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The Journal of Neuroscience, January 1, 2000, 20(1):76-80
Distinction among Neuronal Subtypes of Voltage-Activated Sodium
Channels by µ-Conotoxin PIIIA
Patrick
Safo1,
Tamara
Rosenbaum1,
Anatoly
Shcherbatko1,
Deog-Young
Choi1,
Edward
Han1,
Juan J.
Toledo-Aral3,
Baldomero M.
Olivera4,
Paul
Brehm1, and
Gail
Mandel2
1 Department of Neurobiology and Behavior and
2 Howard Hughes Medical Institute, State University of New
York at Stony Brook, Stony Brook, New York 11794, 3 Department of Physiology and Biophysics, School of
Medicine, University of Seville, 41009 Seville, Spain, and
4 Department of Biology, University of Utah, Salt Lake
City, Utah 84112
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ABSTRACT |
The functional properties of most sodium channels are too similar
to permit identification of specific sodium channel types underlying
macroscopic current. Such discrimination would be particularly advantageous in the nervous system in which different sodium channel family isoforms are coexpressed in the same cell. To test whether members of the µ-conotoxin family can discriminate among known neuronal sodium channel types, we examined six toxins for their ability
to block different types of heterologously expressed sodium channels.
PIIIA µ-conotoxin blocked rat brain type II/IIA (rBII/IIA) and
skeletal muscle sodium current at concentrations that resulted in only slight inhibition of rat peripheral nerve (rPN1) sodium current. Recordings from variant lines of PC12 cells, which selectively express either rBII/IIA or rPN1 channel subtypes, verified that the
differential block by PIIIA also applied to native sodium current. The
sensitivity to block by PIIIA toxin was then used to discriminate
between rBII/IIA and rPN1 sodium currents in NGF-treated PC12 cells in
which both mRNAs are induced. During the first 24 hr of NGF-treatment,
PN1 sodium channels accounted for over 90% of the sodium current.
However, over the ensuing 48 hr period, a sharp rise in the proportion
of rBII/IIA sodium current occurred, confirming the idea, based on
previous mRNA measurements, that two distinct sodium channel types
appear sequentially during neuronal differentiation of PC12 cells.
Key words:
PC12 cells; ion channel; sodium current; growth factor; CNS; PNS
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INTRODUCTION |
Toxins represent potent tools for
distinguishing among the various isoforms of voltage-dependent ion
channels. In particular, conotoxins have been instrumental in
identifying individual subtypes of voltage-dependent calcium channels
(McCleskey et al., 1987 ). Far less progress has been made in
identifying toxins that can be used in an analogous manner to identify
sodium channel subtypes. As is the case for calcium channels, the
macroscopic sodium current in individual neurons often involves the
activation of a complex mixture of sodium channel subtypes. However,
unlike calcium channels, the functional properties of most sodium
channel subtypes are similar at the level of macroscopic current,
precluding discrimination on the basis of functional differences.
Tetrodotoxin sensitivity has served as a standard tool for identifying
certain sodium channel subtypes in both nerve and muscle, and this
toxin has provided important but limited distinction among neuronal
sodium channel types (Campbell, 1992).
Early studies on µ-conotoxin GIIIA offered the promise of further
discrimination between two different sodium channel subtypes that were
both TTX-sensitive (Cruz et al., 1985). GIIIA blocked sodium current
recorded from skeletal muscle (SkM1) at concentrations that had
no discernible effects on neuronal sodium channels. Subsequent studies
of GIIIA have been helpful in understanding the interactions between
toxin- and pore-forming regions of the skeletal muscle SkM1 channel
type (French and Dudley, 1999), but unfortunately, little advance has
been made in identifying conotoxins that serve as discriminators
between neuronal sodium channel types. The recent report that PIIIA
blocked brain type sodium channels but spared the sodium channel
responsible for action potential propagation suggested that this
conotoxin might serve such a purpose (Shon et al., 1998). However, the
PIIIA-insensitive sodium channel responsible for motoneuron action
potential was not known. Our studies took advantage of the fact that
the  subunit of central and peripheral neuron (PN1) sodium
channel types can be heterologously expressed in Xenopus
oocytes to test a battery of conotoxins on skeletal muscle, brain, and
peripheral nerve isoforms. Our findings identify PIIIA as the first
conotoxin that can discriminate between known neuronal sodium channel
subtypes. The application of PIIIA conotoxin to NGF-treated PC12 cells
also provides the first discrimination between PN1 and brain type
II/IIA (rBII/IIA) components of macroscopic sodium current.
