1 Département de Physiologie, Centre Médical Universitaire,
Hôpital Cantonal Universitaire, CH-1211 Geneva 4, Switzerland
2 Division de Recherche Clinique Neuro-Musculaire, Département des
Neurosciences, Cliniques et Dermatologie, Hôpital Cantonal
Universitaire, CH-1211 Geneva 4, Switzerland
Author for correspondence (e-mail:
laurent.bernheim{at}medecine.unige.ch)
Accepted 24 April 2003
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SUMMARY |
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Key words: Ca2+ current, Herg, Muscle differentiation, Myoblast fusion, Window current
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INTRODUCTION |
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Using primary myoblast cultures derived from single human satellite cells
(Baroffio et al., 1993), we
have previously shown that membrane potential and the biophysical properties
of specific ionic channels are important actors in the fusion process. We
found that human myoblasts hyperpolarize before fusion through the sequential
expression of two different K+ channels, ether-à-go-go (EAG)
K+ channels (Bijlenga et al.,
1998
; Occhiodoro et al.,
1998
) and Kir2.1 inward-rectifier K+ channels
(Liu et al., 1998
;
Fischer-Lougheed et al.,
2001
). The hyperpolarization induced by Kir2.1 is a prerequisite
for myoblast fusion to occur, as pharmacological blockade of Kir2.1 channels
(Liu et al., 1998
) or
inhibition of their expression using an antisense RNA vector
(Fischer-Lougheed et al.,
2001
) suppress fusion.
Myoblast fusion is a strictly Ca2+-dependent process
(Shainberg et al., 1969) and
our recent results indicate that the purpose of the fusion-linked
hyperpolarization is to set the resting potential of myoblasts in a range that
allows Ca2+ to enter through
1H T-type Ca2+
channels (Bijlenga et al.,
2000
). These channels are expressed just before fusion, and have
intrinsic properties that produce a substantial permanent Ca2+
current in a defined domain of hyperpolarized membrane potentials, hence the
term window current. This window current is large enough to cause a detectable
increase in intracellular Ca2+ and its inhibition prevents fusion
(Bijlenga et al., 2000
).
The presence of a window current depends on an overlap of the voltage range
for channel activation and inactivation. In human myoblasts, T-type
Ca2+ channels activate near -80 mV, and a maximum window current is
observed at -58 mV (Bijlenga et al.,
2000). At more depolarized voltages (near -40 mV), T channels
inactivate fully and no Ca2+ current persists with time. We
proposed that, by setting the resting membrane potential within the window
range, the hyperpolarization generates the intracellular Ca2+
increase that is necessary for fusion to occur
(Bernheim and Bader, 2002
). At
the time, however, we could not tell whether the coupling between the membrane
potential of myoblasts and their differentiation was tight or not. In other
words, we could not tell whether the Ca2+ signal generated was
interpreted by the differentiation program as being of the all-or-none type,
or whether different Ca2+ influxes could result in different fusion
rates. This is of importance, because if graded Ca2+ signals can be
interpreted by the differentiation program, then other mechanisms able to
change intracellular Ca2+ concentration could in principle modulate
the rate of myoblast fusion.
The only way to evaluate convincingly the coupling between myoblast membrane potential and Ca2+-induced differentiation is to generate a controlled, accurate and long-term small change of membrane potential. Given the profile of the Ca2+ current window domain (from -80 mV to -40 mV with a peak near -60 mV, see Fig. 5A) and the resting potential of fusion-competent myoblasts (-74 mV), our objective was to generate a depolarization of about 10 mV, that would bring the membrane potential nearest to the ideal voltage value to produce the largest window current and consequently the largest Ca2+ signal.
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Fortunately, we observed that myoblasts express yet another member of the K+ channel superfamily: the human ether-à-go-go related gene (HERG; KCNH2 - Human Genome Nomenclature Database) K+ channel. We found that these channels contribute to the resting potential to a lesser extent than Kir2.1 channels, that they can be specifically blocked, and that their inhibition induces a depolarization precisely in the desired window domain.
The results presented here show that an increase in Ca2+ influx parallels an acceleration of myoblast fusion, and demonstrate, within the resolution of the system, that there is a tight coupling between membrane potential, Ca2+ signals and myoblast differentiation.
