1Curriculum in Oral Biology, School of Dentistry and 2Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Wang, Haibin and Gerry S. Oxford. Voltage-Dependent Ion Channels in CAD Cells: A Catecholaminergic Neuronal Line That Exhibits Inducible Differentiation. J. Neurophysiol. 84: 2888-2895, 2000. Cell lines derived from tumors engineered in the CNS offer promise as models of specific neuronal cell types. CAD cells are an unusual subclone of a murine cell line derived from tyrosine hydroxylase (TH) driven tumorigenesis, which undergoes reversible morphological differentiation on serum deprivation. Using single-cell electrophysiology we have examined the properties of ion channels expressed in CAD cells. Despite relatively low resting potentials, CAD cells can be induced to fire robust action potentials when mildly artificially hyperpolarized. Correspondingly, voltage-dependent sodium and potassium currents were elicited under voltage clamp. Sodium currents are TTX sensitive and exhibit conventional activation and inactivation properties. The potassium currents reflected two pharmacologically distinguishable populations of delayed rectifier type channels while no transient A-type channels were observed. Using barium as a charge carrier, we observed an inactivating current that was completely blocked by nimodipine and thus associated with L-type calcium channels. On differentiation, three changes in functional channel expression occurred; a 4-fold decrease in sodium current density, a 1.5-fold increase in potassium current density, and the induction of a small noninactivating barium current component. The neuronal morphology, excitability properties, and changes in channel function with differentiation make CAD cells an attractive model for study of catecholaminergic neurons.
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INTRODUCTION |
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Cultured cell lines of
purely neuronal origin are of immense value in biochemical and
molecular studies of neuron function. They are potentially superior to
primary neuronal cultures as the latter are usually restricted in
numbers, mixed with other types of cells and often fragile in culture.
Furthermore, it is generally difficult to maintain primary neurons
derived from adult animals for long periods of time. Thus the
establishment of immortalized neuronal cell lines, especially those
with specific differentiated neuronal phenotypes is desirable. So far,
several cell lines have been generated for this purpose, such as PC12
cells (Greene and Tischler 1976), P19 cells
(Jones-Villeneuve et al. 1982
), MN9D cells (Choi
et al. 1991
), and MES23.5 cells (Crawford et al.
1992
), but none of these lines derive purely from the CNS nor
exhibit completely differentiated neuronal characteristics.
Through the use of oncogenes driven by cell-specific promoters that
direct tumorigenesis of target defined neurons in transgenic mice, a
new catecholaminergic cell line called CATH.a has been developed
(Suri et al. 1993). This line was derived from tyrosine hydroxylase (TH)-positive tumors induced in transgenic mice carrying the Simian Virus (SV40) large T antigen oncogene under the
transcriptional control of 5' flanking sequences from the rat TH gene.
Tumors derived in this manner are restricted to neuronal populations normally expressing the TH gene, the transcriptional regulation of
which is co-opted to trigger neoplastic growth. CATH.a cells synthesize
dopamine and norepinephrine and express the catecholamine synthetic
enzymes, TH and dopamine
-hydroxylase. In addition, these cells
exhibit neuronal specific molecules such as neurofilaments and
synaptophysin (Suri et al. 1993
) and express
voltage-dependent ion channels including tetrodotoxin (TTX)-sensitive
sodium channels, L- and N-type calcium channels and potassium channels
(Lazaroff et al. 1996
). Although CATH.a cells express a
variety of pan-neuronal markers, these cells do not bear neurites, nor
apparently can they be induced to morphologically differentiate
(Qi et al. 1997
).
CAD cells are a novel subclone recently derived from CATH.a
cells by single cell cloning of spontaneous variants that exhibited short processes not normally seen in the parental line (Qi et al. 1997). In contrast to their parental CATH.a cells,
CAD cells can undergo a reversible differentiation. When cultured in
serum containing medium, CAD cells have an undifferentiated phenotype (Fig. 1A) and proliferate.
