biophysical properties and gating
behavior have not been analyzed.
As a first step toward a better understanding of the role of Cx32 in
the pathogenesis of CMTX, we have studied the functional consequences
of 11 mutations of Cx32 found in CMTX patients by testing their ability
to form intercellular channels in the paired Xenopus oocytes
expression system. Our results indicate that CMTX mutations of Cx32
display drastically distinct functional consequences. Although seven
mutations lacked channel activity, four others were able to generate
intercellular currents of the same order of magnitude as those induced
by wild-type Cx32 (Cx32wt). These functional CMTX mutations showed
altered gating properties, thus suggesting that a more subtle
functional deficit may underlie the development of a similar clinical
phenotype.
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MATERIALS AND METHODS |
Molecular cloning, in vitro transcription, and
translation of Cx32wt and CMTX mutations. Human Cx32wt had been
subcloned previously (Bruzzone et al., 1994b) into the transcription
vector SP64T (Krieg and Melton, 1984). We have analyzed 11 mutations
distributed throughout the connexin molecule (see Fig. 1): R22G, R22P
(Ressot et al., 1996), L56F, L90H (Latour et al., 1997), V95M (Bone et
al., 1995), E102G (Ionasescu et al., 1994), Deletion (Del) 111-116
(Cherryson et al., 1994), P172S (Bergoffen et al., 1993), E208K
(Fairweather et al., 1994), Y211stop (Tan et al., 1996), and R220stop
(Fairweather et al., 1994; Ionasescu et al., 1994). The following
mutations R22G, R22P, and L90Hwere produced by PCR amplification of
the coding region of Cx32, using 100 ng of genomic DNA isolated from
affected CMTX patients as the template. The two primers corresponded to nucleotides 1-24 (sense,
5'-GTACAGATCTATGAACTGGACAGGTTTGTACACC-3') and 832-849 (antisense,
5'-AATTAGATCTTCAGCAGGCCGAGCAGCGGTC-3') of the human Cx32 coding
region (Kumar and Gilula, 1986), with BglII linkers. The
cycling protocol and the rest of the cloning procedure were performed
as described (Bruzzone et al., 1994b). All other mutations were
generated by oligonucleotide-directed site mutagenesis (Ausubel et al.,
1992). Briefly, two oligonucleotides that corresponded to adjacent
regions of Cx32, spanning the site to be mutagenized, were synthesized
for each CMTX mutation. The appropriately modified codons were
contained in the sense primers. Cx32wt, subcloned in the SP64T vector,
was used as the template (5-10 ng) and amplified with Pfu
polymerase (2.5 U; Boehringer Mannheim Biochemicals, Meylan, France),
using a previously described protocol (White et al., 1994).
The sequences of PCR primers are given below; codons with altered
nucleotides and those flanking the 18 bp deletion are underlined. L56F,
sense, 5'-TGC-AAC-ACA-TTC-CAG-CCT-GGC-3'; L56F, antisense, 5'-GAT-GAA-GGA-AGA-TTT-CTC-ATC-3'; V95M, sense,
5'-GCC-ATG-CAC-ATG-GCT-CAC-CAG-3'; V95M, antisense,
5'-CAC-GAG-GAG-AGC-TGG-GGT-GGA-3'; E102G, sense, 5'-CAA-CAC-ATA-GGG-AAG-AAA-ATG-3'; E102G, antisense,
5'-CTG-GTG-AGC-CAC-GTG-CAT-GGC-3'; Del111-116, sense,
5'-CTG-GAG-GAG-GTG-AAG-AGG-CAC-3'; Del111-116, antisense,
5'-GCC-CTC-AAG-CCG-TAG-CAT-TTT-3'; P172S, sense,
5'-GAC-GTC-TAC-TCC-TGC-CCC-3'; P172S, antisense,
5'-GCA-CTT-GAC-CAG-CCG-CAC-3'; E208K, sense, 5'-AAT-GTG-GCC-AAG-GTG-GTG-TAC-3'; E208K, antisense,
5'-GAG-GAT-GAT-GCA-GAT-GCC-AGA-3'; Y211stop, sense,
5'-GAG-GTG-GTG-TAA-CTC-ATC-ATC-3'; Y211stop, antisense,
5'-GGC-CAC-ATT-GAG-GAT-GAT-GCA-3'; R220stop, sense, 5'-TGT-GCC-CGC-TGA-GCC-CAG-CGC-3'; and R220stop, antisense,
5'-GGC-CCG-GAT-GAT-GAGGTA-CAC-3'.
To verify that the CMTX mutations had been incorporated and to ensure
that PCR amplification did not introduce random errors in the Cx32
coding sequence, we sequenced all constructs entirely with Sequenase
(Amersham, Buckinghamshire, UK), following the protocols recommended by
the manufacturer. Constructs were linearized with XbaI, and
capped cRNAs were produced in vitro with SP6 RNA polymerase,
using the mMessage mMachine kit (Ambion, Austin, TX) according to
the manufacturer's instructions. The purity and yield of
transcribed cRNAs were determined either by comparison of the intensity
of ethidium bromide staining to a known RNA standard ladder after
agarose gel electrophoresis or by measuring absorbance at 260 nm.
Aliquots (300-500 ng) of in vitro synthesized connexin cRNAs were translated (1 hr at 37°C) in a rabbit reticulocyte lysate
system (New England Nuclear, Boston, MA), following previously described protocols (Bruzzone et al., 1994a). Radioactive products ( of the reaction volume) were separated by electrophoresis
on a 13% SDS-polyacrylamide gel and visualized by fluorography.