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MATERIALS AND METHODS |
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Electrophysiological recordings
Whole-cell configuration of the patch-clamp technique was used to measure
membrane potential and ionic currents
(Hamill et al., 1981). Signals
were recorded with an Axopatch 200B amplifier. The pipette resistances were
2-5 M
(compensations between 30 and 70% were used). For experiments in
which accurate testing of the membrane potential was required, an extra fire
polishing of the electrode was performed to improve the seal resistance. This
may explain why resting potentials measured in this study are slightly more
hyperpolarized than in a previous study
(Liu et al., 1998
). Cell
capacitances were obtained from direct reading of the whole-cell capacitance
potentiometer of the Axopatch 200B amplifier. Currents were recorded at
20-22°C, low pass-filtered at 1 kHz, and sampled at 5 kHz. To improve
patching procedure, myoblasts were treated with 0.05% trypsin, and replated
1-2 hours before recording. The 30 mM K+ extracellular solution was
made up of (mM): N-methyl-D-glucamine (NMG)-Cl (75), KCl (30),
MgCl2 (2), HEPES (5), NaOH (50), acetic acid (50) and glucose (8).
The pH was adjusted to 7.4 with NMG. The 5 mM K+ extracellular
solution was made up of (mM): N-methyl-D-glucamine (NMG)-Cl (100), KCl (5),
MgCl2 (2), HEPES (5), NaOH (50), acetic acid (50) and glucose (8).
The pH was adjusted to 7.4 with NMG. The intracellular (pipette) solution was
made up of (mM): KCl (120), NaCl (5), MgCl2 (2), HEPES (5), BAPTA
(20), glucose (5) and Mg-ATP (5). The pH was adjusted to 7.3 with KOH.
Dofetilide was a gift from Pfizer, UK. E4031 was purchased from Alomone
Laboratories, Israel.
Northern blot analysis
Total RNA was extracted with Trizol (Invitrogen) according to
manufacturer's instructions. Total RNA (2 µg/lane) was resolved in a 1.5%
agarose gel, transferred to a Zeta-Probe Blotting membrane (BioRad),
crosslinked and hybridized as described by Matter et al.
(Matter et al., 1990). The
membrane was hybridized with a 32P-labeled 1.8 kb probe derived
from the cDNA encoding HERG (Wang et al.,
1998
) and corresponding to nucleotides 2272-4070 (GenBank
Accession Number, U04270).
HERG antisense expressing vector
A bicistronic vector (pEF-IE) was constructed by inserting an IRES-EGFP
(internal ribosomal entry site - enhanced green fluorescent protein) cassette
into the eukaryotic expression vector pEF-BOS
(Uetsuki et al., 1989;
Fischer-Lougheed et al.,
2001
). The antisense vector, pEF-HERG
S-IE, was obtained by
inserting a 237 bp fragment of HERG open reading frame in antisense
orientation into BamHI/SalI sites upstream of the IRES-EGFP.
This sequence corresponds to an N-terminal region of low homology with human
EAG and other related genes, and was generated from an HERG-pcDNA3 clone (kind
gift of G. A. Robertson, University of Wisconsin, Madison) by PCR using
forward and reverse primers 5'-GGCTCATGACACCAAC-3' and
5'-TTGTCCATGGCTGTCACTTC-3'.
Effect of HERG antisense on fusion
Myoblasts were electroporated with 9 pmoles vector/2x106
myoblasts using a GenePulserII (BioRad) as previously described
(Espinos et al., 2001). About
15x106 cells were electroporated, plated in proliferation
medium for 48 hours, then selected for EGFP-expression by cell sorting
(FACStar+, Becton Dickinson), and re-plated in proliferation medium. After
reattachment, fusion was induced with differentiation medium. Cells were fixed
promptly upon appearance of the first myotubes in the control cultures, at day
5, and fusion index determined (the lag time for beginning of fusion increases
in electroporated cells) (Fischer-Lougheed
et al., 2001
).
Pericam vectors
The EGFP sequence from vectors pEF-IE and pEF-HERGS-IE was replaced
by the sequence coding for `inverse-pericam'
(Nagai et al., 2001
). The
inverse-pericam was amplified by PCR using primers containing cloning sites
BamHI and NotI respectively,
5'-GGGGGGATCCAAGCTTGCCACCATG-3' and
5'-GGGGGCGGCCGCGAATTCTTACTTTG-3'.