Remarkably on serum deprivation, CAD cells develop a differentiated
neuronal-like phenotype (Fig. 1B) and stop
proliferation (Qi et al. 1997
). The undifferentiated phenotype
is characterized by round or oval-shaped cells with few if any
processes, while the differentiated phenotype exhibits a spindle-like
shape with long processes morphologically identical to those of many
primary CNS neurons. Ultrastructurally, processes from differentiated
CAD cells have abundant parallel microtubules and intermediate
filaments, and bear varicosities that contain both large dense-core
vesicles and smaller clear vesicles (Qi et al. 1997
).
Interestingly, the SV40 T antigen has been spontaneously lost in CAD
cells, while neuron-specific proteins, such as class III
-tubulin,
GAP-43, and SNAP-25, and synaptotagmin, but not the astrocytic marker
GFAP are expressed in CAD cells. In addition, CAD cells express
enzymatically active TH and accumulate L-DOPA (Qi et
al. 1997
).
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To determine whether CAD cells have the excitability properties
expected for neuronal membranes, we characterized voltage-dependent currents using whole cell recording methods. Furthermore, the reversible differentiation of CAD cells affords the opportunity to
examine the regulation of excitability during differentiation in this
cell line. Differentiation of normal neuron precursors (Spitzer
and Ribera 1998) and other tumor-derived cell lines (e.g., Toselli et al. 1996
) results in changes in channel
expression and function. The parent CATH.a cell line has previously
been shown to express several different voltage-dependent channels (Lazaroff et al. 1996
); however, the inability of that
line to exhibit neuronal morphology prompts the comparison of channel expression in differentiated versus undifferentiated CAD cells. A
preliminary report of some of these findings has appeared in abstract
form (Smith et al. 1997
).
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METHODS |
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Cell culture
CAD cells were grown at 37°C and in 5% CO2 on 25 cm2 tissue culture flasks (Sarstedt, Newton, NC) in Ham's F12/EMEM medium (GIBCO, Grand Island, NY), supplemented with 8% fetal bovine serum (FBS; Sigma, St. Louis, MO) and 1% penicillin/streptomycin (100% stocks, 10,000U/ml penicillin G sodium and 10,000 µg/ml streptomycin sulfate). Cells were passaged every 6-7 days at a 1:20 dilution. To induce morphological differentiation, cells were switched to the same medium without serum supplementation after 12-24 h. For electrophysiological characterization, cells are plated onto poly-lysine-coated plastic coverslips (Fisher, Pittsburgh, PA) and visualized under Hoffman optics during experiments.
Electrophysiological techniques
Standard whole cell patch-clamp methods were employed to record
the resting potential, action potentials, and voltage-dependent currents from individual CAD cells. The resting potential and action
potentials were studied under current clamp, and action potentials were
usually expressed during tonic hyperpolarization of the cell by
injection of 5 to
10 pA currents. Whole cell current responses were
routinely elicited under voltage clamp by depolarizing step voltage
commands (
80 to +60 mV) or repetitive voltage commands to +40 mV. The
holding potential in most experiments was
60 mV, with the exception
of measurements of Ca2+ channel activity where
the holding potential was
80 mV and a -P/4 divided pulse protocol was
employed to eliminate the passive membrane properties. To examine
A-type potassium currents, a family of inactivation prepulses from
100 to +10 mV (280 ms) followed by repetitive steps from holding to
+10 mV was used. Signals were recorded with an Axopatch 1B or 200 amplifier (Axon Instruments, Foster City, CA) and filtered at 5 kHz.
Patch electrodes are pulled from cleaned Drummond N51A glass (Drummond
Scientific, Broomall, PA) on a PP-83 (Narashige, Tokyo) puller. Dental
wax (Kerr Sticky wax, Romulus, MI) was used to coat the electrodes to
minimize the capacitive properties. Electrodes were fire polished with a homemade microforge to a final electrode resistance of 0.5-6 M
.
