Induction of K-Channel Expression in a Neuroblastoma Cell Line Catherine J. Smith-Maxwell,+ Ruth Anne Eatock, and Ted Begenisich* Department of Physiology, University of Rochester Medical Center, Rochester, New York 14642
SUMMARY Whole-cell currents were examined in mouse neuroblastoma cells of the N,AB-1 line. In standard culture medium, N,AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfovide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 p S and 6.4 pS were observed in nutside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene( s ) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA
treatment caused neurite extension in most, but not all, N,AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO /low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N,AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.
INTRODUCTION
zyme activity associatedwith neurotransmitter production (for reviews see Prasad, 1975, and Kimhi, 1981). The induction of long neurites and hypertrophy are often termed morphological diflerentration. We were interested in determining whether such changes are accompanied by changes in the excitability of the neuroblastoma cells. During development, many excitable cells undergo large changes in the types and numbers of ion channels expressed in their surface membranes (Spitzer, 1979; Barish, 1986; Best. McCobb, and Beam, 1988; Hams, Henderson, and Spitzer, 1988; O’Dowd, Ribera, and Spitzer, 1988;Yool, Dionne, and Gruol, 1988; McCobb. Best, and Beam, 1989; Nerbonne and Gurney, 1989). It is known that after being treated to produce morphological differentiation in culture, neuroblastoma cells do express many of the ion channels characteristic of neurons: Na channels, Ca channels, voltage-activated K channels, and Ca-activated K channels (Moolenaar and Spector, 1978; Quandt, 1988). However, little is
Clonal cell lines derived from the mouse neuroblastoma (C- 1300) tumor have been used as in vitro models for examining the process of neuronal differentiation. These cells exhibit many neuronal characteristics. For example, various lines possess the neurotransmitters norepinephrine, dopamine, serotonin. and histamine. In these cells, addition of chemical agents to the culture medium may cause some or all of the following changes that are reminiscent of those that occur during normal neuronal maturation: suppression of cell division, formation of long neurites, hypertrophy, and increased enReceived November 29, 1990; accepted January 16, 1991 Journal of Neurobiology, Vol. 22, No. 4, pp. 327-341 (1991) 0 I991 John Wiley & Sons, Inc. CCC 0022-3034/91/040327-15$04.00 * To whom correspondence should be addressed. t Present address: Department of Molecular and Cellular Physiology. Stanford University Medical Center. Stanford, CA 94305-5425, U.S.A.
32 7
known about currents in neuroblastoma cells that have not been treated with differentiating agents. As a consequence, the relationship between the development of features associated with morphological differentiation and the expression of ion channels has not been well characterized in these cells. We have investigated the ion channel currents in neuroblastoma cells grown in standard culture medium and in medium containing retinoic acid (RA) or dimethylsulfoxide (DMSO) and low serum. These treatments have been shown to cause, in cell culture, some of the changes in morphology and gene expression that are seen during in vivo differentiation ( Kimhi, Palfrey, Spector, Barak, and Littauer, 1976; Bottenstein and Sato, 1979: Seyedin, Pehrson, and Cole, 1981; Haussler et al., 1983; Sidell, Altman, Haussler, and Seeger, 1983; Shea. Fisher. and Sapirstein, 1985; Kubo, 1989). We report that untreated N,AB- 1 cells exhibited voltage-dependent Na currents, but no discernible delayed rectifier K currents, and that treatment with RA or DMSO/low serum caused the expression of delayed rectifier K currents. Moreover, there was no obligatory link between this K-channel expression and the inhibition of cell division, cellular hypertrophy or the elaboration of neurites. Preliminary reports of this study have been presented ( Smith-Maxwell, Eatock, and Begenisich, 1987; Begenisich and Weisenhaus, 1990). METHODS
Cell Culture Many neuroblastoma cell lines have been derived from a murine sympathetic ganglion tumor (C- 1300). One such line is the Neuro-2a cell line (American Type Culture Collection CCL 13l: Klebe and Ruddle, 1969). Thc N,AB- 1 cell line used for this study was subcloned from the Neuro-2a cell line in the laboratory of Dr. Joanna B. Olmsted (University of Rochester), who provided us with the cells. The N,AB-I cells used in this study were grown as monolayer cultures at 37°C in a humidified 5% CO, atmosphere. They were grown in Ham’s F-12 medium, supplemented with 10% heat-inactivated fctal bovinc serum, 2 m M L-glutamine. 50 pg/ml of the antibiotic gentamicin sulfate, and 2.5 kg/ml of Fungizone (Flow Laboratories). The culture medium was replaced three times a week. The cells were passaged every 7-10 days at a 1 :3 to 1:4 dilution. Cells were removed from the culture dishes b y a brief treatment with 0.06% trypsin and 0.02% ethylenediaminetetraacetate (EDTA) at 37°C. The cells
were transferred to 35-mm dishes containing glass coverslips 1-4 days before electrophysiology experiments. All experiments were camed out on cells of approximately the same passage number to minimize changes in cell properties known to occur in many cell lines. Neuroblastoma cells derived from the C-1300 tumor can be morphologically differentiated by a variety of treatments including DMSO (Kimhi et al., 1976),serum deprivation with and without DMSO (Furmanski, 1973; Furmanski and Lubin, 1972), and retinoic acid (Shea et al., 1985). We used two different culture conditions in an effort to morphologically differentiate the N,AB- I cells: ( I ) Retinoic acid: ?J,AB-1 cells were cultured as described above but were treated for 8- 1 1 days with I0 or 20 pLM all-trans retinoic acid. Cells that were treated with RA regenerated neurites within 12 h of replating. ( 2 ) DMSO/low serum: N,AB- I cells were cultured with 1.3%DMSO and 0.3% fetal bovine serum for 7-1 3 days.