Moreover, the time course of appearance of these two channel isoforms
is markedly different and mirrors the differential time course of
induction of the corresponding mRNAs.
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MATERIALS AND METHODS |
Complementary DNAs encoding each of four different sodium
channel subunits [skeletal muscle SkM1 (Trimmer et al.,
1989), rat BII/IIA (rBII/IIA) (provided by A. Goldin, Irvine,
CA) (Auld et al., 1988), human PN1 (hPN1) (provided by F. Hoffman, Munich, Germany) (Klugbauer et al., 1995), and rPN1
(Toledo-Aral et al., 1997)] were used to transcribe RNAs for injection
into Xenopus oocytes. Oocytes were surgically isolated from
anesthetized Xenopus frogs (Nasco, Fort Atkinson, WI) and
enzymatically treated with 10 mg/ml collagenase (Life
Technologies, Grand Island, NY) for 20 min before mechanical
removal of the follicle cell layer. Oocytes were maintained in a
nutrient OR-3 medium composed of 50% L-15 medium, 100 µg/ml
gentamycin, 4 mM glutamine, and 30 mM Na-HEPES (all from Life Technologies) with the
pH adjusted to 7.6 with NaOH. Injected oocytes were maintained at
18°C in OR-3 medium before recording.
Sodium current from oocytes was measured using a two-microelectrode
oocyte voltage clamp (TEV200; Dagan Instruments, Minneapolis, MN). The recording solution contained (in mM):
sodium methanesulfonate 100, NaCl 10, Na HEPES 10, and
CaCl2 2, with the pH adjusted to 7.0. To minimize
voltage errors during the recordings, the microelectrode resistance was
<1 M , and in some recordings the size of the sodium current was
reduced by substituting 90% of the sodium with
N-methyl-D-glucamine. Typically, the
cells were held at  100 mV to remove all inactivation, and the sodium
currents were elicited by 20 msec positive-going pulses. Toxins were
applied at specified concentrations by use of a continuous flow system.
The speed of flow was adjusted to provide complete exchange of solution
within 5 sec. Each test concentration of toxin was continuously applied
until no further block of sodium current was observed. At such time,
the equilibrium block of sodium current was determined on the basis of
the reduction of peak inward current. Equilibrium block by the toxins
was generally achieved within 1 min of application. It is unlikely that
the vitelline membrane posed any significant block to the peptide toxins because a complete and rapid block of current was observed after
application of TTX.
To induce sodium channel synthesis in PC12 cells, three lines
(wild-type PC12, FR3IIIb, and PN1-1) were treated with the appropriate growth factors. The FR3IIIb cell line has been described previously (Lin et al., 1998) and expresses primarily rBII/IIA mRNA. The PN1-1 line was generated by transfection of PC12 cells with pRSVneo. Among the neomycin-resistant clones, one clone (PN1-1) exhibited unusually high levels of PN1 and extremely low levels of rBII/IIA mRNAs, as assessed by Northern blot analysis. To induce sodium channels, wild-type and FR3IIIb lines were treated with 10 ng/ml FGF,
and the PN1-1 line was treated with 100 ng/ml NGF, 2 d before recording. The cells were plated and grown on 35 mm plastic tissue culture dishes. Northern blot analyses were performed as described previously (D'Arcangelo et al., 1993). Hybridizing mRNA was detected using a Molecular Dynamics (Sunnyvale, CA) Phosphorimager.
Whole-cell recordings of PC12 sodium current were made by means of an
Axopatch 200A amplifier (Axon Instruments, Burlingame, CA). The
recording solution contained (in mM): NaMES 140, MgCl2 0.2, CaCl2 0.2, and
Na HEPES 10, adjusted to pH 7.2. The pipette solution contained (in
mM): CsMES 140, CsEGTA 10, and Cs HEPES 10, adjusted to pH
7.2. To record sodium currents, the cells were held at 120 mV and
stepped to positive potentials for 20 msec. Both PC12 cell and oocyte
sodium currents were digitized at 50 kHz and analyzed off-line using
HEKA Pulse and Pulse Fit software (Instrutech, Great Neck, NY).
Capacitive transients were compensated using a combination of manual
compensation on the amplifier and further processing using either a P/4
or P/10 leak subtraction protocol.