Cytoplasmic Ca2+ measurements
Myoblast were transfected by electroporation and plated on 25 mm glass
coverslips in differentiation medium. The inverse-pericam
fluorescence from myoblasts was imaged with a Zeiss Axiovert S100TV microscope
using a 63x plan-Neofluar 1.25 NA oil-immersion objective (Carl Zeiss
AG, Feldbach, Switzerland). Cells were excited at 480±10 nm by an
Optoscan Monochromator (Cairn Research, Faversham, UK) through a 505DCXR
dichroic mirror (Chroma Technology Corp, Brattleboro, VT). Fluorescence
emission at 535 nm (535AF45 Omega Optical, Brattleboro, VT) from the
inverse-pericam was imaged using a cooled, 12 bits TE/CCD interlined
CoolSNAP-HQ camera (Photometrics, Ropper Scientific, Trenton, NJ). Image
acquisition and analysis was performed with Metamorph/Metafluor 4.6 software
(Universal Imaging, West Chester, PA). For convenience, as inverse-pericam
fluoresces less when free Ca2+ increases, pseudo fluorescence ratio
images were calculated by dividing the fluorescence image at time 0
(F0) by the fluorescence image at a given time (F). In this case,
an increase in ratio-image fluorescence (F0/F) reflects an increase
in cytoplasmic Ca2+ concentration.
Statistics
Results are expressed as the means±s.e.m. Statistical analysis was
performed using the Student's t-test.
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RESULTS |
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HERG current in human myogenic cells
A Northern blot analysis revealed the presence of HERG transcripts of 4
kb in undifferentiated myoblasts, in cells triggered to fuse, and in myotubes
(Fig. 1A). An approximate
twofold decrease in HERG mRNA content was observed after induction of
myoblast differentiation but no obvious change in expression was observed
thereafter.
|
Out of 16 fusion-competent myoblasts tested, seven expressed a measurable dofetilide-sensitive K+ current. In these cells, the mean current density recorded during a step to -65 mV from a holding potential of -5 mV was 1.3±0.3 pA/pF (mean capacitance was 37±6 pF). Although dofetilide-sensitive K+ currents were detected in only 44% of the cells tested, we believe that functional channels are synthesized in all fusion-competent myoblasts, but that the current they generate is too small to be detected using the whole-cell voltage-clamp recording techniques (see below).
To confirm that the dofetilide-sensitive current recorded in
fusion-competent myoblasts flows through HERG channels, we compared the
endogenous current with the current produced by an HERG expression vector
(Wang et al., 1998). The
vector was transfected into proliferating myoblasts by electroporation and
current recordings were performed 48 hours later
(Espinos et al., 2001
;
Fischer-Lougheed et al.,
2001
). Fig. 1C
illustrates whole-cell current traces recorded in myoblasts transfected with
the HERG construct. The same voltage protocol as in
Fig. 1B was used. It can be
seen that the current recorded in myoblasts overexpressing HERG is very
similar to the endogenous dofetilide-sensitive current. The only difference is
a 10-fold larger current amplitude, which we attribute to the presence of an
increased number of functional channels in transfected cells.
These results show that HERG transcripts are present in human myogenic cells, and that a K+ current with pharmacological and biophysical properties similar to an identified HERG current can be recorded in fusion-competent myoblasts.
Fusion-competent myoblasts depolarize when HERG current is
inhibited
The possible contribution of HERG channels to the membrane
hyperpolarization that precedes myoblast fusion was examined by measuring the
resting potential of fusion-competent myoblasts before and after application
of dofetilide (Fig. 2A). Only
hyperpolarized fusion-competent myoblasts expressing Kir2.1 inward rectifier
K+ channels were selected for these experiments, as myoblasts that
do not express Kir2.1 channels do not fuse
(Fischer-Lougheed et al.,
2001).
|
These results indicate that HERG channels contribute to the resting membrane potential of myoblasts. They also suggest that all fusion-competent myoblasts express functional HERG channels, as the 20 cells tested were depolarized by either dofetilide or E4031 (note that the probability of measuring a depolarization consecutively in 20 myoblasts with a detectable HERG current is very low, 0.4420, i.e. less than one in 13 million).
