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The GABAAReceptor Is Expressed in Human Neurons Derived from a Teratocarcinoma Cell Line
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
237, 719–723 (1997)
RC977087
The GABAA Receptor Is Expressed in Human Neurons Derived from a Teratocarcinoma Cell Line Toshiyuki Matsuoka,* Takeshi Kondoh,† Norihiko Tamaki,† and Tomoyuki Nishizaki*,1 *Department of Physiology and †Department of Neurosurgery, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan
Received July 3, 1997
NT2 cells, a human teratocarcinoma cell line, are shown to be differentiated in neuron-like cells (NT2-N cells) by treatment with retinoic acid. The present study identified the neurotransmitter receptors expressed in NT2-N cells using patch-clamp recording. Voltage-sensitive Na/ currents, which are specific for neurons, were observed in NT2-N cells but not in NT2 cells, suggesting that NT2-N cells actually function as neurons. Glutamate receptor agonists, N-methyl-Daspartate (NMDA) and kainate, evoked whole-cell currents. In addition, g-aminobutyric acid (GABA) evoked currents and the currents were inhibited by the selective GABAA receptor antagonist, bicuculline. In outside-out patches, GABA elicited single channel currents with two classes of the slope conductance (26 and 50 pS). No current, however, was induced by ACh, serotonin, or dopamine. NT2-N cells, thus, express at least two types of the major excitatory and inhibitory neurotransmitter receptor in the central nervous system, the glutamate and GAGAA receptors, suggesting that these receptors have a crucial role in neurotransmission from the earlier stage of the brain development. q 1997 Academic Press
Numerous studies about physiological function of neurons have been carried out using primary cultured cells or acute dissociated cells of the rodent brain, but there has been very little information about human neurons. NT2-N cells, which are derived from a human teratocarcinoma cell line (NT2 cells) by treatment with retinotic acid (1), are shown to express a variety of neuronal markers such as neuronal cytoskeletal proteins, secretory markers, and surface markers (1). Furthermore, a study demonstrates that the ligand-gated glutamate receptors, the N-methyl-D-aspartate (NMDA) 1
To whom correspondence should be addressed. Fax: /81-78-3415732.
and non-NMDA receptors (2), are expressed in NT2-N cells, suggesting a possibility that the cells are a model for understanding the function of human neurons. It is unknown, however, whether NT2-N cells actually function as neurons or what other neurotransmitter receptors are expressed. The present study aimed at addressing these questions using patch-clamp recording. The results obtained demonstrate that the voltage-dependent Na/ channel, a neuron-specific marker, operates in NT2-N cells and the cells express the g-aminobutyric acid typeA (GABAA) receptors, a major inhibitory neurotransmitter receptor, in addition to the glutamate receptors. METHODS Cell culture. NT2 cells (Stratagene Cloning Systems, CA, USA) were differentiated in NT2-N cells as previously described (1). Briefly, NT2 cells (1 1 106) were plated on 25 cm2-flask and grown in Dulbecco’s modified Eagle’s medium high glucose (DMEM/HG, GIBCO) with 5% fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 mg/ml streptomycin. The cells were treated with retinotic acid (RA) (100 mM) twice a week for five weeks. Afterward, the cells were split at a ratio of 1:6 and regrown in the presence of the mitotic inhibitors, 10 mM fluorodeoxyuridine, 10 mM uridine, and 1 mM cytosine-b-D-arabinofuranoside for 10 days. The cells were treated with 0.53 mM EDTA and mechanically dislodged by tapping. The floating cells were plated on cover slips coated with poly-D-lysin (10 mg/ml) and Materigel (Collaborative Research, 1:40), and grown in DMEM/ HG with 5% FBS. Differentiated cells (NT2-N cells) were used 1-2 weeks after plating. Electrophysiology. Cells were bathed at room temperature (20227C) in a standard extracellular solution containing (in mM) 145 NaCl, 5 KCl, 2.4 CaCl2 , 1.8 glucose, 10 HEPES, and 0.3 1 1003 tetrodotoxin (TTX), pH 7.4. The basic patch electrode-filling solution (in mM) was 150 KCl, 10 BAPTA, and 10 HEPES, pH 7.2. Membrane currents from whole-cell patches and single channel currents from outside-out patches were recorded using an Axopatch-200A amplifier (Axon Instrument, Inc., USA). In the whole-cell voltage-clamp configuration, series resistance (Rs) compensation was made up to Ç95%. The currents were stored on magneto optical disk (MK128D, Mitsubishi-Kasei, Inc., Japan), and analyzed on a laboratory computer using pClamp software (Axon Instrument, Inc.; version 6). Drugs were rapidly applied to whole-cells or excised cells for 1 sec
719
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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BBRC 7087
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6936$$$941
08-11-97 18:55:35
bbrcgs
AP: BBRC
Vol. 237, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Phase-contrast micrographs of undifferentiated NT2 (A) and differentiated NT2-N (B) cells (Original magnification 1 200).