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RESULTS |
Screening of conotoxins on sodium channel subunits expressed in
Xenopus oocytes
Four different sodium channel subunit types were expressed
individually in Xenopus oocytes. All four subunits
exhibited qualitatively similar function in terms of both
voltage-dependence and characteristic slow inactivation of inward
current caused in large part by lack of  -1 sodium channel
subunit in oocytes. To test for block of inward current by specific
conotoxin types, oocytes expressing a single type of subunit were
treated for 5 min at each concentration, after which time the peak
sodium current was determined from the current-voltage relationships (Fig.
1A,B).
Generally, at least seven concentrations were tested for block on each
of three oocytes. The highest concentrations used were, in some cases,
still too low to obtain more than a partial block because of the
insensitivity of the subunit to the particular toxin. Higher
concentrations could not be tested because of the limited availability
of these toxins.
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Figure 1.
Determination of the sensitivity of
heterologously expressed sodium channel isoforms to the conotoxin
PIIIA. A, B, Left panels,
The time-dependent block of inward rPN1 (top 3 traces)
and rBII/IIA (bottom 3 traces) sodium current in
Xenopus oocytes after application of 1 µM
PIIIA conotoxin. The individual sodium currents were elicited by a
voltage step to 10 mV from a holding potential of 100 mV and
acquired at 10 sec intervals. Right panels, The
current-voltage relationships for individual rPN1 (top)
and rBII/IIA (bottom) sodium current before (open
circles) and after (filled circles)
equilibrium block by 1 µM PIIIA conotoxin.
C, The concentration-dependent equilibrium block of
sodium current measured for four different sodium channel isoforms. All
data points reflect the mean ± SD from three different
oocytes (except for the 1 µM SkM1 data point, which
reflected a single measurement). The magnitude of the block was
determined on the basis of the decrease in current measured during a
step to 10 mV.
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The blocking ability of each toxin was determined by fitting the
relationship between peak sodium current versus toxin concentration, according to the equation F = IC50/([Tx] + IC50), where
the equilibrium half-blocking concentration is represented by
IC50, Tx represents the toxin concentration, and
F is the fraction of unblocked peak sodium current (Fig.
1C). The data fit by this relationship represented an
average of three cells for each toxin concentration. The
IC50 for each toxin was then determined from the
midpoint of the fitted curve to the average data or on the basis of the
extrapolated midpoint value. Table 1
indicates the mean IC50 values determined for
individual toxin block of the SkM1, rBII/IIA, hPN1, and rPN1 subunit types. As expected, all four subunit types were sensitive to TTX in the nanomolar range (Table 1). PIIIA and GIIIA both inhibited
SkM1 sodium current in the nanomolar range but contrasted in their
ability to block the neuronal isoforms of sodium channels. GIIIA
required high concentrations to block both rBII/IIA and rPN1,
indicating a general lack of sensitivity on the part of neuronal
isoforms to this toxin. In contrast, PIIIA blocked rBII/IIA at
concentrations intermediate to those of rPN1 and SkM1 (Fig. 1A,B). The
IC50 for PIIIA block of rBII/IIA was 690 nM compared with an extrapolated value of 6.2 µM for block of rPN1 sodium current. MrVIA,
GmVIA, and PVIA each inhibited sodium current, but no differences in
sensitivity were observed among the neuronal sodium channel types
(Table 1).
PIIIA conotoxin distinguishes CNS and PNS components of sodium
current in PC12 cells
Rat PC12 cells, a cell line established from a pheochromocytoma,
acquire the ability to generate sodium-based action potentials after
treatment with NGF, concomitant with the elaboration of other neuronal
phenotypic traits (for review, see Halegoua, 1991). PC12 cells
coexpress two distinct sodium channels in response to treatment with
NGF (Mandel et al., 1988; D'Arcangelo et al., 1993), but the time
course of induction of the two mRNAs is distinct. NGF treatment leads
to a transient rise in mRNA encoding rPN1 channels within the first 24 hr, followed by the appearance of mRNA coding for rBII/IIA channels.
Electrophysiological analyses at times subsequent to the mRNA increases
reveals that induction of sodium current is not associated with changes
in sodium current kinetics (Toledo-Aral et al., 1995).
Our results from Xenopus oocytes expressing either rPN1 or
rBII/IIA sodium channels suggested that PIIIA could provide a tool for
distinguishing between these components of native sodium current in
PC12 cells. To test this idea, variant lines of PC12 cells were
established that expressed, based on mRNA measurements, either rBII/IIA
(FR3IIIb line) or rPN1 (PN1-1) sodium channel types (Fig. 2C). Whole-cell patch-clamp
recordings of FR3IIIb and PN1-1 cells were performed within 48 hr of
treatment with growth factor. The ability of PIIIA conotoxin to block
sodium current was quantitated by the fractional decrease in peak
sodium current in response to application of 1 µM toxin. Based on the findings from channels expressed in Xenopus oocytes, this concentration was
expected to block <10% of rPN1 compared with >75% block of rBII/IIA
sodium current. The results from representative FR3IIIb and PN1-1
cells are shown in Figure 2, A and B. PIIIA
blocked only 13% of peak sodium current in the PN1-1 cells,
consistent with expression of the rPN1 sodium channel type. In the
FR3IIIb cells, 77% block was observed, consistent with expression of
rBII/IIA sodium channels (Fig.