The observation that an inhibition of HERG channels depolarizes fusion-competent myoblasts by 10 mV implies that functional HERG channels are physiologically continuously activated at a hyperpolarized resting potential of -64 mV. The experiments described in Fig. 2B,C were performed to demonstrate that a steady activation of HERG channels is indeed present at this hyperpolarized potential. The superfusion medium contained 5 mM K+ concentration to mimic physiological conditions, and the electrophysiological recordings were performed in myoblasts overexpressing HERG channels to increase the magnitude of the current (under physiological conditions the current is small in native cells; in Fig. 1B, we had to set the extracellular K+ concentration to 30 mM in order to visualize the current). Fig. 2B shows examples of dofetilide-sensitive current traces, and Fig. 2C shows current-to-voltage and conductance-to-voltage relationships. The relationship at steady-state demonstrates that a HERG conductance able to carry a steady outward K+ current under physiological conditions is present at voltages between -15 and -80 mV. This observation confirms that the biophysical properties of the channel are compatible with the role we propose for HERG, namely to assist Kir2.1 channels in driving the resting membrane potential of fusion-competent myoblasts near -74 mV.
Inhibition of HERG channels increases the rate of myoblast
fusion
Given the results described in the previous section, we were now in a
position to test the effect of a 10 mV depolarization of myoblasts on their
ability to fuse. When transferred to serum-free differentiation medium, human
myoblasts fuse into multinucleated myotubes within 48 hours. If there is a
tight coupling between membrane potential, Ca2+ signal and the
differentiation program, the fusion rate should increase.
It can be seen in Fig. 3
that, under control condition, less than 4% of the myoblasts have fused after
24-27 hours of exposure to differentiation medium
(Fig. 3A,B, circles). A lag
time of 1 day was always observed in human myoblasts triggered to fuse in
culture. By contrast, in sister cultures treated with methanesulfonanilide
agents, large multinucleated myotubes were already observed after 1 day in
differentiation medium (see pictures in
Fig. 3). In the presence of
dofetilide (10 µM), 38% of the myoblasts have fused after 24 hours
(Fig. 3A, triangles). A similar
result was observed with E4031 (10 µM). When this drug was added to the
culture medium, the fusion index was 42% after 27 hours
(Fig. 3B, triangles). These
effects of dofetilide and E4031 were observed in three independent
experiments. Additional support for the hypothesis that there is a tight
coupling between membrane potential and myoblast fusion was provided by
testing the effects of increasing concentrations of dofetilide on the fusion
rate. We found that 0.1 µM increased the fusion rate by about 50% of the
maximum, and that the maximum increase is reached at 1 µM
(Fig. 3A, inset). Thus,
methanesulfonanilide agents clearly accelerate myoblast fusion in a
dose-dependent manner and, as these agents are relatively specific, this
effect may be linked to an inhibition of HERG channels. The final
(steady-state) fusion index, however, was not affected by the blockade of
HERG.
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In conclusion, these experiments with the antisense vector confirm that the rate of myoblast fusion is increased by a 10 mV depolarization. They also further confirm that dofetilide and E4031 are acting specifically on HERG channels.
Inhibition of HERG channel increases the cytoplasmic Ca2+
concentration
We mentioned that, in human myoblasts, T-type Ca2+ channels
activate near -80 mV and the peak window Ca2+ current occurs at -58
mV (Bijlenga et al., 2000).
When HERG channels are activated, the resting potential of fusion-competent
myoblasts is approximately -74 mV (`Rp' in
Fig. 5A). This means that the
window Ca2+ current is relatively small (
2.1 fA/pF). Full
inhibition of HERG channel activity depolarizes myoblasts by
10 mV
(horizontal arrow in Fig. 5A).
According to the biophysical properties of T-type Ca2+ channels
(Fig. 5A), this 10 mV
depolarization will increase the magnitude of the window T-type
Ca2+ current from 2.1 to 7.7 fA/pF (the shift from -74 to -64 mV is
in a rising phase of the window Ca2+ current), and this should lead
to a substantial increase in cytoplasmic Ca2+ concentration.
The experiments described in Fig.
5B,C show that, as expected, inhibiting HERG channel activity
raises the cytoplasmic Ca2+ concentration in fusion-competent
myoblasts. Free Ca2+ fluctuations were assessed using the
`inversepericam' fluorescent indicator
(Nagai et al., 2001).