by air pressure microinjector (PV 830, Pneumatic Picopump, World Precision Instrument, Inc., USA).
RESULTS Neuronal function in NT2-N cells. Differentiated NT2 cells (NT2-N cells) displayed morphological appearance completely distinct from undifferentiated cells (Fig. 1A,B). RA-untreated cells appeared as phase-dark and flat cells (Fig. 1A). In contrast, RAtreated cells displayed phase-bright, small, roundshaped appearance associated with multiple processes (Fig. 1B). Thick untapering long processes and
thin tapering processes were much alike to the axons and dendrites of neurons, respectively. To assess whether NT2-N cells functionally serve as neurons, voltage pulses from 080 to /40 mV from a holding potential of 080 mV for 50 msec were applied to cells in TTX-free extracellular solution. Inward whole-cell currents were evoked by the depolarization-steps in NT2-N cells (Fig. 2A) and the bell-shaped current/ voltage relation obtained was typical for that of the Na/ channel (Fig. 2B). Otherwise, no current was observed in NT2 cells (Fig. 2A), indicating that the voltage-dependent Na/ channel was expressed in NT2-N cells concomitantly with cell differentiation; in other
720
AID
BBRC 7087
/
6936$$$942
08-11-97 18:55:35
bbrcgs
AP: BBRC
Vol. 237, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
words, NT2-N cells possessed a functional characteristic of neurons. Functional expression of the neurotransmitter receptors in NT2-N cells. Whole-cell patch-clamp was made to NT2 and NT2-N cells. In NT2-N cells, kainate and NMDA produced inward currents in standard and Mg 2/-free extracellular solution, respectively (Fig. 3), suggesting that the non-NMDA (the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate) and NMDA receptors were expressed in NT2-N cells. In addition, GABA evoked currents and this was inhibited in the presence of the selective GABAA receptor antagonist, bicuculline (Fig. 3), indicating that NT2-N cells express the GABAA receptor as well. In contrast, undifferentiated NT2 cells had no response to kainate, NMDA or GABA (Fig. 3), providing an idea that signal for cell differentiation induced by RA was also involved in expression of these receptors. To further identify expression of the other neurotransmitter receptors in NT2-N cells, ACh, serotonin, or dopamine FIG. 3. Expression of the glutamate and GABAA receptors in NT2-N cells. Kainate (100 mM) was applied to NT2 and NT2-N cells for 1 sec. NMDA (100 mM) was applied to cells in Mg 2/-free media containing 5 mM glycine. GABA (100 mM) was applied to cells in the presence and absence of bicuculline (20 mM). The holding potential was 080 mV. Inward currents correspond to downward deflections.
was applied to cells, but none of them produced currents (data not shown). Properties of single channel currents through the GABAA receptors expressed. GABA produced single channel currents with two classes of the conductance levels (Fig. 4A,B). The single channel current/voltage relation gave the slope conductances of 26 and 50 pS and each conductance level accounted for 87 and 13%, respectively, of the currents carried through the channel (Fig. 4C). The mean channel opening time in the high conductance events (1.38 { 0.57 msec) was longer than that in the low conductance events (0.84 { 0.63 msec). DISCUSSION
FIG. 2. Expression of the voltage-dependent Na/ channel in NT2-N cells. 50-msec voltage pulses from 080 to /40 mV in 20-mV increments from a holding potential of 080 mV were applied to NT2 and NT2-N cells in TTX-free media (A). The evoked current/voltage relation in NT2-N cells is shown in B.