2A,B). The current-voltage
relationships for both cell lines indicated no shift in
voltage-dependent properties of sodium current associated with the
block of inward current (Fig.
2A,B). The fractional blocks for
seven individual FR3IIIb cells and for nine different PN1-1 cells are
shown in Figure 2D. The overall average for FRIIIb cells corresponded to 87% compared with an average value of 13% for
PN1-1 cells.
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Figure 2.
Determination of the sensitivity of native rPN1
and rBII/IIA sodium channels to inhibition by conotoxin PIIIA.
A, Left panels, Sodium currents recorded
PN1-1 and FR3IIIb cells in the absence and presence of 1 µM PIIIA conotoxin. Small block of rPN1 current
(top) contrasts with the large block of rBII/IIA current
(bottom). The individual sodium currents were elicited
by a voltage step to 10 mV from a holding potential of 100 mV and
were acquired at 10 sec intervals. Right panels, The
current-voltage relationships for individual rPN1 (top)
and rBII/IIA (bottom) sodium currents before
(open circles) and after (filled
circles) equilibrium block by 1 µM PIIIA
conotoxin. C, Northern blot analyses of the FR3IIIb
versus PN1-1 cell lines using a sodium channel isoform-specific probe.
The measurements of mRNA were made after 5 hr of NGF treatment for the
PN1-1 cells and after 72 hr of FGF treatment for the FR3IIIb cells.
D, A histogram comparing the amount of PIIIA conotoxin
block for individual PN1-1 and FR3IIIb cells. The percentage
inhibition was determined on the basis of block of inward current by 1 µM PIIIA conotoxin during a step depolarization to 10
mV from a holding potential of 100 mV.
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PIIIA conotoxin was then applied, at a fixed 1 µM
concentration, to wild-type PC12 cells to quantitate the relative
contributions of rBII/IIA and rPN1 sodium channels to the macroscopic
current. Previous measurements of mRNA indicated that rPN1 mRNA peaked during the first 12 hr after NGF treatment, whereas rBII/IIA mRNA was
induced after 48-72 hr. Therefore, we quantitated the fractional block
of sodium current by PIIIA at 24 hr time points after NGF treatment.
Based on the findings from the variant lines, 1 µM conotoxin was expected to block an average of 13% of rPN1 current and
an average of 87% rBII/IIA sodium current. During the initial 24 hr,
an average of 13 ± 12% of the NGF-induced sodium current in
wild-type PC12 cells was blocked by PIIIA, pointing to rPN1 sodium
current (Fig. 3). The amount of
PIIIA-sensitive current increased to an average of 39 ± 15% at
48 hr and to 63 ± 16% at 72 hr, supporting the idea that the
relative proportion of rBII/IIA current was increasing over time. The
increase in proportion of PIIIA-sensitive sodium current was not
associated with any significant increase in sodium current density
(Fig. 3). In a separate experiment, the PIIIA-sensitive current
measured 13% at 24 hr and 51% at 48 hr (data not shown).
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Figure 3.
Time-dependent changes in the fractional
inhibition of PC12 sodium current by PIIIA conotoxin after growth
factor induction. The sodium current density (mean ± SD pA/pF )
is shown for 24, 48, and 72 hr after growth factor treatment. The
shaded area represents the current that is
PIIIA-insensitive, and the solid area represents the
amount of current that is PIIIA-sensitive. The fractional sensitivity
of overall sodium current to PIIIA toxin is indicated as a percentage
at each time point (see Results for SD). Each time point
represents the average of nine cells.