`Pericams' are a family of GFP-based Ca2+ indicators, which allow
long-term Ca2+ measurements without compartmentalization of the
probe. Although it does not allow measuring absolute Ca2+
concentration, the `inverse pericam' was chosen because its high
Ca2+ affinity permits to monitor accurately small cytoplasmic
Ca2+ changes around basal level. It can be seen in
Fig. 5B (circles) that, in
response to 5 µM dofetilide, cytoplasmic Ca2+ rises within a few
minutes and remains elevated. The mean increase of fluorescence ratio was
7.3±1.4% in the 22 myoblasts tested
(Fig. 5C). To verify that the
dofetilide-induced Ca2+ increase was indeed due to HERG channel
inhibition, the same protocol was applied to myoblasts transfected with an
HERG antisense RNA vector (AS-HERG). For this purpose, a bicistronic vector
expressing both HERG antisense and inverse-pericam was constructed. The
expected result was that, although basal Ca2+ might be higher,
dofetilide should not increase cytoplasmic Ca2+ in myoblasts unable
to synthesize HERG channels. Fig.
5B (square) shows that, in a myoblast expressing both antisense
and inverse-pericam, dofetilide did not affect cytoplasmic Ca2+
concentration as the fluorescence ratio remained stable in the nine myoblasts
tested (-0.4±1.3%, Fig.
5C). Note that this result is significantly different from the
fluorescence ratio measured in control cells (7.3±1.4%,
P=0.003).
Taken together, these results indicate that the inhibition of HERG channel activity, via a 10 mV depolarization of myoblast membrane potential, increases cytoplasmic Ca2+ concentration and consequently accelerates the rate of human myoblast fusion.
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DISCUSSION |
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The present work demonstrates that (1) there is a coupling between membrane potential and differentiation, and 2) the result of this coupling can be an acceleration of a differentiation process, myoblast fusion. This demonstration was possible as reproducible small depolarizations could be induced in myoblasts cultures by full blockade of a K+ current, HERG. Incidentally, this is the first report of the presence of this current in myoblasts and we shall briefly put our observations in perspective in the next section.
Functional HERG channels are expressed in human myogenic cells
The HERG channel is a member of the voltage-gated ether-à-go-go
K+ channel family. HERG channels are characterized by a slow
current activation and deactivation, paired with a fast C-type inactivation
mechanism (Trudeau et al.,
1995). These unusual characteristics confer to HERG its peculiar
electrophysiological properties. The HERG gene was shown to be expressed in
the heart (Wymore et al.,
1997
), and mRNA for HERG is present in a number of different
species and tissues (Wymore et al.,
1997
; Arcangeli et al.,
1995
; Arcangeli et al.,
1997
; Shi et al.,
1997
; Bianchi et al.,
1998
; Zhou et al.,
1998
; Overholt et al.,
2000
).
We found that the HERG gene is expressed in proliferating myoblasts,
fusion-competent myoblasts and in myotubes. From our northern blot analysis,
it appears that the amount of mRNA declines after the induction of
differentiation. This may explain the reported absence of detectable signal in
human skeletal muscle by Curran et al.
(Curran et al., 1995).
However, using the more sensitive assay of RNase protection, ERG presence
could be demonstrated in rat muscle
(Wymore et al., 1997
).
The presence of functional HERG channels in myogenic cells was assessed
using electrophysiological tools and pharmacological agents.
Methanesulfonanilide antiarrhythmic agents were used, such as dofetilide and
E4031, which block HERG channels with a well documented specificity
(Sanguinetti and Jurkiewicz,
1990). In myoblasts, both drugs inhibit a K+ current
that has the typical activation and inwardly rectifying properties of the HERG
current. The expression of HERG channels in human myogenic cells is confirmed
by the comparison of the endogenous dofetilide-sensitive K+ current
to the corresponding current flowing through cloned HERG channels
overexpressed after electroporation of proliferating human myoblasts. The
biophysical properties of both currents are undistinguishable, strongly
suggesting that they reflect the activity of the same channel. We also tested
the effect of antisense RNA directed against the endogenous
dofetilide-sensitive K+ current. Unfortunately, the very small
current amplitude (as shown earlier, 56% of the cells have no detectable
current under conditions that favor the visualization of HERG, i.e. 30 mM
extracellular K+) precluded interpretation of the results of these
experiments.