Voltage-Clamp Techniques
Macroscopic membrane currents were obtained with the whole-cell variant of the patch-clamp technique, and single-channel currents were measured from outside-out patches (Hamill, Marty, Neher, Sakmann, and Sigworth, 1981 ). Electrode resistances were between 3 and 7 Ma. Because the Kchannel currents we studied were typically 0.20). Currents in RA-treated cclls with neuritcs werc not significantly different from currents in RA-treated cells without neurites ( p > 0.20).
’
rites, most did not, and we did not quantify such changes. The effects of DMSO on the morphology of other neuroblastoma cells are described in Kimhi et al. ( 1976). Whole-Cell Currents. As in RA-treated cells, significant voltage- and time-dependent outward currents were consistently observed in cells treated with DMSO/low serum. Figure 5 shows such currents in a cell treated with DMSO/low serum for 12 days. Of 26 cells treated with DMSO/low serum that were examined, all exhibited currents similar to those in Figure 5. K-Channel Pharmacology
Addition of the K-channel blockers, TEA or 4-AP, to the external solution blocked the outward currents recorded from N,AB-1 cells treated with either RA or DMSO/low serum. In Figure 6, current-voltage relations from RA-treated cells show reversible block of the outward current by 20 m M TEA [Fig. 6 (A)] and by 20 m M 4-AP [Fig. 6 (B)]. In five RA-treated cells, 20 m M TEA reduced the outward current at +60 mV by 78% k 7%. 20 m M 4-AP in the external solution blocked the outward current at +60 mV by 86% in one cell and 94% in a second cell. In cells treated with DMSO/low serum, 10 mMTEA in the external medium reversibly blocked 89% k 5% ( 3 ) of the outward current; an example is shown in Figure 7A. 4-AP applied externally was effective also.
The outward currents at +60 mV were reversibly reduced by 71% in 1 mM4-AP ( l ) , 81%in 10 m M 4-AP ( 1 ), and 84 k 4% ( 3 ) in 20 m M 4-AP. Block by 10 m M 4-AP at several potentials is illustrated in Figure 7B. These results clearly identify most of the outward current in treated N,AB-l cells as current through K channels. The I-V relations of Figures 6 and 7 also show that under both treatment regimes, the threshold for activation of outward current was about -20 mV. Single-Channel K Currents In outside-out patches from the cell bodies of treated N,AB-l cells, we frequently observed two types of channels that passed outward current at depolarized potentials [Fig. 8 (A)]. The two channels were easily identified by their current amplitudes and were expressed by both populations of treated N,AB- 1 cells. As can be seen in Figure 8B, the current amplitudes from cells grown in RA were indistinguishable from current amplitudes from cells grown in DMSO/low-serum medium. At +60 mV, the single-channel current of one channel was 2.4 1 f 0.06 pA while that of the other channel was 1.02 0.02 PA. Some patches contained both channel types: in others, only one or the other channel was present. The smaller conductance channel was found more often. In about 30%-40% of excised patches, no channels were found. The larger conductance channels appeared to open earlier in response to membrane depolarization and to have shorter open times than the smaller conductance channels [Fig. 8( A)]. From linear regression of data pooled from both treatment populations [ Fig. 8 (B)], the slope conduc-
*
Table 2 Outward Current in Untreated N,AB-1 Cells as a Function of Time in Culture
10 20 38 39 44 45 47
39.8 f 12.0 9.3 20.7 k 8.7 19.9 + 4.0 4.6 4.0 35.8 36.4 + 2.3
*
Note: Abbreviations as in Table 1. Days in culture since thawing. ’Outward current at the end of a 40-ms voltage step to +60 mV, after subtraction of currents that scaled linearly with voltage.
K-Channel E.ypression in Neuroblastoma Cells
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T r e a t m e n t time (days) Figure 3 Time course of neurite extension caused by retinoic acid. The fraction of cells with neurites longer than one cell diameter is plotted as a function of treatment duration. Each point represents a count of at least 150 cells. and usually 250-400 cells. The cells were treated with either 10 F M RA ( 0 , O ) or 20 pM RA (I). To check whether replating influenced neurite outgrowth, we compared cells that were repkdtcd on days 4 and 9 (I 0, )with cells that were not replated (0).(Retinoic acid was present throughout. whether or not cells were replated.) Neurite outgrowth followed the same time course for cells that were replated and for cells that were not replated.
tances of the two channel types were estimated as 15.0 pS and 6.4 pS. The apparent reversal potentials from extrapolation of the linear regression fits to the data in Figure 8B were -96 mV for the large conductance channel and -92 mV for the small conductance channel. While recognizing that data from this restricted voltage range may not provide the best estimate of the reversal potentials, it is worth noting that these estimates are very close to the equilibrium potential for K' (-90 mV) and not to that for C1- (-45 mV).