Preparation of Xenopus oocytes. Ovarian lobes
were removed surgically under cold anesthesia from Xenopus
laevis females purchased from the colony of the Institut für
Entwicklungsbiologie (Hamburg, Germany). Oocytes (stage V-VI) were
defolliculated after collagenase treatment and processed for the paired
oocyte expression assay as previously described (Dahl, 1992). After
collagenase digestion and defolliculation, all subsequent steps were
performed at 18°C in modified Barth's (MB) medium as previously
described (Swenson et al., 1989), except in experiments using Cx46, in
which the final concentration of calcium was adjusted to 2.9 mM with CaCl2 (Ebihara and Steiner, 1993). To
eliminate the possible contribution of endogenous intercellular
channels to the measured conductance (Barrio et al., 1991; Bruzzone et
al., 1993), we injected the oocytes with an antisense oligonucleotide
(3 ng/oocyte) corresponding to a portion of the coding sequence of
Xenopus Cx38 mRNA (5'-CTGACTGCTCGTCTGTCCACACAG-3'). After an
overnight incubation at 18°C, each antisense-treated oocyte was
injected with 40 nl of either water or appropriate dilutions of the
various cRNAs. Microinjected oocytes were immersed for a few minutes in
hypertonic solution to strip the vitelline envelope (Methfessel et al.,
1986), transferred to Petri dishes containing MB medium, and manually
paired with the vegetal poles apposed.
Labeling of connexin proteins and Western blotting.
Metabolic labeling of oocytes was performed as previously
described (Bruzzone et al., 1993), except that each cell received 2 µCi of L-[35S]methionine (ICN
Pharmaceuticals, Costa Mesa, CA) together with water or cRNA (120-200
ng). Approximately 1/2 of one oocyte was loaded onto each lane
of a 13% SDS-polyacrylamide gel; after migration, labeled proteins
were visualized by fluorography. For Western blotting, oocytes injected
in parallel with the same cRNA aliquots used for electrophysiological
experiments were transferred to chilled Eppendorf tubes containing
lysis buffer [(in mM) 5 Tris, 5 EDTA, and 5 EGTA, pH 8.0, plus 10 µg each of chymostatin, leupeptin, and pepstatin] and
homogenized to prepare a crude membrane fraction, as previously
described (White et al., 1994). For each experimental condition two
oocyte equivalents per lane were loaded on a 13% SDS-polyacrylamide
gel. After separation, proteins were transferred (1 hr at 50 V) to
nitrocellulose membranes (Schleicher & Schuell, Keene, NH) at 4°C
with precooled buffer (1.92 M glycine, 248 mM Tris-base, and 20% methanol). Transferred proteins were visualized by
staining the membranes with 0.2% (w/v) Ponceau S, and the position of
molecular weight standards was marked with a needle. All subsequent steps were performed at room temperature, using the reagents provided with the Western-Light kit (Tropix, Bedford, MA) according to the
manufacturer's recommendations. The culture supernatant of an
anti-Cx32 monoclonal antibody, designated as M12.13 (Goodenough et al.,
1988), was used at a 1:50 dilution. After three washes, the membranes
were reacted for 30 min with an alkaline phosphatase-conjugated goat
anti-mouse IgG plus IgM (H+L) (Tropix) used at a final dilution of
1:2500, and the protein bands were visualized by chemiluminescence.
Electrophysiological measurements of junctional currents.
Intercellular communication was quantitated by double voltage
clamp (Spray et al., 1981) 24-48 hr after pairing. Electrodes had a resistance of 0.5-2 M and were filled with (in M) 3 KCl, 10 EGTA, and 10 HEPES, pH 7.4. Voltage clamping of oocyte pairs
was performed with two GeneClamp 500 amplifiers (Axon Instruments,
Foster City, CA) controlled by a PC-compatible computer (Kenitec,
Taiwan) via a Digidata 1200 interface (Axon Instruments). pCLAMP 6.0 software (Axon Instruments) was used to program stimulus and data
collection paradigms. Current outputs were filtered at 10 Hz, and the
sampling interval was 7.5 msec. For simple measurements of junctional
conductance, both cells of a pair were clamped initially at  40 mV to
ensure zero transjunctional potential, and alternating pulses of ± 10-20 mV were imposed to one cell. Current delivered to the cell
clamped at 40 mV during the voltage pulse was equal in magnitude to
the junctional current and was divided by the voltage to yield the conductance. Transjunctional potentials of increasing amplitude and
opposite polarity were generated by hyperpolarizing or depolarizing one
cell in 10 mV increments while clamping the second cell at 40 mV;
steady-state currents were measured 30 sec after the imposition of
voltage steps. Then the calculated conductance was normalized to its
value at ± 10 mV and plotted against the transjunctional potential. Data were fit to a Boltzmann equation of the form: Gjss = (Gjmax Gjmin)/{1 + exp(A[V V0])} + Gjmin, where
Gjss is the steady-state conductance,
Gjmax is maximum conductance, Gjmin is minimum conductance, A is
the cooperativity constant, and V0 is the
voltage at which the decrease in Gjss is
half-maximal (Spray et al., 1981). The mean conductance of oocyte pairs
selected for analysis of voltage sensitivity was 4.5 ± 0.4 µS
(n = 49), thereby ensuring adequate control of
transjunctional potential and avoiding the risk of overestimating the
actual Gj at steady state (Wilders and Jongsma,
1992). The kinetics of voltage-dependent transitions of junctional
conductance were fit with Clampfit functions in pCLAMP (Axon
Instruments), using the following equation: An × exp{ (t K)/ n} + C, where is the time constant reported for each
component (n), K is the time at the start of the
fit region, A is the current amplitude evaluated at the time
t = K, and C is the steady-state
asymptote. Experimental time constants () were calculated for
transjunctional voltages 50 mV, which corresponded to values
resulting in a consistent voltage-dependent closure of channels
composed of either Cx32wt or functional CMTX mutations. The
sensitivity of junctional conductance to cytoplasmic acidification was
tested by perfusing the incubation dish (4.2 ml/min; total dish volume,
5 ml) with MB medium equilibrated with 100% CO2 for 10 min, after which the perfusion medium was switched to normal MB to
allow junctional conductance to recover. Junctional conductance was
measured in response to alternating ± 10 mV pulses applied, for 1 sec at 1 min intervals, to one cell and normalized to the average
conductance values recorded for 3 min previous to the start of
perfusion with 100% CO2.