NT2 cells were differentiated in morphologically distinct cells (NT2-N cells) with multiple processes, much similar to the dendrites and axons of normal neurons, by treatment with RA. NT2-N cells are shown to be reactive to a variety of anti-neuron-specific antibodies (1). There has been, however, no functional evidence for neurons in NT2-N cells. The glutamate receptors such as the NMDA and AMPA/kainate receptors were thought to be expressed selectively in neurons. Recent studies demonstrated that these receptors are shown to operate also in glial cells (3,4), and therefore, electrophysiological definition of neurons presently depends
721
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BBRC 7087
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6936$$$942
08-11-97 18:55:35
bbrcgs
AP: BBRC
Vol. 237, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Single channel currents evoked by GABA. Outside-out patches were made from NT2-N cells. GABA (5 mM)-induced single channel currents are illustrated in A and the distribution of the currents is shown in B. The holding potential was 080 mV. GABAevoked single channel currents were recorded at holding potentials varying from 0100 to 40 mV and two classes of the slope conductances were obtained with the current/voltage relations (C).
on only whether cells have the voltage-sensitive Na/ channels or not. Indeed, voltage-sensitive Na/ currents were observed in NT2-N cells but not in NT2 cells, suggesting that differentiated NT2 cells function as neurons. Then, the question to address is: what signal mediated by RA leads to differentiation into neurons. The effect of RA is recognized to be mediated by the RA receptor and retinoid X receptor families of nuclear transcription factors (5). The relevant signal transduction pathway, however, is unknown, and further experiments need to be carried out to make clear this question.
It is notably that RA-treated NT2 cells expressed at least two types of the major excitatory and inhibitory neurotransmitter receptors in the central nervous system, the glutamate and GAGAA receptors concomitant with their differentiation into NT2-N cells. This may imply that the glutamate and GAGAA receptors are the most primitive. In contrast, the finding that NT2-N cells had no response to ACh, serotonin, or dopamine suggests that these neurotransmitter receptors, which are highly distributed in the limbic system involving higher-order function of the brain, are expressed in the later stage of the brain development. There has been no report to demonstrate that the GABAA receptor operate in NT2-N cells, although the functional expression of the glutamate receptors such as the NMDA and AMPA/kainate receptors was confirmed (2). Structurally, the GABAA receptor is composed of a heterooligomeric complex and forms a chloride ion channel (6). In single channel studies using cultured mouse spinal neurons, the GABAA receptor channels are characterized by at least four different levels of the slope conductances (44, 30, 19, and 12 pS, 27.2 and 15.9 pS, or 27 and 19 pS) and the most frequently observed conductance states are 27-30 pS (7-9). This is in good agreement with a main conductance state of 26 pS in the present study, suggesting a possibility that the properties of the GABAA receptor channels obtained with rodent neurons are generalized to those of the human GABAA receptor channels. At least five different classes of the GABAA receptor subunits, a (1-6), b (1-4), g (1-3), d, and r are cloned (6,10). Two forms of the g2 subunit (g2S and g2L), which are generated by RNA alternative splicing, was found in the brain of cattle, rats, mice, and humans (11,12). Interestingly, the g2S subunit is expressed at a fairly constant level during brain development while huge increase of the g2L subunit, which contains a protein kinase C consensus phosphorylation site (11), is found with maturation (13). Of great interest are studies aimed at assessing which combinations of the subunits are expressed in NT2-N cells and whether the g2S to g2L subunit switch is observed in NT2-N cells. NT2-N, thus, may be a model for understanding the regulatory mechanism for the expression of the neurotransmitter receptors. In conclusion, the results presented here clearly demonstrate that NT2 cells differentiate in NT2-N cells, expressing neuronal functions, by treatment with RA. REFERENCES 1. Pleasure, P. J., Page, C., and Lee, V. M.-Y. (1992) J. Neurosci. 12, 1802–1815. 2. Younkin, D. P., Tang, C.-M., Hardy, M., Reddy, U. R., Shi, Q.-Y., Pleasure, S. J., Lee, V. M.-Y., and Pleasure, D. (1993) Proc. Natl. Acad. Sci. USA 90, 2174–2178.