Cell Size and Current Density Table 1 summarizes the magnitudes of the outward currents from the different cell groups examined; the figures given are the means of currents measured at the end of 40-ms pulses to +60 mV. Cells treated with rctinoic acid or DMSO/low serum had four to seven times more current than did untreated N,AB- 1 cells.
To examine the possibility that differences in current magnitude between cell groups reflect differences in cell size rather than in channel density, we estimated cell surface area from capacitance measurements for some cells. The average capacitance of eight untreated (round) N,AB-l cells was 11.O & 0.8 pF, equivalent to that for a smooth, spherical cell about 18.7 pm in diameter, assuming 1 pF/cm2. This value is similar to the estimate of 15 pm that Notter and Lcary ( 1987) obtained for untreated N,AB- 1 cells using flow cytometry. The average capacitance of 20 cells treated with retinoic acid for 8-1 1 days was 12.8 & 1.1 pF, suggesting that this treatment produced little, if any, hypertrophy ( p > 0.2). In contrast, cells maintained in DMSO/low serum did hypertrophy. The average capacitance after 1 2- 13 days of treatment was 19.9 k 4.0 pF ( 6 ) , about 80% greater than the capacitance of the untreated cells ( p < 0.05). For those cells whose capacitance was mea-
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I Figure 5 Currents from a cell treated with DMSO/low serum for 12 days. Currents were elicited by 40-ms pulses to 0, +20, +40, +60 mV from a holding potential of -70 mV. The bath and pipette solutions are the same as those in Figure I . As in Figure 1 B, the bath solution contained 300 n M TTX to block Na currents. Calibration: 100 pA, 5 ms.
sured, we normalized the outward currents measured at +60 mV by dividing by cell capacitance. The average current densities for untreated cells, RA-treated cells with neurites, RA-treated cells without neurites, and DMSO/low-serum-treated cells were 2.6 k 0.5 (12), 9.5 f 1.5 (13), 8.8 k 1.1 (7 j and 12.8 1.8 ( 12j pA/pF, respectively. Thus, the average current density for cells treated with RA or DMSO/low serum was significantly greater
*
335
than that for untreated N,AB- 1 cells ( p < 0.00 1 ) . Also, there was no significant difference in current density between RA-treated cells with neurites and those without neurites ( p > 0.2 j . Thus, it appears that the increased outward current of treated cells resulted largely from increased channel density. The magnitude of the small outward current in untreated N,AB-1 cells was very similar to that seen in treated N,AB- 1 cells in the presence of TEA or 4-AP. At +60 mV, the current density in treated N2AB-1 cells in I0 or 20 m M TEA or 4-AP was 1.8 t 0.4 ( 12) pA/pF. This value does not differ significantly from the current density of 2.6 -t 0.5 (12) pA/pF obtained at +60 mV from untreated N2AB1 cells ( p > 0.2). This suggests that all of the treatment-induced outward current could be blocked by TEA or 4-AP and, therefore, was K-channel current. DISCUSSION When raised in standard culture medium, N,AB-l cells exhibited large voltage-dependent Na-channel currents, but lacked significant voltage- and timedependent outward currents. The absence of such currents was a stable property of the cells when monitored over time in cell culture. Both RA and DMSO/low-serum treatments induced voltage- and time-dependent outward currents in the N,AB-l cells. These were identified as K-channel currents because of their susceptibility to TEA and 4-AP. The F2A and DMSO/lowserum treatments were chosen because they are known to enhance the expression of features associated with a differentiated neuronal phenotype. RA caused up to 75% of the cells to form long neurites, while DMSO/low serum inhibited cell division and caused hypertrophy. However, none of these changes was obligatorily linked to the appearance of the K-channel currents. ( 1) Neurite extension: RA-treated cells both with and without neu-
Figure 4 Membrane currents from two RA-treated cells with different morphologies. The cells were treated with 20 ~ L L MRA for 8 days. The bath and pipette solutions were the same as those used in Figure 1 (no TTX was added). In both ( A ) and (B), currents were evoked by 40-ms pulses to 0, +20, +40. and +60 mV from a holding potential of -60 mV with a 10-ms prepulse to - 100 mV. ( A ) Currents from a cell with neurites. Calibration: 2.50 PA, 5 ms. A cell of this type is indicated by the arrow in the photograph below the current records. ( B ) Currents from a round cell. Calibration: 100 pA, 5 ms. A cell of this type is indicated by the arrow in the photograph below.