Statistical analysis. Results are shown as the mean ± SEM. Two population comparisons were made with Student's unpaired
t test; p values of 0.05 or less were considered
to be significant.
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RESULTS |
Biochemical characterization and expression of CMTX mutations in
Xenopus oocytes
The CMTX mutations and their approximate localization on Cx32 are
illustrated in Figure 1. The constructs
were used as templates for in vitro transcription and the
subsequent translation of the produced cRNAs, using a rabbit
reticulocyte lysate in the presence of
L-[35S]methionine. The radioactive
products were separated by SDS-gel electrophoresis and detected by
fluorography (Fig. 2A).
Translation reactions that contained cRNAs encoding a CMTX missense
mutation (Fig. 2A, lanes 2-7, 9, 10)
produced a predominant band of the same apparent size as that of the
wild-type Cx32 protein (Fig. 2A, lane 1).
The deletion of six amino acids at residues 111-116 (Fig.
2A, lane 8) did not change appreciably the
migration of the construct under the experimental conditions that were
used. As expected, translation reactions that contained cRNAs for
either Y211stop or R220stop (Fig. 2A, lanes
11-12, respectively), the two mutations resulting in premature
stop codons, produced proteins migrating with a faster electrophoretic
mobility that was in agreement with their predicted molecular mass.
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Figure 1.
Schematic representation and topology relative to
the plasma membrane of Cx32, with the approximate location of the CMTX
mutations analyzed: two were in the first transmembrane domain
(TM1), one in the first extracellular loop
(EC1), one in the second transmembrane domain
(TM2), three in the middle cytoplasmic region, one in
the second extracellular loop (EC2), and three in the
C-terminal portion (C).
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Figure 2.
CMTX mutations of Cx32 are expressed by
Xenopus oocytes injected with in vitro
transcribed cRNAs. A, In vitro
translation of cRNAs of Cx32wt and CMTX mutations. Each cRNA directed
the synthesis of a major polypeptide product that, in the case of the
two mutations predicting a premature stop codon, migrated with the
expected faster electrophoretic mobility. B, Western
blots of Xenopus oocytes membranes. In oocytes that
received cRNAs for either wild type or 10 of 11 CMTX mutations of Cx32,
the antibody M12.13 (Goodenough et al., 1988) reacted with a major
protein product that exhibited the expected SDS-gel mobility between 22 and 28 kDa. In addition, the antibody recognized a protein band with
slower electrophoretic mobility, most likely a dimer of Cx32. No major
proteins were detected in control, water-injected oocytes (lane
13), as well as in membranes prepared from oocytes injected
with the Del111-116 mutation (lane 8).
C, Metabolic labeling of Xenopus oocytes.
Oocytes injected with cRNAs produced high levels of the encoded
proteins (lanes 1-3), which were easily detected above
the background of endogenous proteins synthesized by water-injected
cells (lane 4). Experimental conditions are
indicated at the top of each lane. The
molecular mass (in kDa) and migration of protein standards are
indicated on the left edge of each gel.
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We next verified the translation efficacy of the CMTX constructs in
Xenopus oocytes by Western blotting of membrane fractions (Fig. 2B). The monoclonal antibody M12.13 (Goodenough
et al., 1988) identified a major polypeptide band of ~27 kDa in
oocytes injected with cRNAs encoding either Cx32wt or CMTX mutations, except in the case of cells receiving Del111-116, for which no bands
were recognized by the antibody (Fig. 2B, lane
8). This failure could have been attributable either to a very
rapid degradation of the synthesized protein in a cellular environment
or to the disruption of the epitope recognized by M12.13, which is
known to be exposed on the cytoplasmic side (Goodenough et al., 1988). To distinguish between this alternative, we metabolically labeled oocyte proteins with L-[35S]methionine
and analyzed aliquots of homogenates for incorporation of label into
total protein. Oocytes injected with cRNA for Del111-116 produced high
levels of the encoded mutation (Fig. 2C, lane 3) that were comparable to those observed with the injection of a similar
amount of cRNA encoding Cx32wt (Fig. 2C, lane 1).