722
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BBRC 7087
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6936$$$942
08-11-97 18:55:35
bbrcgs
AP: BBRC
Vol. 237, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3. Muller, T., Grosche, J., Ohlemeyer, C., and Kettenmann, E. (1993) NeuroReport 4, 671–674. 4. Ikeuchi, Y., Nishizaki, T., and Matsuoka, T. (1995) Biochem. Biophys. Res. Commun. 217, 811–816. 5. Moasser, M. M., Reuter, V. E., and Dmitrovsky, E. (1995) Oncogene 10, 1537–1543. 6. Burt, D. R., and Kamatchi, G. L. (1991) FASEB J. 5, 2916–2923. 7. Bormann, J., Hamill, O. P., and Sakmann, B. (1987) J. Physiol. 385, 243–286. 8. Macdonald, R. L., Rogers, C. J., and Twyman, R. E. (1989) J. Physiol. 410, 479–499.
9. Porter, N. M., Twyman, R. E., Uhler, M. D., and Macdonald, R. L. (1990) Neuron 5, 789–796. 10. DeLorey, T. M., and Olsen, R. W. (1992) J. Biol. Chem. 267, 16747–16750. 11. Whiting, P., McKernan, R. M., and Iversen, L. L. (1990) Proc. Natl. Acad. Sci. USA 87, 9966–9970. 12. Kofuji, P., Wang, J. B., Moss, S. J., Huganir, R. L., and Burt, D. R. (1991) J. Neurochem. 56, 713–715. 13. Wang, J. B., and Burt, D. R. (1991) Brain Res. Bull. 27, 731– 735.
723
AID
BBRC 7087
/
6936$$$942
08-11-97 18:55:35
bbrcgs
AP: BBRC
237, 719–723 (1997)
RC977087
The GABAA Receptor Is Expressed in Human Neurons Derived from a Teratocarcinoma Cell Line Toshiyuki Matsuoka,* Takeshi Kondoh,† Norihiko Tamaki,† and Tomoyuki Nishizaki*,1 *Department of Physiology and †Department of Neurosurgery, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan
Received July 3, 1997
NT2 cells, a human teratocarcinoma cell line, are shown to be differentiated in neuron-like cells (NT2-N cells) by treatment with retinoic acid. The present study identified the neurotransmitter receptors expressed in NT2-N cells using patch-clamp recording. Voltage-sensitive Na/ currents, which are specific for neurons, were observed in NT2-N cells but not in NT2 cells, suggesting that NT2-N cells actually function as neurons. Glutamate receptor agonists, N-methyl-Daspartate (NMDA) and kainate, evoked whole-cell currents. In addition, g-aminobutyric acid (GABA) evoked currents and the currents were inhibited by the selective GABAA receptor antagonist, bicuculline. In outside-out patches, GABA elicited single channel currents with two classes of the slope conductance (26 and 50 pS). No current, however, was induced by ACh, serotonin, or dopamine. NT2-N cells, thus, express at least two types of the major excitatory and inhibitory neurotransmitter receptor in the central nervous system, the glutamate and GAGAA receptors, suggesting that these receptors have a crucial role in neurotransmission from the earlier stage of the brain development. q 1997 Academic Press
Numerous studies about physiological function of neurons have been carried out using primary cultured cells or acute dissociated cells of the rodent brain, but there has been very little information about human neurons. NT2-N cells, which are derived from a human teratocarcinoma cell line (NT2 cells) by treatment with retinotic acid (1), are shown to express a variety of neuronal markers such as neuronal cytoskeletal proteins, secretory markers, and surface markers (1). Furthermore, a study demonstrates that the ligand-gated glutamate receptors, the N-methyl-D-aspartate (NMDA) 1
To whom correspondence should be addressed. Fax: /81-78-3415732.