336
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Figure 7 TEA and 4-AP block of outward whole-cell currents from cells treated with DMSO/low serum. Currents at the end of 40-ms voltage steps are plotted as a function of membrane potential as in Figure 6. The bath and pipette solutions were as given in Figure 6 (no TTX was added to the bath solution). Na currents were eliminated by applying 50-ms dcpolarizing pulses to -30 mV before the test pulse. ( A ) Currents measured from a cell maintained in DMSO/low serum for 8 days. Currents were obtained before ( A), during (a),and after (V)exposure to 10 m M TEA. The holding potential was -60 mV. (B) Currents measured from a cell grown in DMSO/low serum medium for 10 days. Currents were obtained before (A),during (a),and after (V)exposure to 10 m M 4-AP. The holding potential was -80 mV.
K-Channel Expression in Neuroblastoma C d s
rites had similar K-current densities; similar currents were seen in DMSO /low-serum-treated cells, which did not form long neurites; and untreated cells with neurites did not have K-channel currents. ( 2 ) Cessalion of cell division: The Kchannel currents were exhibited by rapidly dividing RA-treated cells and by nonmitotic cells treated with DMSO/ low serum, but not by rapidly dividing untreated cells. ( 3 ) Hypertrophy: While both treatments caused the expression of K-channel currents, only DMSO/low serum caused hypertrophy. Thus, the treatments appear to affect the regulation of multiple genes and gene products. While little is known about how DMSO and low-serum treatments exert their effects, recent progress has been made toward understanding the mechanisms of RA action. RA has been shown to bind with high affinity to a group of receptors (RAR ) that are part of a superfamily of ligand-inducible transcriptional enhancer factors (Giguere, Ong, Segui, and Evans, 1987; Petkovich, Brand, Krust, and Chambon, 1987; Benbrook, Lernhardt, and Pfahl, 1988; Brand et al., 1988; Krust, Kastner, Petkovich, Zelent, and Chambon, 1989; Ragsdale, Petkovich. Gates. Chambon. and Brockes, 1989; Zelent, Krust, Petkovich, Kastner, and Chambon, 1989). The superfamily includes receptors for steroid hormones, vitamin D, and thyroid hormones (reviewed in Evans, 1988). The different RA-Rs described show different temporal and tissue-specific patterns of expression in which more than one type may be expressed in any tissue. In particular, undifferentiated cells of an embryonal carcinoma cell line (F9 ) express two types of RA-R, and treatment with RA causes the expression of a third form (Zelent et al., 1989). It is possible that the different forms of RA-R may subserve different functions and regulate the expression of different gene products. Also, at the rather high concentrations of RA used in our experiments, it is possible that receptors for related compounds such as retinol are activated as well, leading to an even greater diversity of functional changes (Brand et al., 1988). The two types of channels found in treated N,AB- 1 cells have properties that closely resemble two voltage-dependent K channels described in DMSO-treated N 1E- 1 15 cells (Quandt, 1988). Although direct comparison of single-channel conductances is not possible due to the different temperatures and ionic conditions used, the ratio of the current amplitudes for the two K-channel subtypes in N,AB-1 cells is comparable to the ratio of
337
current amplitudes through the voltage-dependent K channels in N 1E- I 1 5 cells. Quandt found singlechannel K conductances of 35.0 pS and 14.0 pS in DMSO-treated N 1E- I I 5 cells, while we found values of 15.0 pS and 6.4 pS in treated N,AB-l cells. The two channel types in N1E-115 cells also have distinctive kinetic properties: the large conductance channel opens at depolarized membrane potentials with a shorter latency and mean open time than does the small conductance channel. A similar distinction between the small and large conductance channels of treated N,AB- 1 cells is suggested by single-channel records [Fig. 8 (A)]. The K-channel currents in the treated N2AB-1 cells appear to be in the broad category of delayed rectifier K currents, which includes all voltdge-dependent K currents that are neither transient (Acurrents) nor Cd2+-activated,for the following reasons. (1) The currents did not inactivate during 40-ms depolarizing pulses and therefore are not considered to be A currents. (2) The low level of free internal Ca2+in these experiments suggests that the K-channel currents were not Ca2+-activated. A high concentration of EGTA ( 1 1 mM) in the pipette solution buffered the free internal Ca2' concentration to below 10 nM. In over half of the experiments, the free internal Ca2+concentration was further reduced by the presence of 20 rn M Fin the pipette solution. Finally, the extracellular concentration of Ca2+was relatively low (2 mM). Under such conditions, Ca'+-activated K current is suppressed in, for example, cultured rat muscle cells (Barrett, Magleby, and Pallotta, 1982), bovine chromaffin cells (Marty and Neher, 1985), cultured chick ciliary ganglion neurons (Gardner, 1986), PC-12 cells (Hoshi and Aldrich, 1988), and DMSO-treated N1E-115 cells (Quandt, 1988). ( 3 ) The voltage dependence and rates of activation were in the range expected for delayed rectifier K channels. The nature of the small outward currents in the untreated cells and of the small residual currents in treated cells after addition of 10 or 20 mMTEA or 4-AP has not been determined. The mean density of the residual, unblocked current did not differ significantly from the mean density of the outward current in untreated cells. Therefore, it seems likely that the outward current in the treated cells comprises both K-channel current that was induced by the treatments and a small component that is also present in the untreated cells. In a preliminary study of 10 cells of the parent Neuro-2a clone, grown in standard medium, we
338
SmithMaxwell et (11
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SUMMARY Whole-cell currents were examined in mouse neuroblastoma cells of the N,AB-1 line. In standard culture medium, N,AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfovide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 p S and 6.4 pS were observed in nutside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene( s ) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA
treatment caused neurite extension in most, but not all, N,AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO /low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N,AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.