Electrode tips were filled by dipping into a relatively simple chloride
salt solution of the major cation for an experiment (e.g., KCl, HEPES,
MgCl2) and then backfilled with the desired patch solution (see Recording solutions and drugs). The
experimental chamber containing cells was viewed with a Nikon Diaphot
inverted microscope (Nikon, Japan) at ×400 under Hoffman optics and
was continuously superfused at ~1 ml/min with external solutions. We
routinely achieved gigaseals of 1 to ~10 G
on CAD cells. All experiments were performed at room temperature (21-23°C). The access
resistance values typically ranged from 2 to 7 M
. The current
responses were normalized by the cell capacitance (pA/pF) to account
for variation in cell size and geometry.
Recording solutions and drugs
All experimental solutions were adjusted to pH 7.4 and
osmolarity of ~300 mosM. The standard external solution (SES)
contained (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose. Calcium currents
were measured with barium as a charge carrier. The barium external
solution (BaES) contained (in mM) 125 NaCl, 10 TEA-Cl, 10 BaCl2, 1 MgCl2, 10 HEPES,
and 10 glucose. To eliminate sodium currents during potassium and
calcium current measurements, 300 nM tetrodoxin (TTX) was added to
external and drug solutions. Two internal solutions were used.
"K1.2," which was used in most experiments unless the internal
solution was specified, contained (in mM) 130 K-Aspartate, 20 KCl, 10 HEPES, 10 glucose, 1 MgCl2·6 H2O, and 1 EGTA.
"Cs1" contained (in mM) 145 CsCl, 10 HEPES, 10 glucose, 0.1 GTP, 2 MgATP, and 1 EGTA. All drugs were stored as stock solutions at 4°C
and then diluted in external solution on the day of the experiment.
Stock solutions of TTX (Alomone Labs, Jerusalem, Israel),
tetraethylammonium chloride (TEA-Cl; Eastman Kodak, Rochester, NY) and
3,4-diaminopyridine (3,4-DAP; Aldrich, Milwaukee, WI) were made in
distilled water at a concentration of 0.1, 100, and 100 mM,
respectively. Ten millimolar nimodipine stock solution was made in 95%
ethanol and stored at 20°C. Drugs were applied from a multi-barrel
pipette array built from 3 µl Drummond Microcap tubes (Drummond
Scientific, Broomall, PA) connected to syringe reservoirs via a
switching valve.
Data acquisition and analysis
Whole cell currents were sampled via a Digidata 1200b interface using Axotape and pClamp 6.0 software (Axon Instruments, Foster City, CA). Data files are then imported into SigmaPlot and SigmaStat (SPSS, Chicago, IL) for display or analysis. Statistical significance for comparative data was determined by a Student's t-test and two-way ANOVA at a probability value <0.05.
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RESULTS |
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Resting membrane potentials and action potentials of CAD cells
A hallmark of neuronal cells, the firing of action potentials
underlies efficient integration and transfer of electrical signals. To
investigate the resting membrane potential and excitability of CAD
cells, we performed current-clamp studies on both undifferentiated and
differentiated phenotypes. In both phenotypes, the resting potentials
were nearly identical, with mean values of 40 ± 1 mV (mean ± SE, n = 8) for undifferentiated CAD cells
and
41 ± 1 mV (n = 13) for differentiated CAD
cells. Due to the relatively low resting potentials, only a few cells
(1 of 7 undifferentiated CAD cells, data not shown; and 1 of 11 differentiated CAD cells, Fig. 1D) exhibited spontaneous
firing of action potentials at the resting membrane potential. However,
hyperpolarization of the membrane potential to about
80 mV by steady
injection of a small negative current permitted spontaneous firing in
every cell examined (n = 6, undifferentiated CAD, Fig.
1C; and n = 10, differentiated CAD, Fig.
1E). These results suggest that both phenotypes of CAD cells
are excitable, but that they rarely are capable of excitation at their
normally rather depolarized resting potential. This situation likely
reflects two features of the ion channel complement expressed in these
cells. First, under voltage-clamp conditions, CAD cells do not display
significant inwardly rectifying currents when hyperpolarized to levels
as negative as
120 mV (data not shown). Second, more than 90% of the
sodium channels in CAD cells are inactivated at
40 mV (Fig. 4), thus making action potential generation problematic.