and non-NMDA receptors (2), are expressed in NT2-N cells, suggesting a possibility that the cells are a model for understanding the function of human neurons. It is unknown, however, whether NT2-N cells actually function as neurons or what other neurotransmitter receptors are expressed. The present study aimed at addressing these questions using patch-clamp recording. The results obtained demonstrate that the voltage-dependent Na/ channel, a neuron-specific marker, operates in NT2-N cells and the cells express the g-aminobutyric acid typeA (GABAA) receptors, a major inhibitory neurotransmitter receptor, in addition to the glutamate receptors. METHODS Cell culture. NT2 cells (Stratagene Cloning Systems, CA, USA) were differentiated in NT2-N cells as previously described (1). Briefly, NT2 cells (1 1 106) were plated on 25 cm2-flask and grown in Dulbecco’s modified Eagle’s medium high glucose (DMEM/HG, GIBCO) with 5% fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 mg/ml streptomycin. The cells were treated with retinotic acid (RA) (100 mM) twice a week for five weeks. Afterward, the cells were split at a ratio of 1:6 and regrown in the presence of the mitotic inhibitors, 10 mM fluorodeoxyuridine, 10 mM uridine, and 1 mM cytosine-b-D-arabinofuranoside for 10 days. The cells were treated with 0.53 mM EDTA and mechanically dislodged by tapping. The floating cells were plated on cover slips coated with poly-D-lysin (10 mg/ml) and Materigel (Collaborative Research, 1:40), and grown in DMEM/ HG with 5% FBS. Differentiated cells (NT2-N cells) were used 1-2 weeks after plating. Electrophysiology. Cells were bathed at room temperature (20227C) in a standard extracellular solution containing (in mM) 145 NaCl, 5 KCl, 2.4 CaCl2 , 1.8 glucose, 10 HEPES, and 0.3 1 1003 tetrodotoxin (TTX), pH 7.4. The basic patch electrode-filling solution (in mM) was 150 KCl, 10 BAPTA, and 10 HEPES, pH 7.2. Membrane currents from whole-cell patches and single channel currents from outside-out patches were recorded using an Axopatch-200A amplifier (Axon Instrument, Inc., USA). In the whole-cell voltage-clamp configuration, series resistance (Rs) compensation was made up to Ç95%. The currents were stored on magneto optical disk (MK128D, Mitsubishi-Kasei, Inc., Japan), and analyzed on a laboratory computer using pClamp software (Axon Instrument, Inc.; version 6). Drugs were rapidly applied to whole-cells or excised cells for 1 sec
719
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 7087
/
6936$$$941
08-11-97 18:55:35
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AP: BBRC
Vol. 237, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Phase-contrast micrographs of undifferentiated NT2 (A) and differentiated NT2-N (B) cells (Original magnification 1 200).
by air pressure microinjector (PV 830, Pneumatic Picopump, World Precision Instrument, Inc., USA).