Thus, the monoclonal antibody M12.13 recognizes an epitope in the
middle cytoplasmic loop of Cx32 that is destroyed by the deletion of amino acid residues 111-116. In addition, the injection of L90H cRNA
induced the synthesis of a specific protein band (Fig. 2C, lane 2) of the same intensity as that of Cx32wt. This result
indicates that the lower immunoreactivity noticed in Western blots
(Fig. 2B, lane 5) is not attributable to a
reduced translatability of this cRNA but suggests a conformational
change that would affect the antibody epitope in the cytoplasmic
portion. Because the L90H mutation occurs in the second transmembrane
segment, this observation is compatible with the idea of interdomain
interactions in connexins (Verselis et al., 1994; White and Bruzzone,
1996; Wang and Peracchia, 1997; Zhou et al., 1997). Thus,
Xenopus oocytes support the efficient biosynthesis of all
CMTX mutations of Cx32.
Functional expression of CMTX mutations
Intercellular channels are defined as homotypic, when both
connexons are composed of the same connexin, or heterotypic, when each
connexon contains a different connexin. The ability of CMTX mutations
to form homotypic channels was tested by using the paired Xenopus oocyte expression system. Although Cx32 is not known
to interact with the endogenous Xenopus Cx38 (Swenson et
al., 1989; Werner et al., 1989; White et al., 1995), all experiments
were performed with oocytes pretreated with antisense oligonucleotides to ensure that the observed currents were the result of the exogenously supplied connexins. As previously reported (Barrio et al., 1991; Bruzzone et al., 1993), water-injected cells showed no detectable coupling under these conditions (Fig.
3).
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Figure 3.
Expression of CMTX mutations of Cx32 in homotypic
oocyte pairs reveals two opposite behaviors. Antisense-treated oocytes
(see Materials and Methods) were injected with the specified cRNAs and
paired in homotypic configuration for 24-48 hr before junctional
conductance was measured by a dual voltage-clamp procedure. Four of the
CMTX mutations retained channel-forming activity that was
macroscopically indistinguishable from that of Cx32wt. Data were pooled
from at least three independent experiments and are presented as the
mean ± SEM of the number of pairs indicated for each
condition.
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Injection of cRNAs for seven (R22G, R22P, L90H, V95M, P172S, E208L,
Y211stop) of the 11 mutations that were analyzed did not induce the
formation of homotypic junctional channels, because the levels of
conductance that were measured never exceeded background values (Fig.
3). In contrast, the following four mutationsL56F, E102G,
Del111-116, and R220stopefficiently assembled homotypic channels and
induced conductance levels of the same order of magnitude as those
developed by homotypic pairs expressing Cx32wt (Fig. 3).
Homotypic CMTX channels display distinct
voltage-gating properties
Although four CMTX mutations retained the ability to form
functional channels, it remained possible that some of their electrical properties and gating behavior were altered. To address this issue, we
first examined the response of homotypic L56F, E102G, Del111-116, and
R220stop channels to voltage gating (Fig.
4, Table
1). Typical transjunctional currents and
plots of the relationship between steady-state junctional conductance
(Gj) and transjunctional voltage (Vj) are presented in Figure 4. Voltage
steps lasted 30 sec to allow currents to approach equilibrium values.
The junctional currents of Cx32wt pairs decayed slowly over time for
potentials >40 mV (Figs. 4A, 5A), in
agreement with previous studies that used the rodent homologs (Barrio
et al., 1991; Rubin et al., 1992; Suchyna et al., 1993; Bruzzone et
al., 1994a; Wang and Peracchia, 1996). Conductance values measured at
the end of the imposed pulses were normalized to those recorded at the
lower transjunctional potential (i.e., ±10 mV) and were plotted
against the increasing Vj of either polarity. As
previously reported (Bruzzone et al., 1994a), the
Gj/Vj relationship
showed a slight asymmetry, with positive potentials inducing a greater
channel closure over the duration of the voltage step (Fig.
4F). This asymmetry was quantitated by fitting the
Gj/Vj relationship
for positive and negative polarity to Boltzmann equations, for which
the parameters are given in Table 1.
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Figure 4.
Intercellular channels formed by CMTX mutations of
Cx32 exhibit distinct voltage dependence. Antisense-treated oocytes
(see Materials and Methods) were injected with the specified cRNAs and
then paired homotypically for measurements of junctional currents,
using a dual voltage-clamp procedure.
A-E, Time-dependent decay of junctional
currents (Ij) induced by
transjunctional voltage (Vj) steps of
opposite polarity applied in 10 mV increments. Currents from oocytes
expressing Cx32wt (A) decayed with a slow time
course for Vj 40 mV. CMTX mutations
(B-E) showed changes in voltage-induced
channel closure that were particularly pronounced in the case of
Del111-116 (D).
F-J, Plots describe the relationship of
Vj to steady-state junctional conductance
(Gj), normalized to the values
obtained at ±10 mV. Smooth lines represent the best
fits to Boltzmann equations for which the parameters are given in Table
1. G-J, For the sake of comparison,
dashed lines show the Boltzmann curve of Cx32wt. Results
are shown as the mean ± SEM of the following number of oocyte
pairs: Cx32wt, n = 7; L56F, n = 5; E102G, n = 7; Del111-116, n = 6; R220stop, n = 4.
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Table 1.
Comparison of Boltzmann parameters of intercellular
channels composed of either Cx32wt or functional CMTX mutations
expressed in paired Xenopus oocytes
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Oocyte pairs expressing channels made by the functional CMTX mutations
showed a voltage dependence that appeared to be more pronounced in
comparison to that of Cx32wt (Fig. 4B-E).
In particular, Del111-116 homotypic pairs exhibited a marked
sensitivity to voltage, with junctional currents decaying symmetrically
at Vj 30 mV and a value of transjunctional
potential at which the transition between maximal and minimal
conductance was halfway (V0) of 40 mV
(Fig. 4D, Table 1). Finally, the extent of channel
closure was more complete, as reflected in the value of minimal
conductance (Gjmin), which was only 10%
of the maximal level measured at the lowest Vj
(Fig. 4I, Table 1). The changes in voltage dependence of the other CMTX mutations were also evident in the values of V0 and Gjmin (Table 1).