General properties of voltage-dependent Na+ and K+ currents in CAD cells
Since both phenotypes of CAD cells were excitable, we investigated
and compared the voltage-dependent currents underlying the excitation
in both phenotypes. On depolarization we observed a transient inward
current and a delayed outward current in all cells examined including
both undifferentiated (n = 50) and differentiated (n = 53) phenotypes. Representative responses to both
brief (15 ms) and long (150 ms) voltage steps are shown in Fig.
2 for an undifferentiated CAD cell
(A and B), and a differentiated CAD cell
(C and D). Regardless of phenotype, the transient
inward current in CAD cells activated between 30 and
20 mV and
reached its peak at 0 to +10 mV (Fig. 2, E and F;
) consistent with the typical voltage-dependent
Na+ current recorded in other excitable cells and
suggesting no significant changes in activation properties on
differentiation. One distinguishing characteristic of most neuronal
Na+ channels is their sensitivity to TTX. The
transient inward currents in the parental CATH.a cells were shown to be
TTX sensitive (Lazaroff et al. 1996
). We therefore
challenged CAD cells with 300 nM TTX, which totally eliminated the
transient inward currents in both undifferentiated (n = 39) and differentiated (n = 45) conditions (data not
shown).
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The delayed outwardly rectifying current, most prominent in
differentiated CAD cells, was observed at depolarizations above 20 to
0 mV (Fig. 2, E and F;
), and is comparable to
other delayed rectifier K+ currents. Transient
K+ currents (A-type K+
current) have been shown to play a role in regulating the frequency of
action potentials in neurons (Rogawski 1985
;
Segal et al. 1984
). While the kinetic properties of
outward currents elicited by standard voltage step families did not
suggest a significant inactivating current component, we further
explored the expression of such channels using a prepulse inactivation
voltage protocol (see METHODS). The absence of any
transient component evoked by depolarizations from holding potentials
as negative as
120 mV and the absence of appreciable attenuation of
currents following prepulses of increasing depolarization indicate that
few if any A-type K+ channels are expressed in
these cells (undifferentiated n = 8, differentiated
n = 5; data not shown).
Differentiation of CAD cells induces a reciprocal change of Na+ and K+ current expression
As suggested by the data in Fig. 2, differentiation might alter the relative expression of sodium and potassium channels. Assessment of our accumulated data from both differentiated and undifferentiated cells supports this idea and reveals a reciprocal change in the functional expression level of the two types of current. Specifically, in undifferentiated CAD cells, Na+ currents were rather large and K+ currents were relatively small. In contrast, on differentiation, Na+ currents were dramatically decreased while K+ currents were significantly increased (Fig. 2, E and F). Thus on differentiation of CAD cells, Na+ and K+ currents underwent reciprocal changes. To quantify this change, the average Na+ and K+ currents measured at 0 and +60 mV, respectively, were compared (Fig. 3). Normalizing the currents by cell capacitance to account for differences in cell size, we found that the differentiation of CAD cells resulted in an approximate 4-fold decline in Na+ current density (P < 0.001) and an approximate 1.5-fold increase in K+ current density (P < 0.001).
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The dramatic reduction in sodium current density could arise from a
number of different mechanisms, including changes in channel expression
or gating properties. As currents were usually elicited from holding
potentials of 60 or
80 mV, one particularly obvious possibility
would be a shift in the voltage dependence of steady-state sodium
inactivation such that more channels might simply be inactivated at
these potentials in differentiated neurons. This mechanism has been
shown to primarily account for an increase in sodium conductance on
differentiation of another neuronal line, the human SH-SY5Y line
(Toselli et al. 1996
). We examined this possibility by
comparing steady-state inactivation of sodium channels induced by 50-ms
prepulses from
120 to +10 mV in undifferentiated versus differentiated CAD cells. All cells were internally dialyzed with Cs1
(see METHODS) to remove interference from K currents. As
shown in Fig. 4, no significant change in
either the position or voltage sensitivity of the resulting
steady-state inactivation relationship is seen with differentiation of
CAD cells. Thus changes in gating properties of sodium channels do not
appear to underlie the reduction in current density.