RESULTS Neuronal function in NT2-N cells. Differentiated NT2 cells (NT2-N cells) displayed morphological appearance completely distinct from undifferentiated cells (Fig. 1A,B). RA-untreated cells appeared as phase-dark and flat cells (Fig. 1A). In contrast, RAtreated cells displayed phase-bright, small, roundshaped appearance associated with multiple processes (Fig. 1B). Thick untapering long processes and
thin tapering processes were much alike to the axons and dendrites of neurons, respectively. To assess whether NT2-N cells functionally serve as neurons, voltage pulses from 080 to /40 mV from a holding potential of 080 mV for 50 msec were applied to cells in TTX-free extracellular solution. Inward whole-cell currents were evoked by the depolarization-steps in NT2-N cells (Fig. 2A) and the bell-shaped current/ voltage relation obtained was typical for that of the Na/ channel (Fig. 2B). Otherwise, no current was observed in NT2 cells (Fig. 2A), indicating that the voltage-dependent Na/ channel was expressed in NT2-N cells concomitantly with cell differentiation; in other
720
AID
BBRC 7087
/
6936$$$942
08-11-97 18:55:35
bbrcgs
AP: BBRC
Vol. 237, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
words, NT2-N cells possessed a functional characteristic of neurons. Functional expression of the neurotransmitter receptors in NT2-N cells. Whole-cell patch-clamp was made to NT2 and NT2-N cells. In NT2-N cells, kainate and NMDA produced inward currents in standard and Mg 2/-free extracellular solution, respectively (Fig. 3), suggesting that the non-NMDA (the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate) and NMDA receptors were expressed in NT2-N cells. In addition, GABA evoked currents and this was inhibited in the presence of the selective GABAA receptor antagonist, bicuculline (Fig. 3), indicating that NT2-N cells express the GABAA receptor as well. In contrast, undifferentiated NT2 cells had no response to kainate, NMDA or GABA (Fig. 3), providing an idea that signal for cell differentiation induced by RA was also involved in expression of these receptors. To further identify expression of the other neurotransmitter receptors in NT2-N cells, ACh, serotonin, or dopamine FIG. 3. Expression of the glutamate and GABAA receptors in NT2-N cells. Kainate (100 mM) was applied to NT2 and NT2-N cells for 1 sec. NMDA (100 mM) was applied to cells in Mg 2/-free media containing 5 mM glycine. GABA (100 mM) was applied to cells in the presence and absence of bicuculline (20 mM). The holding potential was 080 mV. Inward currents correspond to downward deflections.
was applied to cells, but none of them produced currents (data not shown). Properties of single channel currents through the GABAA receptors expressed. GABA produced single channel currents with two classes of the conductance levels (Fig. 4A,B). The single channel current/voltage relation gave the slope conductances of 26 and 50 pS and each conductance level accounted for 87 and 13%, respectively, of the currents carried through the channel (Fig. 4C). The mean channel opening time in the high conductance events (1.38 { 0.57 msec) was longer than that in the low conductance events (0.84 { 0.63 msec). DISCUSSION
FIG. 2. Expression of the voltage-dependent Na/ channel in NT2-N cells. 50-msec voltage pulses from 080 to /40 mV in 20-mV increments from a holding potential of 080 mV were applied to NT2 and NT2-N cells in TTX-free media (A). The evoked current/voltage relation in NT2-N cells is shown in B.
NT2 cells were differentiated in morphologically distinct cells (NT2-N cells) with multiple processes, much similar to the dendrites and axons of normal neurons, by treatment with RA. NT2-N cells are shown to be reactive to a variety of anti-neuron-specific antibodies (1). There has been, however, no functional evidence for neurons in NT2-N cells. The glutamate receptors such as the NMDA and AMPA/kainate receptors were thought to be expressed selectively in neurons. Recent studies demonstrated that these receptors are shown to operate also in glial cells (3,4), and therefore, electrophysiological definition of neurons presently depends
721
AID
BBRC 7087
/
6936$$$942
08-11-97 18:55:35
bbrcgs
AP: BBRC
Vol. 237, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Single channel currents evoked by GABA. Outside-out patches were made from NT2-N cells. GABA (5 mM)-induced single channel currents are illustrated in A and the distribution of the currents is shown in B. The holding potential was 080 mV. GABAevoked single channel currents were recorded at holding potentials varying from 0100 to 40 mV and two classes of the slope conductances were obtained with the current/voltage relations (C).
on only whether cells have the voltage-sensitive Na/ channels or not. Indeed, voltage-sensitive Na/ currents were observed in NT2-N cells but not in NT2 cells, suggesting that differentiated NT2 cells function as neurons. Then, the question to address is: what signal mediated by RA leads to differentiation into neurons. The effect of RA is recognized to be mediated by the RA receptor and retinoid X receptor families of nuclear transcription factors (5). The relevant signal transduction pathway, however, is unknown, and further experiments need to be carried out to make clear this question.