The apparent increase of Vj dependence of mutant
E102G was statistically significant (p < 0.02)
for voltage steps greater than ±70 mV. Further differences became
apparent by analyzing the kinetics of Vj-induced
transitions of Gj (Fig. 5). The time course of current decay
followed a single exponential relation (see traces in Fig. 4) and was
symmetrical for positive and negative Vj >50
mV. Moreover, time constants were faster with increasing
Vj for either Cx32wt or CMTX mutations (Fig. 5).
The calculated values are comparable to those recently reported for mammalian Cx45 (Barrio et al., 1997). The voltage dependence of time
constants for E102G was similar to that of Cx32wt (compare Fig.
5A and C), whereas it was significantly faster
(p < 0.05) for L56F at the higher
transjunctional potentials (more than or equal to +60 and 80 mV).
Junctional currents evoked by Del111-116 (Fig. 5D) and
R220stop (Fig. 5E) decayed with time constants that were up
to fivefold faster than those calculated for Cx32wt at the same
transjunctional potentials. These differences were statistically significant (p < 0.02), with the exception of
the +50 mV pulse of R220stop. Thus, homotypic channels composed of
L56F, E102G, Del111-116, and R220stop differ from Cx32wt in both the
sensitivity and time course of their response to imposed
Vj steps.
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Figure 5.
Time constants of voltage-dependent channel
closure. For all experimental conditions, the time course of current
decay followed a monoexponential relation (see traces in Fig. 4). Time
constants () were plotted versus positive and negative
transjunctional potentials, corresponding to values that induced a
consistent closure of channels composed of either Cx32wt or functional
CMTX mutations. The values of were markedly faster for Del111-116
(D) and R220stop (E) than
for Cx32wt (A). Results are shown as the
mean ± SEM of the same number of pairs as indicated in Figure 4.
When the error bars are not visible, SEM are within the size of the
symbol.
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Heterotypic channels between Cx32wt and CMTX mutations exhibit
novel properties
We have examined these functional differences further by
investigating the ability of CMTX mutations to interact with Cx32wt and
the voltage sensitivity of the resulting heterotypic channels. All four
functional mutations displayed the ability to interact with Cx32wt
(Table 2). In the experiments analyzing
voltage dependence, the cell expressing the CMTX mutation was held at a
constant voltage, whereas the other one, expressing the wild-type
protein, was depolarized or hyperpolarized. Thus, positive potentials
denote that the right-hand cell, i.e., Cx32wt, was relatively positive,
and, vice versa, negative potentials denote that the cell expressing a
CMTX mutation is relatively positive (Fig.
6).
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Table 2.
The four functional CMTX mutations interact with Cx32wt and
retain its same selective compatibility in heterotypic channel
formation
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Figure 6.
The formation of heterotypic channels between
Cx32wt and CMTX mutations reveals unexpected electrical properties.
Experimental conditions were as described in the legend to Figure 4.
A-D, Time-dependent decay of junctional
currents (Ij) induced by
transjunctional voltage (Vj) steps
applied in 10 mV increments. When Cx32wt was paired to L56F
(A), E102G (B), or R220stop
(D), currents exhibited a mainly symmetrical
reduction for Vj 30 mV. In contrast,
Del111-116/Cx32wt heterotypic channels (C) were
characterized by a marked rectification and asymmetry of the
voltage-gating behavior. E-H, Plots of
mean steady-state conductance versus
Vj, normalized to the values obtained
with a driving force of ±10 mV. Vj is
defined as positive for depolarization of the right-hand cell (e.g.,
Cx32wt) relative to the left-hand cell (e.g., either one of the CMTX
mutations) and vice versa. Solid lines represent the
best fits to Boltzmann equations for which the parameters are given in
Table 1. Dashed lines represent the predicted
conductance of heterotypic channels on the basis of the properties of
homotypic channels. Results are shown as the mean ± SEM of the
following number of oocyte pairs: L56F/Cx32wt, n = 4; E102G/Cx32wt, n = 5; Del111-116/Cx32wt,
n = 6; R220stop/Cx32wt, n = 5.
|
|
Heterotypic channels composed of L56F/Cx32wt (Fig.
6A,E), E102G/Cx32wt (Fig.
6B,F) or R220stop/Cx32wt
(Fig. 6D,H) were
characterized by a time-dependent channel closure over the duration of
the voltage step, leading to a reduced steady-state conductance for
transjunctional potentials of either polarity. Comparison of the
experimentally obtained
Gj/Vj plots (Fig.
6E-H, solid lines) with the
predicted equilibrium conductance, if the properties displayed in
homotypic configurations had been maintained (Fig.
6E-H, dashed lines), revealed
only a minor deviation in voltage sensitivity when L56F and E102G
partnered Cx32wt. In general, there was a tendency for Cx32wt to
increase and for CMTX mutations to decrease their voltage sensitivity
in heterotypic configuration. These changes, which were more
pronounced in the case of R220stop/Cx32wt (Fig.
6D,H), were quantitated
by fitting the data to Boltzmann equations (see Table 1).