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Two distinct populations of K+ channels were identified in both phenotypes
While A-type channels do not appear to contribute to the total outward K+ currents, we were interested in further exploring the nature of the channels contributing to these currents. 3,4-DAP and tetraethylammonium (TEA+) are both voltage-dependent K+ channel blockers with distinct pharmacological mechanisms. We found that the delayed rectifier K+ currents in both differentiated (Fig. 5, A and B) and undifferentiated CAD cells (data not shown) were sensitive to both 3,4-DAP and TEA. However, when applying a repetitive +40-mV voltage step, we observed that the dynamics of block and unblock by the two drugs were distinct. TEA+ almost instantaneously blocked K+ current; likewise, the reversal of block on removing TEA+ was also instantaneous and complete. The addition of 3,4-DAP following equilibration with TEA+ failed to produce further block (Fig. 5C). In contrast to TEA+, both block and unblock of 3,4-DAP were much slower, although the block, as for TEA+, was also completely reversible. In this case when TEA+ was added following of the block by 3,4-DAP, an additional rapid reduction in current was observed (Fig. 5D).
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These kinetic differences suggest that the CAD cell K+ channels can be subdivided into distinct subpopulations according to their pharmacological sensitivity to 3,4-DAP. A statistical comparison of block of delayed rectifying current in differentiated CAD cells by the two drugs revealed that nearly all K current (86.0 ± 2.7%) was blocked by TEA+, and a subset of those channels was also sensitive to 3,4-DAP (55.1 ± 4.4%). Similar percentages were found for block of the smaller currents observed in undifferentiated CAD cells (TEA+ sensitive 85.7 ± 1.5%, 3,4-DAP sensitive 45.4 ± 2.5%), indicating that while differentiation appears to change the magnitude of total K+ current (see Figs. 2 and 3), it does not change the relative contribution of the pharmacologically different K+ channel populations.
Ba2+ currents in CAD cells
In neuronal cells, calcium currents play a
crucial role in signal transduction, neurotransmitter secretion,
neuronal maturation, and differentiation. In CATH.a cells, N-type and
L-type Ca2+ currents have been previously
identified (Lazaroff et al. 1996). Given that CAD cells
are excitable and express both Na+ and
K+ currents, we were interested in investigating
Ca2+ currents. We enhanced calcium channel
currents by using 10 mM Ba2+ as the charge
carrier, and Cs+ as the only internal monovalent
cation to eliminate the K+ currents. In both
undifferentiated (Fig. 6A) and
differentiated (Fig. 6C) CAD cells, an inward current with
characteristics expected for a voltage-dependent calcium channel
current was routinely observed. The dominant current component in both
phenotypes was a transient current that completely inactivated in
undifferentiated cells. In contrast, differentiated cells exhibited an
additional steady-state component maintained for the duration of the
voltage step. The current-voltage (I-V) curves of the peak
Ba2+ currents showed that regardless the
phenotype, the threshold for activation was around
50 mV and the
maximum value occurred at about
20 to
10 mV (Fig. 6, E
and F,
). The inward Ba2+ currents
could be completely and reversibly blocked by 50 µM Cd2+ in both phenotypes (data not shown). To
compare Ba2+ currents between the two phenotypes,
the maximum currents at 0 mV were measured and normalized to the cell
size, yielding nearly identical current densities of 7 ± 1 pA/pF
(n = 24) and 6 ± 1 pA/pF (n = 17)
in undifferentiated and differentiated phenotypes, respectively
(P > 0.05).
