Voltage-Dependent Ion Channels in CAD Cells: A Catecholaminergic Neuronal Line That Exhibits Inducible Differentiation

Haibin Wang1 and Gerry S. Oxford2

 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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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Fig. 1. Interference contrast images of CAD cells maintained in serum containing medium (A, 48 h postplating) and CAD cells following removal of serum (B, 7 days postserum). Note the increase in neurite formation and the development of putative synaptic varicosities following serum deprivation. On hyperpolarization of the membrane potential to around -80 mV, spontaneous firing of action potentials was routinely observed in both undifferentiated (C) and differentiated (E) CAD cells. In both phenotypes, some cells occasionally exhibited spontaneous firing activities at the normal resting potential; a representative recording from a differentiated CAD cell is shown (D). The bars indicate a time scale of 1 min in each case.

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega . 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 GOmega on CAD cells. All experiments were performed at room temperature (21-23°C). The access resistance values typically ranged from 2 to 7 MOmega . 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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; triangle ) 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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Fig. 2. Voltage-dependent ionic currents in CAD cells in response to voltage steps from -80 to +60 mV. Representative responses to both brief (15 ms, A and C) and long (150 ms, B and D) commands are shown. Undifferentiated CAD cells express large, transient sodium currents, but relatively small outward potassium currents (A and B). In contrast, differentiation of CAD cells results in much smaller sodium currents, but a dramatic increase in outward potassium currents (C and D). Current-voltage (I-V) curves indicate that transient sodium currents were activated near approximately -30 mV and reached the peak at 0-10 mV (E and F, triangle ); outward potassium currents were elicited when the cells were depolarized above approximately -20 mV and increased monotonically with depolarization (E and F, ).

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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Fig. 3. Average sodium and potassium current densities for undifferentiated (UD) and differentiated (D) CAD cells. Bars represent mean amplitudes (±SE) for sodium currents at 0 mV () and potassium currents at +60 mV (). The number of cells in each case is indicated above each bar. The P values for statistical comparison (t-test) between differentated and undifferentiated phenotypes have been indicated also. On differentiation, a reciprocal change of Na+ and K+ currents was observed. Specifically, sodium currents decreased by approximately 4-fold, and potassium currents increased by approximately 1.5-fold.

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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Fig. 4. Steady-state inactivation of sodium channels is unchanged by differentiation of CAD cells. Peak sodium currents at 0 mV were measured following 50-ms prepulses from -120 to +10 mV, normalized to the maximum current in each cell, averaged and plotted as a function of the prepulse voltage (mean ± SE) for undifferentiated (open circle , n = 9) and differentiated (, n = 9) phenotypes. A 1-ms interval to -90 mV between prepulse and test pulse ensured resetting of the activation gating between trials. Curves are Marquardt-Levenberg least-squares fits of the equation INa(norm) = 1/{1 + exp[(VV0.5)/k]}, where V is the prepulse voltage, V0.5 is the midpoint voltage of the inactivation curve, and k is the voltage sensitivity in mV. Fit parameters for undifferentiated cells are V0.5 = -64.3 mV, k = 7.7 mV, and for differentiated cells V0.5 = -60.8 mV, k = -8.9 mV.

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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Fig. 5. Voltage-dependent potassium currents manifested in CAD cells are sensitive to both external tetraethylammonium chloride (TEA+) and 3,4-diaminopyridine (3,4-DAP). A: families of delayed rectifier K+ currents in a differentiated CAD cell before (left) and during (right) exposure to 10 mM TEA+. B: families of K+ currents in another differentiated CAD cell before (left) and during (right) exposure to 1 mM 3,4-DAP. The bar indicates 150-ms time scale. C and D: the kinetics of block and unblock of K+ channels at +40 mV by TEA+ and 3,4-DAP suggest 2 pharmacologically distinct populations of channels. TEA+ rapidly and reversibly blocks both types of channel. In contrast, 3,4-DAP interacts more slowly with an apparent subpopulation of channels. open circle , the K+ currents at control solution; , the K+ currents at either 10 mM TEA+ or 1 mM 3,4-DAP; triangle , the K+ currents at 10 mM TEA+ plus 1 mM 3,4-DAP.

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, triangle ). 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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Fig. 6. Inward barium currents through voltage-dependent calcium channels were manifested in both undifferentiated (A) and differentiated (C) CAD cells dialyzed with Cs+ internal solutions and bathed in 10 mM barium external solution (BaES) with 300 nM tetrodotoxin (TTX). Current families elicited by voltage steps from -80 to +60 mV from a holding potential of -80 mV. In the undifferentiated phenotype, the Ba2+ currents exhibited a relatively fast activation followed by a significant inactivation and were almost completely eliminated by 10 µM nimodipine (B). In contrast, the differentiated phenotype exhibited an identical nimodipine-sensitive component, but an additional nimodipine-resistant sustained component was clearly identified with a relatively slow activation (D). In both phenotypes, the threshold for the peak Ba2+ currents was about -60 mV, and the peak occurred at about -20 to -10 mV (E, undifferentiated; F, differentiated, triangle ). The I-V curve of the nimodipine-resistant component indicates that it increases on differentiation and activates at more positive membrane potentials (E and F, ). G: the nimodipine-sensitive component (total current - current after nimodipine, ) was comparable in both phenotypes, while a 2-fold increase of nimodipine-resistant component () occurred on differentiation. UD and D stand for undifferentiated and differentiated, respectively. The numbers of observations are indicated on top of the each individual bar. # P > 0.05; * P < 0.02.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1C subunit is expressed by CAD cells and caused strong acceleration of the Ba2+ current inactivation (Soldatov et al. 1998). In addition, some beta  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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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