In contrast, heterotypic Del111-116/Cx32wt channels were markedly
asymmetrical (Fig. 6C,G), showing rectification,
i.e., decreased conductance for relatively positive and increased
conductance for relatively negative potentials of the wild-type
injected cell (Furshpan and Potter, 1959; Auerbach and Bennett, 1969;
Jaslove and Brink, 1986; Giaume et al., 1987). On the one hand,
depolarizing voltage steps to the cell expressing Cx32wt evoked
junctional currents that were closed by voltage with a faster time
course and at a much lower threshold than that observed for the
corresponding homotypic channels (compare Figs.
6C,G and
4A,F). On the other hand,
depolarizing voltage steps applied to the Del111-116 injected cell
resulted in steady-state currents that were not inhibited by
transjunctional potentials up to 60 mV and showed a slow
voltage-dependent closure at the higher Vj (Fig.
6C,G). The dramatic deviation of Del111-116/Cx32wt heterotypic channels from the properties predicted on the basis of their behavior in homotypic oocyte pairs (Fig. 6G, dashed lines) was reflected in the Boltzmann
parameters given in Table 1. This analysis reinforces the concept that
novel gating properties result from the docking of connexons composed
of distinct connexins.
CMTX mutations retain the pattern of heterotypic compatibility
of Cx32wt
Because the formation of intercellular channels is a selective
process that is also dependent on connexin compatibility (Bruzzone et
al., 1993; Elfgang et al., 1995; White et al., 1995), we investigated the ability of the four functional CMTX mutations to interact with
other connexins in heterotypic configurations. We paired oocytes
expressing either one of the functional CMTX mutations with Cx26, Cx46,
or Cx50, which have been shown to be compatible with Cx32wt (Barrio et
al., 1991; White et al., 1995). Interestingly, Cx26 and Cx46 have an
overlapping pattern of expression with Cx32 (Paul, 1986, 1995); more
importantly, Cx32 and Cx46 are expressed in Schwann cells (Chandross et
al., 1996a,b). Heterotypic oocyte pairs resulted in the development of
robust junctional conductance, indicating that the CMTX mutations
(L56F, E102G, Del111-116, and R220stop) are compatible with Cx26,
Cx46, and Cx50 (Table 2). As expected, CMTX mutations failed to form
channels with Cx40 (Table 2), indicating that they are restricted in
their ability to interact functionally with other connexins and retain
the same pattern of heterotypic compatibility as that of Cx32wt
(Elfgang et al., 1995; White et al., 1995).
CMTX mutations are pH-sensitive
To study the susceptibility of the functional CMTX mutations to
low pH-induced uncoupling (Turin and Warner, 1977), we have used a
common acidification protocol that consisted of perfusing oocyte pairs
with a 100% carbon dioxide-saturated medium (Werner et al., 1991;
White et al., 1994; Wang et al., 1996). It has been shown recently
that, under these experimental conditions, cytoplasmic pH decreases in
oocytes within 5-7 min of exposure to CO2 from a level of
7.58 to 6.38 and does not decline further, even after prolonged
perfusion with the same medium (Wang and Peracchia, 1997). Thus, we
routinely have adopted a 10 min acidification protocol, followed by a
recovery period, to compare the pH sensitivity of CMTX mutations under
maximal uncoupling conditions. The values of junctional conductance
were normalized to those recorded for 2-3 min before starting the
perfusion with the CO2-equilibrated MB solution.
As previously reported (Swenson et al., 1989; Werner et al., 1991; Wang
et al., 1996), homotypic channels composed of Cx32wt exhibited pH
sensitivity (Fig. 7A). The
time course showed an initial, transient increase, followed by a marked
inhibition of junctional conductance, which decreased to <20% of the
initial value at the end of the 10 min perfusion with 100%
CO2 gassed medium. The extent of uncoupling for human
Cx32wt was greater than that observed for the rat counterpart (cf.
Swenson et al., 1989; Werner et al., 1991; Wang et al., 1996). The
reduction in conductance was fully reversible on switching the
perfusion medium to normal buffer. After a delay of ~2 min,
conductance decreased at an average rate of 10.2%/min and recovered at
a rate of 14.2%/min (Fig. 7A). Oocytes expressing the
mutations L56F (Fig. 7B) and R220stop (Fig. 7E)
displayed an uncoupling behavior relatively similar to that of Cx32wt,
with conductance decreasing at an average rate of 8.2 and 10.2% per
minute, respectively, whereas the rates of recovery after maximal
inhibition of junctional conductance were 14.8%/min for L56F and
19.4%/min for R220stop.
View larger version (15K):
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Figure 7.
CMTX mutations in the middle cytoplasmic loop of
Cx32 increase the sensitivity of intercellular channels to cytoplasmic
acidification. Oocyte pairs were perfused with modified Barth's (MB)
solution equilibrated with 100% CO2 for 10 min
(horizontal bar), after which the perfusion medium was
returned to normal MB to allow the junctional conductance to recover.
Junctional conductance was normalized to the average conductance values
recorded for 3 min before the start of perfusion with 100%
CO2. In A, the dashed line
represents the average values of junctional conductance measured
between pairs expressing Cx32wt during perfusion with normal MB
(n = 3). In the case of E102G (C;
n = 5) and Del111-116 (D;
n = 4), both the rate and extent of pH-dependent
uncoupling were enhanced greatly. The pH sensitivity of L56F
(B; n = 5) and R220stop
(E; n = 5) was similar to that of
Cx32wt (A; n = 5). Results are shown
as the mean ± SEM of the specified number of pairs.
|
|
The time course of pH-induced uncoupling of the two other functional
mutations, E102G (Fig. 7C) and Del111-116 (Fig.