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To further identify the Ca2+ channel subtypes
underlying these different kinetic components, we challenged the cells
with 10 µM nimodipine, an L-type Ca2+ channel
blocker. In undifferentiated phenotype, 10 µM nimodipine was able to
almost completely and reversibly block the transient Ba2+ currents (Fig. 6B), suggesting
that L-type channels are the only channels functionally expressed in
undifferentiated CAD cells. In the differentiated phenotype, nimodipine
also completely blocked the transient current component; however, a
distinct nimodipine-resistant component remained with a relatively
slow, maintained activation (Fig. 6D). This
nimodipine-resistant component did not activate until about 30 mV and
peaked at about
10 to 0 mV (Fig. 6F,
). Subtracting the
nimodipine-resistant currents from the total Ba2+
currents, we calculated the nimodipine-sensitive
Ba2+ current density, which was comparable
between undifferentiated and differentiated phenotypes (Fig.
6G,
, P > 0.05). In contrast, the much
smaller nimodipine-resistant component was increased by twofold on
differentiation (Fig. 6G,
, P < 0.02).
As the nimodipine-resistant component only accounted for a very small
portion of the total Ba2+ currents, we did not
characterize it in further using other pharmacological tools. Hence, we
conclude that in both phenotypes, the major Ca2+
channel component is L-type; nevertheless, on differentiation, expression of another distinct nimodipine-resistant
Ca2+ channel type is induced.
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DISCUSSION |
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The parental CATH.a cell line has been extensively investigated
due to its CNS origin and promise as a potential catecholaminergic neuronal line. The three major subtypes of opioid receptor have been
found in CATH.a cells (Baraban et al. 1995;
Bouvier et al. 1998
), and K+
channels in these cells can be regulated by kappa-opioid receptors (Baraban et al. 1995
). Also reports indicate that both
dopamine (Masserano et al. 1996
; Smith et al.
1998
) and nitric oxide donors (Smith et al.
1998
) can induce time- and dose-dependent apoptosis in CATH.a
cells. However, these cells do not exhibit neuronal morphology, and
there are no reports of spontaneous action potential activity despite
the expression of voltage-dependent Na+,
K+, and Ca2+ currents
(Lazaroff et al. 1996
). The novel CAD subclone, however, appears to maintain the expression of ion channels, while losing the
oncogene that drove the original tumor production for the parental line
and gaining the ability to express neuronal morphology with inducible differentiation.
We have confirmed that serum deprivation induces morphological
differentiation in CAD cells. On differentiation, extensive neurite
outgrowth and synaptic varicosities were evidenced (Fig. 1). Other
tumor-derived cell lines have been previously shown to express a
neuronal morphological phenotype on differentiation induced by agents
such as nerve growth factor (e.g., PC-12 cells) or retinoic acid (e.g.,
SH-SY5Y cells). Furthermore, in these two cell lines, such
neuronal-like differentiation also results in an increase in the
functional expression of voltage-gated sodium channels (Greene
and Tischler 1976; Toselli et al. 1996
). In
examining the corresponding changes in ion current expression on CAD
cell differentiation, we made an unexpected observation. In CAD cells there is a reciprocal change of Na+ and
K+ currents on differentiation reflecting a
4-fold decrease of Na+ current and
1.5-fold increase of K+ current. There are at
least three possible mechanisms that could contribute to these changes.
First, changes in gating properties, most importantly channel
inactivation behavior, through posttranslational modifications might
effect a change in open probability and thus current density. This
mechanism is unlikely, however, in the case of CAD cell differentiation
as the steady-state sodium inactivation behavior is not changed with
differentiation (Fig. 4) and no significant inactivating K channels
were observed in either phenotype. This is quite different from the
observation of increases in sodium current documented during retinoic
acid induced differentiation of SH-SY5Y cells (Toselli et al.
1996
). In that case, the steady-state sodium inactivation curve
was found to be shifted by nearly 25 mV to the right along the voltage
axis. This shift coupled with the holding potential used in those
experiments can account for most, if not all, of the increase in sodium
conductance seen with differentiation.