INTRODUCTION
zyme activity associatedwith neurotransmitter production (for reviews see Prasad, 1975, and Kimhi, 1981). The induction of long neurites and hypertrophy are often termed morphological diflerentration. We were interested in determining whether such changes are accompanied by changes in the excitability of the neuroblastoma cells. During development, many excitable cells undergo large changes in the types and numbers of ion channels expressed in their surface membranes (Spitzer, 1979; Barish, 1986; Best. McCobb, and Beam, 1988; Hams, Henderson, and Spitzer, 1988; O’Dowd, Ribera, and Spitzer, 1988;Yool, Dionne, and Gruol, 1988; McCobb. Best, and Beam, 1989; Nerbonne and Gurney, 1989). It is known that after being treated to produce morphological differentiation in culture, neuroblastoma cells do express many of the ion channels characteristic of neurons: Na channels, Ca channels, voltage-activated K channels, and Ca-activated K channels (Moolenaar and Spector, 1978; Quandt, 1988). However, little is
Clonal cell lines derived from the mouse neuroblastoma (C- 1300) tumor have been used as in vitro models for examining the process of neuronal differentiation. These cells exhibit many neuronal characteristics. For example, various lines possess the neurotransmitters norepinephrine, dopamine, serotonin. and histamine. In these cells, addition of chemical agents to the culture medium may cause some or all of the following changes that are reminiscent of those that occur during normal neuronal maturation: suppression of cell division, formation of long neurites, hypertrophy, and increased enReceived November 29, 1990; accepted January 16, 1991 Journal of Neurobiology, Vol. 22, No. 4, pp. 327-341 (1991) 0 I991 John Wiley & Sons, Inc. CCC 0022-3034/91/040327-15$04.00 * To whom correspondence should be addressed. t Present address: Department of Molecular and Cellular Physiology. Stanford University Medical Center. Stanford, CA 94305-5425, U.S.A.
32 7
known about currents in neuroblastoma cells that have not been treated with differentiating agents. As a consequence, the relationship between the development of features associated with morphological differentiation and the expression of ion channels has not been well characterized in these cells. We have investigated the ion channel currents in neuroblastoma cells grown in standard culture medium and in medium containing retinoic acid (RA) or dimethylsulfoxide (DMSO) and low serum. These treatments have been shown to cause, in cell culture, some of the changes in morphology and gene expression that are seen during in vivo differentiation ( Kimhi, Palfrey, Spector, Barak, and Littauer, 1976; Bottenstein and Sato, 1979: Seyedin, Pehrson, and Cole, 1981; Haussler et al., 1983; Sidell, Altman, Haussler, and Seeger, 1983; Shea. Fisher. and Sapirstein, 1985; Kubo, 1989). We report that untreated N,AB- 1 cells exhibited voltage-dependent Na currents, but no discernible delayed rectifier K currents, and that treatment with RA or DMSO/low serum caused the expression of delayed rectifier K currents. Moreover, there was no obligatory link between this K-channel expression and the inhibition of cell division, cellular hypertrophy or the elaboration of neurites. Preliminary reports of this study have been presented ( Smith-Maxwell, Eatock, and Begenisich, 1987; Begenisich and Weisenhaus, 1990). METHODS
Cell Culture Many neuroblastoma cell lines have been derived from a murine sympathetic ganglion tumor (C- 1300). One such line is the Neuro-2a cell line (American Type Culture Collection CCL 13l: Klebe and Ruddle, 1969). Thc N,AB- 1 cell line used for this study was subcloned from the Neuro-2a cell line in the laboratory of Dr. Joanna B. Olmsted (University of Rochester), who provided us with the cells. The N,AB-I cells used in this study were grown as monolayer cultures at 37°C in a humidified 5% CO, atmosphere. They were grown in Ham’s F-12 medium, supplemented with 10% heat-inactivated fctal bovinc serum, 2 m M L-glutamine. 50 pg/ml of the antibiotic gentamicin sulfate, and 2.5 kg/ml of Fungizone (Flow Laboratories). The culture medium was replaced three times a week. The cells were passaged every 7-10 days at a 1 :3 to 1:4 dilution. Cells were removed from the culture dishes b y a brief treatment with 0.06% trypsin and 0.02% ethylenediaminetetraacetate (EDTA) at 37°C. The cells
were transferred to 35-mm dishes containing glass coverslips 1-4 days before electrophysiology experiments. All experiments were camed out on cells of approximately the same passage number to minimize changes in cell properties known to occur in many cell lines. Neuroblastoma cells derived from the C-1300 tumor can be morphologically differentiated by a variety of treatments including DMSO (Kimhi et al., 1976),serum deprivation with and without DMSO (Furmanski, 1973; Furmanski and Lubin, 1972), and retinoic acid (Shea et al., 1985). We used two different culture conditions in an effort to morphologically differentiate the N,AB- I cells: ( I ) Retinoic acid: ?J,AB-1 cells were cultured as described above but were treated for 8- 1 1 days with I0 or 20 pLM all-trans retinoic acid. Cells that were treated with RA regenerated neurites within 12 h of replating. ( 2 ) DMSO/low serum: N,AB- I cells were cultured with 1.3%DMSO and 0.3% fetal bovine serum for 7-1 3 days.