It is notably that RA-treated NT2 cells expressed at least two types of the major excitatory and inhibitory neurotransmitter receptors in the central nervous system, the glutamate and GAGAA receptors concomitant with their differentiation into NT2-N cells. This may imply that the glutamate and GAGAA receptors are the most primitive. In contrast, the finding that NT2-N cells had no response to ACh, serotonin, or dopamine suggests that these neurotransmitter receptors, which are highly distributed in the limbic system involving higher-order function of the brain, are expressed in the later stage of the brain development. There has been no report to demonstrate that the GABAA receptor operate in NT2-N cells, although the functional expression of the glutamate receptors such as the NMDA and AMPA/kainate receptors was confirmed (2). Structurally, the GABAA receptor is composed of a heterooligomeric complex and forms a chloride ion channel (6). In single channel studies using cultured mouse spinal neurons, the GABAA receptor channels are characterized by at least four different levels of the slope conductances (44, 30, 19, and 12 pS, 27.2 and 15.9 pS, or 27 and 19 pS) and the most frequently observed conductance states are 27-30 pS (7-9). This is in good agreement with a main conductance state of 26 pS in the present study, suggesting a possibility that the properties of the GABAA receptor channels obtained with rodent neurons are generalized to those of the human GABAA receptor channels. At least five different classes of the GABAA receptor subunits, a (1-6), b (1-4), g (1-3), d, and r are cloned (6,10). Two forms of the g2 subunit (g2S and g2L), which are generated by RNA alternative splicing, was found in the brain of cattle, rats, mice, and humans (11,12). Interestingly, the g2S subunit is expressed at a fairly constant level during brain development while huge increase of the g2L subunit, which contains a protein kinase C consensus phosphorylation site (11), is found with maturation (13). Of great interest are studies aimed at assessing which combinations of the subunits are expressed in NT2-N cells and whether the g2S to g2L subunit switch is observed in NT2-N cells. NT2-N, thus, may be a model for understanding the regulatory mechanism for the expression of the neurotransmitter receptors. In conclusion, the results presented here clearly demonstrate that NT2 cells differentiate in NT2-N cells, expressing neuronal functions, by treatment with RA. REFERENCES 1. Pleasure, P. J., Page, C., and Lee, V. M.-Y. (1992) J. Neurosci. 12, 1802–1815. 2. Younkin, D. P., Tang, C.-M., Hardy, M., Reddy, U. R., Shi, Q.-Y., Pleasure, S. J., Lee, V. M.-Y., and Pleasure, D. (1993) Proc. Natl. Acad. Sci. USA 90, 2174–2178.
722
AID
BBRC 7087
/
6936$$$942
08-11-97 18:55:35
bbrcgs
AP: BBRC
Vol. 237, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3. Muller, T., Grosche, J., Ohlemeyer, C., and Kettenmann, E. (1993) NeuroReport 4, 671–674. 4. Ikeuchi, Y., Nishizaki, T., and Matsuoka, T. (1995) Biochem. Biophys. Res. Commun. 217, 811–816. 5. Moasser, M. M., Reuter, V. E., and Dmitrovsky, E. (1995) Oncogene 10, 1537–1543. 6. Burt, D. R., and Kamatchi, G. L. (1991) FASEB J. 5, 2916–2923. 7. Bormann, J., Hamill, O. P., and Sakmann, B. (1987) J. Physiol. 385, 243–286. 8. Macdonald, R. L., Rogers, C. J., and Twyman, R. E. (1989) J. Physiol. 410, 479–499.
9. Porter, N. M., Twyman, R. E., Uhler, M. D., and Macdonald, R. L. (1990) Neuron 5, 789–796. 10. DeLorey, T. M., and Olsen, R. W. (1992) J. Biol. Chem. 267, 16747–16750. 11. Whiting, P., McKernan, R. M., and Iversen, L. L. (1990) Proc. Natl. Acad. Sci. USA 87, 9966–9970. 12. Kofuji, P., Wang, J. B., Moss, S. J., Huganir, R. L., and Burt, D. R. (1991) J. Neurochem. 56, 713–715. 13. Wang, J. B., and Burt, D. R. (1991) Brain Res. Bull. 27, 731– 735.
723
AID
BBRC 7087
/
6936$$$942
08-11-97 18:55:35
bbrcgs
AP: BBRC