7D), was dramatically different from that of wild type.
Plotting the mean decrease in junctional conductance of these mutations
during perfusion with 100% CO2 showed a fast drop that
resulted in a complete uncoupling within 5 min of acidification.
Whereas the average rate of uncoupling was 22.2 and 35.0% per minute
for E102G and Del111-116, respectively (i.e., over twofold greater
than for Cx32wt), the effect was reversible and junctional conductance recovered fully on cessation of perfusion with carbon dioxide-saturated buffer. Recovery occurred more promptly for E102G after the perfusion medium was switched to normal MB (11.8%/min; Fig. 7C),
whereas there was a 6-7 min lag before the recoupling of oocyte pairs expressing Del111-116 started, at an average rate of 12.6%/min (Fig.
7D).
| |
DISCUSSION |
The present study demonstrates that the naturally occurring CMTX
mutations of Cx32 fall into two separate groups: those associated with
a complete loss of function and those that retain the ability to form
functional gap junction channels. Our findings confirm the previous
observation that the R220stop mutation exhibits functional competence
(Rabadan-Diehl et al., 1994; Omori et al., 1996; Wang and Peracchia,
1997) and extend it by establishing that the maintenance of
channel-forming activity is shared by several CMTX mutations.
It has been reported that mice lacking Cx32 (Nelles et al., 1996)
develop a late-onset peripheral neuropathy with similarities to that
observed in CMTX patients (Anzini et al., 1997). These findings
establish the causative role of Cx32 in the pathogenesis of CMTX and
suggest that the loss of Cx32 channels in Schwann cells may be
sufficient to induce the cellular abnormalities and demyelination that
accompany this disease. The results of our functional analysis are
compatible with those of Cx32 null mice, in that we find that a
majority of mutations that have been tested have lost the ability to
form channels. In addition, we have shown that the group of mutations
retaining functional competence exhibits altered gating properties.
Although changes in voltage sensitivity may not be relevant to the
pathophysiology of CMTX, because Cx32 forms reflexive gap junctions in
Schwann cells, our data raise the possibility that more subtle
abnormalities of channel regulation eventually may lead to a functional
deficit. This hypothesis is consistent with the independent
observations of Oh and colleagues (1997), who recently have
characterized two functional CMTX mutations with altered channel
permeability.
The effect of CMTX mutations on Cx32 function
Structure-function studies of connexins have postulated that the
major cytoplasmic domains, which are unique in sequence and length, are
critical for the control of channel gating (Bruzzone et al., 1996;
Yeager and Nicholson, 1996). In search of mutations retaining
functional activity, we have chosen to study those identified by us
(Ressot et al., 1996; Latour et al., 1997) and to focus on mutations
occurring in the cytoplasmic portions.
Three of the functional mutations analyzed in this studyE102G,
Del111-116, and R220stopoccur in these cytoplasmic regions. These
mutations display increased sensitivity to transjunctional voltage and,
in the case of Del111-116 and R220stop, faster time constants of
channel closures. Differences in voltage dependence between Cx32wt and
R220stop were not observed in a previous study (Rabadan-Diehl et al.,
1994). It should be noted that, in the same study, Cx32wt channels were
not gated by transjunctional voltage, an observation at variance with
the findings of several other groups (Barrio et al., 1991; Suchyna et
al., 1993; Bruzzone et al., 1994a; Bukauskas et al., 1995). It is
difficult, therefore, to analyze the results of Rabadan-Diehl and
colleagues (1994) in the context of voltage sensitivity.
The molecular determinants of voltage gating have not been elucidated
completely, but it has been proposed that more than one region of the
connexin molecule is involved in this process (Rubin et al., 1992;
Verselis et al., 1994; White and Bruzzone, 1996). Our findings
demonstrate that the amino acids deleted in the middle cytoplasmic loop
and C terminus modulate the voltage-induced closure of Cx32wt channels.
A similar conclusion has been reached by swapping equivalent domains
between connexins with different voltage sensitivity, because the
degree of voltage dependence correlates with the identity of the middle
cytoplasmic portion (Wang and Peracchia, 1996). Moreover, the
rectification induced by Del111-116, when paired heterotypically with
Cx32wt, is also suggestive of specific changes of channel activity. It
is known that connexins can modify their voltage-gating properties when forming heterotypic channels (Barrio et al., 1991; Rubin et al., 1992;
White and Bruzzone, 1996). Data obtained in the transfected cell
expression system suggest that rectification can be explained by
changes of single channel conductance (Bukauskas et al., 1995) that are
the consequence of differences in the relative ionic permeability of
the connexins forming the heterotypic channel (Cao et al., 1998). Thus,
it is possible that the ionic preference of Del111-116 channels may
differ from that of Cx32wt, although only studies of this mutation at
the single channel level will resolve this issue.
The two functional mutations of the middle cytoplasmic loop, E102G and
Del111-116, exhibited an increased sensitivity to intracellular acidification. These channels closed faster and, in contrast to those
made of Cx32wt, completely. Recent work has demonstrated that the
middle cytoplasmic loop (Wang and Peracchia, 1996; Wang et al., 1996)
and the C-terminal portion (Morley et al., 1995; Ek-Vitorín et
al., 1996; Wang and Peracchia, 1997) contain sequences that are
critical for the pH sensitivity of Cx32 and Cx43. The deletion of amino
acids 111-116 results in a net loss of a positive charge (two
histidines, positive, and one aspartic acid, negative, are lost), but a
similar effect of pH is observed with the mutation E102G, which results
in the loss of a single acidic residue. Although these experiments do
not address the issue of interdomain interactions, they clearly
reinforce the preeminence of the middle cytoplasmic loop in the
sensitivity of channel gating to acidification.