Voltage-Clamp Techniques
Macroscopic membrane currents were obtained with the whole-cell variant of the patch-clamp technique, and single-channel currents were measured from outside-out patches (Hamill, Marty, Neher, Sakmann, and Sigworth, 1981 ). Electrode resistances were between 3 and 7 Ma. Because the Kchannel currents we studied were typically 0.20). Currents in RA-treated cclls with neuritcs werc not significantly different from currents in RA-treated cells without neurites ( p > 0.20).
’
rites, most did not, and we did not quantify such changes. The effects of DMSO on the morphology of other neuroblastoma cells are described in Kimhi et al. ( 1976). Whole-Cell Currents. As in RA-treated cells, significant voltage- and time-dependent outward currents were consistently observed in cells treated with DMSO/low serum. Figure 5 shows such currents in a cell treated with DMSO/low serum for 12 days. Of 26 cells treated with DMSO/low serum that were examined, all exhibited currents similar to those in Figure 5. K-Channel Pharmacology
Addition of the K-channel blockers, TEA or 4-AP, to the external solution blocked the outward currents recorded from N,AB-1 cells treated with either RA or DMSO/low serum. In Figure 6, current-voltage relations from RA-treated cells show reversible block of the outward current by 20 m M TEA [Fig. 6 (A)] and by 20 m M 4-AP [Fig. 6 (B)]. In five RA-treated cells, 20 m M TEA reduced the outward current at +60 mV by 78% k 7%. 20 m M 4-AP in the external solution blocked the outward current at +60 mV by 86% in one cell and 94% in a second cell. In cells treated with DMSO/low serum, 10 mMTEA in the external medium reversibly blocked 89% k 5% ( 3 ) of the outward current; an example is shown in Figure 7A. 4-AP applied externally was effective also.
The outward currents at +60 mV were reversibly reduced by 71% in 1 mM4-AP ( l ) , 81%in 10 m M 4-AP ( 1 ), and 84 k 4% ( 3 ) in 20 m M 4-AP. Block by 10 m M 4-AP at several potentials is illustrated in Figure 7B. These results clearly identify most of the outward current in treated N,AB-l cells as current through K channels. The I-V relations of Figures 6 and 7 also show that under both treatment regimes, the threshold for activation of outward current was about -20 mV. Single-Channel K Currents In outside-out patches from the cell bodies of treated N,AB-l cells, we frequently observed two types of channels that passed outward current at depolarized potentials [Fig. 8 (A)]. The two channels were easily identified by their current amplitudes and were expressed by both populations of treated N,AB- 1 cells. As can be seen in Figure 8B, the current amplitudes from cells grown in RA were indistinguishable from current amplitudes from cells grown in DMSO/low-serum medium. At +60 mV, the single-channel current of one channel was 2.4 1 f 0.06 pA while that of the other channel was 1.02 0.02 PA. Some patches contained both channel types: in others, only one or the other channel was present. The smaller conductance channel was found more often. In about 30%-40% of excised patches, no channels were found. The larger conductance channels appeared to open earlier in response to membrane depolarization and to have shorter open times than the smaller conductance channels [Fig. 8( A)]. From linear regression of data pooled from both treatment populations [ Fig. 8 (B)], the slope conduc-
*
Table 2 Outward Current in Untreated N,AB-1 Cells as a Function of Time in Culture
10 20 38 39 44 45 47
39.8 f 12.0 9.3 20.7 k 8.7 19.9 + 4.0 4.6 4.0 35.8 36.4 + 2.3
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Note: Abbreviations as in Table 1. Days in culture since thawing. ’Outward current at the end of a 40-ms voltage step to +60 mV, after subtraction of currents that scaled linearly with voltage.
K-Channel E.ypression in Neuroblastoma Cells
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T r e a t m e n t time (days) Figure 3 Time course of neurite extension caused by retinoic acid. The fraction of cells with neurites longer than one cell diameter is plotted as a function of treatment duration. Each point represents a count of at least 150 cells. and usually 250-400 cells. The cells were treated with either 10 F M RA ( 0 , O ) or 20 pM RA (I). To check whether replating influenced neurite outgrowth, we compared cells that were repkdtcd on days 4 and 9 (I 0, )with cells that were not replated (0).(Retinoic acid was present throughout. whether or not cells were replated.) Neurite outgrowth followed the same time course for cells that were replated and for cells that were not replated.
tances of the two channel types were estimated as 15.0 pS and 6.4 pS. The apparent reversal potentials from extrapolation of the linear regression fits to the data in Figure 8B were -96 mV for the large conductance channel and -92 mV for the small conductance channel. While recognizing that data from this restricted voltage range may not provide the best estimate of the reversal potentials, it is worth noting that these estimates are very close to the equilibrium potential for K' (-90 mV) and not to that for C1- (-45 mV).