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DISCUSSION |
Our findings indicate that PIIIA µ-conotoxin permits distinction
between two major sodium channel types whose origins are predominantly
the CNS (rBII/IIA) or PNS (rPN1). Although the difference in
sensitivities between the rPN1 and rBII/IIA channel types was only an
order of magnitude, the difference was sufficient to reveal the
distinct current contributions in PC12 cells undergoing differentiation by NGF. One question that arises is whether the conotoxin sensitivity measured for channels expressed in oocytes is the same as that for
native sodium channels in mammalian cells. We addressed this question
through quantitative comparisons of conotoxin sensitivity for both rPN1
and rBII/IIA subunit types. Comparisons of the sensitivities to
PIIIA conotoxin show excellent agreement between oocytes and PC12
cells. A fixed 1 µM concentration of PIIIA led to a 10%
block of rPN1 in oocytes compared with 13% in PC12 cells. Similar
agreement was indicated by the 75 and 87% block of rBII/IIA in oocytes
and PC12 cells, respectively.
Our findings provide the first direct evidence that two structurally
distinct sodium channel types are coexpressed in PC12 cells. To date,
the idea that two sodium channel isoforms underlie the NGF-induced
sodium current in PC12 cells has been based solely on inference from a
variety of indirect measurements. Assignment of sodium current to
either rPN1 or rBII/IIA channels in PC12 cells relied on the
differences in time courses for the appearance of respective mRNA
species. For example, because a brief treatment with NGF selectively
triggers upregulation of rPN1 mRNA, but not rBII/IIA mRNA, it was
possible to record sodium currents at times when only PN1 sodium
channels should be expressed (Toledo-Aral et al., 1995). However,
precedent also existed for induction of sodium channel mRNA in PC12
cells without expression of functional sodium channels (Ginty et al.,
1992). The existence of intracellular pools of subunits in neurons
(Schmidt et al., 1985) further prevented unequivocal assignment
of induction of a specific sodium channel mRNA with macroscopic
current. Thus, the ability to discriminate between functional channels
on the cell surface provides a more direct measurement of signaling
effects on specific sodium channel types.
In addition to discriminating between rPN1 and rBII/IIA functional
channels in PC12 cells, the differential toxin sensitivity provided an
estimation of the proportions of the two channel types after NGF
treatment. The proportions, based on functional estimates, compared
favorably with the estimates based on mRNA measurements. Previous mRNA
measurements after pulsed NGF treatment of PC12 cells indicated that
mRNA coding for rPN1 first appeared at ~1 hr, peaked at 5 hr, and
declined to near zero values during the first 24 hr period. In
contrast, in the continued presence of NGF, both sodium channel mRNAs
are induced, but mRNA coding for rBII/IIA mRNA appears over a much
slower time course than rPN1, peaking after several days instead of
after 5 hr. Consequently, our first functional estimate corresponded to
a time when the dominant mRNA species corresponded to rPN1. At 24 hr,
only 13% of the sodium current was PIIIA-sensitive in each of two
experiments. This value agreed with the predicted amount of block based
on a pure rPN1 type sodium current as determined by the PN1-1 variant cell line. Therefore, it is likely that the current observed during the
first 24 hr is, in fact, contributed almost exclusively by the rPN1
channel type. During the subsequent 48 hr period, we observed a steep
increase in the proportion of PIIIA-sensitive current, once again
consistent with the observed increase in mRNA encoding rBII/IIA sodium
channels. Furthermore, in these experiments, the increase in proportion
occurred without significant changes in sodium channel density. Thus,
the switch to predominantly rBII/IIA sodium channel function likely
reflects a decrease in rPN1 mRNA in addition to accumulation of
rBII/IIA mRNA.
In conclusion, PIIIA has provided unequivocal evidence for the
contributions of two distinct sodium channel types to TTX-sensitive macroscopic sodium current in PC12 cells. More importantly, the time
course studies indicate that the functional appearance of the
peripheral nerve and brain type sodium currents are well predicted by
the time course of the individual mRNA species. The functional significance of two channel types exhibiting different time courses of
expression during differentiation is not known. However, further studies using PIIIA µ-conotoxin to dissect the individual
contribution by each channel type in vivo should help
provide answers to this important question.
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FOOTNOTES |
Received Aug. 19, 1999; revised Sept. 28, 1999; accepted Oct. 12, 1999.
This work was supported by National Institutes of Health (NIH)
Grant PO1 NS 34375 and by NIH Minority Access Grant GM08655 to
P.S. G.M. is an investigator of the Howard Hughes Medical
Institute. J.T.-A. was supported by a fellowship from the Multiple
Sclerosis Society (RG 2959A1/T).
Correspondence should be addressed to Gail Mandel, Howard Hughes
Medical Institute, Department of Neurobiology and Behavior, State
University of New York at Stony Brook, Stony Brook, NY 11794. E-mail:
gmandel{at}notes1.cc.sunysb.edu.
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ABSTRACT
INTRODUCTION