The second mechanism possibly underlying the changes in current density in CAD cells is a change in the level of expression of genes coding for the relevant channel types. Without knowing which isoforms of voltage-dependent Na+ or K+ channels are expressed by CAD cells, we have not attempted to assess this possibility. Third, it is possible that the level of Na+ channel gene expression was comparable between these two phenotypes, but the cellular distribution of channels was altered on differentiation. In undifferentiated CAD cells, all Na+ channels are necessarily located at the soma as no neurites are expressed; however, when cells are differentiated, there may be a redistribution of Na+ channels coincident with the growth of processes. During voltage clamp, control of the membrane potential in the remote neurite compartments may well be less than adequate, and the degree of depolarization induced in distal neurites may be diminished and hence channel activation compromised. If sodium channels were preferentially expressed in the neurite compartment, their contribution to currents during voltage-clamp measurements might be significantly reduced in differentiated CAD cells. In addition, there was an increase of K+ channel expression on differentiation, but the relative expression ratio of different pharmacological subpopulations was unchanged. To address the possible mechanisms involved, further experiments assessing channel mRNA expression or direct assessment of currents in neurites are required. Whether the current density changes reflect changes in expression or redistribution, both phenotypes were capable of generating action potentials, thus the changes observed may not have functional consequences.
The pharmacology of CAD cell K currents revealed by
TEA+ and 3,4-DAP is consistent with other studies
(Kirsch et al. 1986, 1991
; Purali
and Rydqvist 1992
; Wagoner and Oxford 1990
). Two classes of K channel exist in these neurons. One is sensitive to
TEA+ alone, while the other is sensitive to both
blockers. Not only do current magnitudes reveal these two classes, but
the characteristic differences in blocking kinetics confirm two
populations of channel, both of which exhibit delayed rectifier
properties. Neither A-type channels nor inwardly rectifying K channels
(e.g., Kir2 family) significantly contribute to K currents in CAD cells.
High-voltage-activated Ca2+ channels have also
been identified in both phenotypes. The overwhelming fraction of the
barium currents in both phenotypes was blocked by nimodipine, thus
being classified as reflecting L-type Ca2+
channels. On differentiation, a distinct nimodipine-resistant component
was induced. Shifts in expression of different
Ca2+ channel types with the different neuronal
developmental stages has been reported previously
(Chameau et al. 1999; Desmadryl et al.
1998
). While the induced divalent conductance seen with
differentiation did not exhibit inactivation, it was insensitive to
nimodipine and thus not a conventional L-type channel. The nature of
this conductance was difficult to explore reliably with routine
pharmacology (e.g., selective toxins) due to its small magnitude.
Contrary to the conventional neuronal L-type Ca2+
channel, which exhibits little if any inactivation when
Ba2+ was used as the charge carrier, the L-type
Ca2+ channel in both CAD cell phenotypes
manifested a significant inactivation even Ba2+
was used. That might suggest that a particular splice variant of
1C subunit is expressed by CAD cells and
caused strong acceleration of the Ba2+ current
inactivation (Soldatov et al. 1998
). In addition, some
subunits have been reported to modulate both voltage-dependent and
Ca2+-dependent Ca2+ channel
inactivation kinetics (Cens et al. 1999
).
From the functional perspective, our studies suggest that the CAD cell line exhibits the most important features of a purely CNS-derived catecholaminergic neuronal cell making it a useful tool for in vitro study. Differentiation is not a simple morphological change, but a change of channel expression and perhaps distribution. Further studies on CAD cells may help us to better understand the physiological functions of voltage-dependent ion channels in neuronal differentiation, and yield further understanding of molecular and electrophysiological nature of CNS-derived catecholaminergic neurons.
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ACKNOWLEDGMENTS |
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The authors express appreciation to Dr. Dona Chikaraishi for providing the CAD cells used in this study. Additionally, the authors thank E. Kuzhikandathil, D. Smith, and A. Kohn for constructive comments throughout the course of this work and R. Khan for technical assistance in cell culture.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-18788 to G. S. Oxford.
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FOOTNOTES |
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Address for reprint requests: G. S. Oxford, Dept. of Cell and Molecular Physiology, University of North Carolina, Box 7545, 452 Medical Science Research Bldg., Chapel Hill, NC 27599 (E-mail: gsox{at}med.unc.edu).
Received 10 February 2000; accepted in final form 11 August 2000.
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REFERENCES |
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