Cell Size and Current Density Table 1 summarizes the magnitudes of the outward currents from the different cell groups examined; the figures given are the means of currents measured at the end of 40-ms pulses to +60 mV. Cells treated with rctinoic acid or DMSO/low serum had four to seven times more current than did untreated N,AB- 1 cells.
To examine the possibility that differences in current magnitude between cell groups reflect differences in cell size rather than in channel density, we estimated cell surface area from capacitance measurements for some cells. The average capacitance of eight untreated (round) N,AB-l cells was 11.O & 0.8 pF, equivalent to that for a smooth, spherical cell about 18.7 pm in diameter, assuming 1 pF/cm2. This value is similar to the estimate of 15 pm that Notter and Lcary ( 1987) obtained for untreated N,AB- 1 cells using flow cytometry. The average capacitance of 20 cells treated with retinoic acid for 8-1 1 days was 12.8 & 1.1 pF, suggesting that this treatment produced little, if any, hypertrophy ( p > 0.2). In contrast, cells maintained in DMSO/low serum did hypertrophy. The average capacitance after 1 2- 13 days of treatment was 19.9 k 4.0 pF ( 6 ) , about 80% greater than the capacitance of the untreated cells ( p < 0.05). For those cells whose capacitance was mea-
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I Figure 5 Currents from a cell treated with DMSO/low serum for 12 days. Currents were elicited by 40-ms pulses to 0, +20, +40, +60 mV from a holding potential of -70 mV. The bath and pipette solutions are the same as those in Figure I . As in Figure 1 B, the bath solution contained 300 n M TTX to block Na currents. Calibration: 100 pA, 5 ms.
sured, we normalized the outward currents measured at +60 mV by dividing by cell capacitance. The average current densities for untreated cells, RA-treated cells with neurites, RA-treated cells without neurites, and DMSO/low-serum-treated cells were 2.6 k 0.5 (12), 9.5 f 1.5 (13), 8.8 k 1.1 (7 j and 12.8 1.8 ( 12j pA/pF, respectively. Thus, the average current density for cells treated with RA or DMSO/low serum was significantly greater
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335
than that for untreated N,AB- 1 cells ( p < 0.00 1 ) . Also, there was no significant difference in current density between RA-treated cells with neurites and those without neurites ( p > 0.2 j . Thus, it appears that the increased outward current of treated cells resulted largely from increased channel density. The magnitude of the small outward current in untreated N,AB-1 cells was very similar to that seen in treated N,AB- 1 cells in the presence of TEA or 4-AP. At +60 mV, the current density in treated N2AB-1 cells in I0 or 20 m M TEA or 4-AP was 1.8 t 0.4 ( 12) pA/pF. This value does not differ significantly from the current density of 2.6 -t 0.5 (12) pA/pF obtained at +60 mV from untreated N2AB1 cells ( p > 0.2). This suggests that all of the treatment-induced outward current could be blocked by TEA or 4-AP and, therefore, was K-channel current. DISCUSSION When raised in standard culture medium, N,AB-l cells exhibited large voltage-dependent Na-channel currents, but lacked significant voltage- and timedependent outward currents. The absence of such currents was a stable property of the cells when monitored over time in cell culture. Both RA and DMSO/low-serum treatments induced voltage- and time-dependent outward currents in the N,AB-l cells. These were identified as K-channel currents because of their susceptibility to TEA and 4-AP. The F2A and DMSO/lowserum treatments were chosen because they are known to enhance the expression of features associated with a differentiated neuronal phenotype. RA caused up to 75% of the cells to form long neurites, while DMSO/low serum inhibited cell division and caused hypertrophy. However, none of these changes was obligatorily linked to the appearance of the K-channel currents. ( 1) Neurite extension: RA-treated cells both with and without neu-
Figure 4 Membrane currents from two RA-treated cells with different morphologies. The cells were treated with 20 ~ L L MRA for 8 days. The bath and pipette solutions were the same as those used in Figure 1 (no TTX was added). In both ( A ) and (B), currents were evoked by 40-ms pulses to 0, +20, +40. and +60 mV from a holding potential of -60 mV with a 10-ms prepulse to - 100 mV. ( A ) Currents from a cell with neurites. Calibration: 2.50 PA, 5 ms. A cell of this type is indicated by the arrow in the photograph below the current records. ( B ) Currents from a round cell. Calibration: 100 pA, 5 ms. A cell of this type is indicated by the arrow in the photograph below.