The fourth functional mutant, L56F, is a missense mutation in the first
extracellular loop. This mutation retained the same pattern of
heterotypic compatibility as Cx32wt. Together with the observation that
the CMTX mutations of the second extracellular loop that have been
analyzed so far are loss-of-function (Bruzzone et al., 1994b;
Deschênes et al., 1997; Yoshimura et al., 1998; this study),
these data are consistent with the permissive role attributed to the
first extracellular segment in the process of interconnexon pairing
(White and Bruzzone, 1996). We have noticed, however, that increasing
dilutions of the injected L56F resulted in a drastic loss of homotypic
channel formation, whereas the same amounts of cRNA were able to
interact efficiently with Cx32wt as well as with Cx26, Cx46, and Cx50,
which are compatible partners of Cx32 (Elfgang et al., 1995; White et
al., 1995). Thus, a mutation in the first extracellular loop of Cx32
can influence interconnexon affinity.
Cx32 mutations and the cellular mechanism of CMTX
The functional consequences of Cx32 mutations seem to be of two
kinds: those that result in loss of function and those that retain
channel-forming ability. In the case of the nonfunctional group, we
have reported previously that three such mutations were found
predominantly at the cell surface of paired Xenopus oocytes (Bruzzone et al., 1994b). If these mutations also were targeted correctly to the incisures of Schmidt-Lanterman and paranodal loops of
Schwann cells in vivo, they should be unable to dock and/or
gate into the open configuration. Alternatively, the mutated Cx32 may
be either rapidly degraded or intracellularly trapped. A defective
connexin trafficking, leading to a conspicuous cytoplasmic accumulation, has been reported for several CMTX mutations transfected in mammalian cell lines (Omori et al., 1996; Deschênes et al., 1997). These disparities between Xenopus oocytes and
mammalian cell lines, which regard only nonfunctional mutations, have
been interpreted as the consequence of cell-type and species
differences (Deschênes et al., 1997). The blockade of the radial
pathway normally provided by Cx32 channels across the cytoplasm would, in turn, perturb the ability of Schwann cell to respond to axonal signals. In contrast, the group of functional mutations poses the
challenge of understanding how the pathological changes characteristic of CMTX develop in vivo. Although these results have been
obtained in an expression system that uses an amphibian cell, there has been a good agreement in the data derived from either
Xenopus oocytes or mammalian cells with respect to connexin
physiology (Elfgang et al., 1995; Veenstra et al., 1995; White et al.,
1995; Cao et al., 1998). Moreover, three mutations, S26L, M34T, and R220stop, have been found to be functional in both the amphibian and
mammalian expression systems (Rabadan-Diehl et al., 1994; Omori et al.,
1996; Oh et al., 1997; this study). Thus, channel-forming ability is an
intrinsic property of several CMTX mutations. Alternatively, in the
case of functional mutations, there could be additional changes in the
noncoding region, as reported by Ionasescu and colleagues (1996), that
inhibit gene transcription. We speculate, however, that some CMTX
mutations retain functional competence in their natural cellular
environment and that a more subtle abnormality is responsible for the
development of the peripheral neuropathy.
Our initial characterization has revealed that functional mutations
exhibit altered voltage dependence and pH gating. Other characteristics
could be affected, because channels composed of different connexins are
endowed with distinct properties with regard to unitary conductance,
ionic permeability, and size selectivity (Steinberg et al., 1994;
Elfgang et al., 1995; Koval et al., 1995; Veenstra, 1996; Cao et al.,
1998). It has been reported recently that two CMTX mutations, S26L and
M34T, produce functional channels with reduced permeability to
molecules that can permeate Cx32wt channels (Oh et al., 1997). If
similar alterations are shared by the group of functional mutations,
the trafficking of cellular metabolites and signaling molecules would
be perturbed, resulting in a functional deficit. It is conceivable that
signals that originate from the axon fail to reach the nucleus of a
myelinating Schwann cell, leading to the extinction of specific genes
that control the expression of myelin proteins (Mirsky and Jessen,
1996; Scherer, 1997). The presence of a group of functional mutations
should help in understanding the cellular basis of CMTX disease by
identifying the specific molecules that need to be exchanged via Cx32
channels but that are excluded from the mutated ones.
| |
FOOTNOTES |
Received Nov. 17, 1997; revised March 18, 1998; accepted March 20, 1998.
This work was supported by grants from Institut Pasteur and Association
Française contre les Myopathies (to R.B); European Leukodystrophies Association (to A.D. and D.P-D.); European Community BIOMED 2, ACCV n.4, and Centre National de la Recherche Scientifique (to A.D.); and a predoctoral fellowship from European Leukodystrophies Association (to C.R.). We thank Christian Giaume, Piotr Bregestovski, and members of the Bruzzone and Dubois-Dalcq labs for their helpful comments on this manuscript and Wilfrid Bergeret for preparing one
construct. We give special thanks to David Paul for continuous encouragement and the generous gift of the M12.13 antibody, to Henri
Korn for making his lab space available to house our frogs, and to
Daniel Eugène and Daniel Brusciano for help in setting up the
data acquisition system and introducing us to pCLAMP software.
Correspondence should be addressed to Dr. Roberto Bruzzone, Unité
de Neurovirologie et Régénération du Système
Nerveux, Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France.
| |
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Abstract
Introduction