HERG channels are present in all fusion-competent myoblasts and
contribute to their resting potential
We showed that HERG channels contribute to the resting potential of
fusion-competent myoblasts. Their contribution is lesser than that of Kir2.1
channels, as a full blockade depolarizes myoblasts only by 10 mV instead of
30-40 mV for Kir2.1 channels (Liu et al.,
1998; Fischer-Lougheed et al.,
2001
). The existence of rather specific blocking agents for HERG
channels (dofetilide and E4031) and the use of HERG antisense allowed us to
estimate that HERG channels contribute
14% (10 out of 74 mV) of the
K+ current required to drive the membrane potential of myoblasts to
-74 mV.
We detected HERG current in only 44% of the cells tested. Nevertheless, we suggest that all fusion-competent myoblasts have functional HERG channels contributing to their resting potential. This is based on the observation that every fusion-competent cell specifically tested for its resting potential (n=37) was depolarized when HERG channel activity or expression was reduced (22 myoblasts treated with dofetilide, six with E4031 and nine myoblasts transfected with HERG antisense). The probability is extremely low (0.4428 or one in 9.6 billion) that the depolarizing effect of dofetilide and E4031 observed in a total of 28 cells would have resulted from recording exclusively from myoblasts expressing a detectable HERG current.
We can explain the fact that we did not always detect an HERG current
during voltage-clamp of fusion-competent myoblasts by the low number of
overall K+ channels expressed in these cells
(Fischer-Lougheed et al.,
2001). Indeed, if the input resistance of a myoblast at rest is in
the order of 2 G
, a 10 mV depolarization would be produced by blocking
an HERG current of 5 pA. Such currents may not always be detected due to the
noise associated with the whole-cell voltage-clamp technique and the
superfusion of solutions, or small instabilities during the long protocols
required for recording the series of dofetilide-sensitive currents necessary
for establishing the current-to-voltage relationship of HERG.
It is important to mention that, although HERG blockade allowed us to demonstrate the coupling between membrane potential and myoblast differentiation, we neither demonstrate nor disprove here that HERG is physiologically involved in modulating myoblast differentiation. However, when one considers the effect of HERG inhibition, it appears that HERG channels contribute to set the myoblast membrane potential in a region of the T-type Ca2+ current window domain distant from the maximum window current, i.e. in a region where increments and decrements in window Ca2+ current signals by membrane potential modulation are possible. This could be used by myoblasts to adapt their fusion rate in response to environmental conditions during muscle growth or repair.
K+ channels, hyperpolarization, window current and
differentiation
Many studies in several preparations have linked modulation of the resting
membrane potential by K+ channels with differentiation
(Ma et al., 1998;
Pancrazio et al., 1999
;
Mauro et al., 1997
;
Puro et al., 1989
;
Lewis and Cahalan, 1990
;
Pappone and Ortiz-Miranda,
1993
), but a precise mechanism linking membrane potential and
differentiation was not characterized. We demonstrate here that a small
voltage change in the domain of the T-type Ca2+ window current
affects the differentiation of myoblasts.
The T-type Ca2+ channel 1 subunit genes [
1G,
1H,
1I (Perez-Reyes,
1999
)] encode channels that are all endowed with a steady-state
inward window Ca2+ current activated at negative potentials, i.e.
close to the resting membrane potential
(McRory et al., 2001
). It has
been suggested that a window current may play a role in many cellular events,
such as neuritogenesis at the onset of neuronal differentiation
(Chemin et al., 2002
),
aldosterone or atrial natriuretic factor secretion
(Lotshaw, 2001
;
Leuranguer et al., 2000
),
pancreatic ß-cell apoptosis in diabetes
(Wang et al., 1999
), or
motoneuron death in spinobulbar muscular atrophy
(Sculptoreanu et al., 2000
).
These examples of possible physiological and pathophysiological implications
of window T-type Ca2+ currents suggest that fine tuning of the
Ca2+ influx via a window current is not solely an attribute of
differentiating myoblasts and that it may participate to a greater diversity
of cell functions than thus far examined.
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ACKNOWLEDGMENTS |
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Footnotes |
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