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Figure 7 TEA and 4-AP block of outward whole-cell currents from cells treated with DMSO/low serum. Currents at the end of 40-ms voltage steps are plotted as a function of membrane potential as in Figure 6. The bath and pipette solutions were as given in Figure 6 (no TTX was added to the bath solution). Na currents were eliminated by applying 50-ms dcpolarizing pulses to -30 mV before the test pulse. ( A ) Currents measured from a cell maintained in DMSO/low serum for 8 days. Currents were obtained before ( A), during (a),and after (V)exposure to 10 m M TEA. The holding potential was -60 mV. (B) Currents measured from a cell grown in DMSO/low serum medium for 10 days. Currents were obtained before (A),during (a),and after (V)exposure to 10 m M 4-AP. The holding potential was -80 mV.
K-Channel Expression in Neuroblastoma C d s
rites had similar K-current densities; similar currents were seen in DMSO /low-serum-treated cells, which did not form long neurites; and untreated cells with neurites did not have K-channel currents. ( 2 ) Cessalion of cell division: The Kchannel currents were exhibited by rapidly dividing RA-treated cells and by nonmitotic cells treated with DMSO/ low serum, but not by rapidly dividing untreated cells. ( 3 ) Hypertrophy: While both treatments caused the expression of K-channel currents, only DMSO/low serum caused hypertrophy. Thus, the treatments appear to affect the regulation of multiple genes and gene products. While little is known about how DMSO and low-serum treatments exert their effects, recent progress has been made toward understanding the mechanisms of RA action. RA has been shown to bind with high affinity to a group of receptors (RAR ) that are part of a superfamily of ligand-inducible transcriptional enhancer factors (Giguere, Ong, Segui, and Evans, 1987; Petkovich, Brand, Krust, and Chambon, 1987; Benbrook, Lernhardt, and Pfahl, 1988; Brand et al., 1988; Krust, Kastner, Petkovich, Zelent, and Chambon, 1989; Ragsdale, Petkovich. Gates. Chambon. and Brockes, 1989; Zelent, Krust, Petkovich, Kastner, and Chambon, 1989). The superfamily includes receptors for steroid hormones, vitamin D, and thyroid hormones (reviewed in Evans, 1988). The different RA-Rs described show different temporal and tissue-specific patterns of expression in which more than one type may be expressed in any tissue. In particular, undifferentiated cells of an embryonal carcinoma cell line (F9 ) express two types of RA-R, and treatment with RA causes the expression of a third form (Zelent et al., 1989). It is possible that the different forms of RA-R may subserve different functions and regulate the expression of different gene products. Also, at the rather high concentrations of RA used in our experiments, it is possible that receptors for related compounds such as retinol are activated as well, leading to an even greater diversity of functional changes (Brand et al., 1988). The two types of channels found in treated N,AB- 1 cells have properties that closely resemble two voltage-dependent K channels described in DMSO-treated N 1E- 1 15 cells (Quandt, 1988). Although direct comparison of single-channel conductances is not possible due to the different temperatures and ionic conditions used, the ratio of the current amplitudes for the two K-channel subtypes in N,AB-1 cells is comparable to the ratio of
337
current amplitudes through the voltage-dependent K channels in N 1E- I 1 5 cells. Quandt found singlechannel K conductances of 35.0 pS and 14.0 pS in DMSO-treated N 1E- I I 5 cells, while we found values of 15.0 pS and 6.4 pS in treated N,AB-l cells. The two channel types in N1E-115 cells also have distinctive kinetic properties: the large conductance channel opens at depolarized membrane potentials with a shorter latency and mean open time than does the small conductance channel. A similar distinction between the small and large conductance channels of treated N,AB- 1 cells is suggested by single-channel records [Fig. 8 (A)]. The K-channel currents in the treated N2AB-1 cells appear to be in the broad category of delayed rectifier K currents, which includes all voltdge-dependent K currents that are neither transient (Acurrents) nor Cd2+-activated,for the following reasons. (1) The currents did not inactivate during 40-ms depolarizing pulses and therefore are not considered to be A currents. (2) The low level of free internal Ca2+in these experiments suggests that the K-channel currents were not Ca2+-activated. A high concentration of EGTA ( 1 1 mM) in the pipette solution buffered the free internal Ca2' concentration to below 10 nM. In over half of the experiments, the free internal Ca2+concentration was further reduced by the presence of 20 rn M Fin the pipette solution. Finally, the extracellular concentration of Ca2+was relatively low (2 mM). Under such conditions, Ca'+-activated K current is suppressed in, for example, cultured rat muscle cells (Barrett, Magleby, and Pallotta, 1982), bovine chromaffin cells (Marty and Neher, 1985), cultured chick ciliary ganglion neurons (Gardner, 1986), PC-12 cells (Hoshi and Aldrich, 1988), and DMSO-treated N1E-115 cells (Quandt, 1988). ( 3 ) The voltage dependence and rates of activation were in the range expected for delayed rectifier K channels. The nature of the small outward currents in the untreated cells and of the small residual currents in treated cells after addition of 10 or 20 mMTEA or 4-AP has not been determined. The mean density of the residual, unblocked current did not differ significantly from the mean density of the outward current in untreated cells. Therefore, it seems likely that the outward current in the treated cells comprises both K-channel current that was induced by the treatments and a small component that is also present in the untreated cells. In a preliminary study of 10 cells of the parent Neuro-2a clone, grown in standard medium, we
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