Regulation of Kv1 subunit expression in oligodendrocyte progenitor cells and their role in G1兾S phase progression of the cell cycle R. Chittajallu, Y. Chen, H. Wang, X. Yuan, C. A. Ghiani*, T. Heckman, C. J. McBain, and V. Gallo†‡ Laboratory of Cellular and Synaptic Neurophysiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-4495 Communicated by Erminio Costa, University of Illinois, Chicago, IL, December 26, 2001 (received for review December 7, 2001)
Proliferative oligodendrocyte progenitor cells (OPs) express large, delayed outward-rectifying Kⴙ currents (IK), whereas nondividing immature and mature oligodendrocytes display much smaller IK. Here, we show that up-regulation of IK occurs in G1 phase of the cell cycle in purified cultured OPs and is the result of an RNA synthesis-dependent, selective increase of the Kⴙ channel subunit proteins Kv1.3 and Kv1.5. In oligodendrocyte cells acutely isolated from developing rat brain, a decrease of cyclin D expression is observed as these cells mature along their lineage. This is accompanied by a decrease in Kv1.3 and Kv1.5 subunit expression, suggesting a role for these subunits in the proliferative potential of OPs in situ. IK expressed in OPs in subventricular zone and developing white matter in acutely isolated slice preparations were selectively blocked by antagonists of Kv1.3, illustrating the functional presence of this subunit in situ. Interestingly, Kv1.3 block inhibited S-phase entry of both purified OPs in culture and in tissue slice cultures. Thus, we employ both in vitro and in situ experimental approaches to show that (i) RNA-dependent synthesis of Kv1.3 and Kv1.5 subunit proteins occurs in G1 phase of the OP cell cycle and is responsible for the observed increase in IK, and (ii) currents through Kv1.3-containing channels play a crucial role in G1兾S transition of proliferating OPs.
phase of the cell cycle at which these alterations occur; (ii) investigate the mechanism(s) responsible for the increase in IK; and (iii) identify the subunits that underlie the observed increase in the functional expression of K⫹ channels. In addition to a purely in vitro approach, we also investigate the presence of Kv subunits and K⫹ channels in OPs in slice preparations to link the proliferative potential of these cells in situ to the expression of IK and to confirm that previous observations in the culture system are relevant to a scenario more closely associated with an in vivo setting.
O
Purified OP Cell Cultures and Organotypic Tissue Slice Cultures. Rat
ligodendrocytes, a major class of macroglia, are responsible for myelination in the central nervous system and primarily originate from highly proliferative oligodendrocyte precursor cells (OPs) in a spatially restricted central nervous system area termed the subventricular zone (SVZ; refs. 1 and 2). Under the influence of extrinsic trophic signals (3, 4), OPs migrate, proliferate, and the majority differentiate (by means of a number of intermediate cell stages) into myelinating oligodendrocytes. A correlation exists between expression of the delayed, outward-rectifying voltage-gated K⫹ currents (IK) and the proliferative potential of oligodendrocyte lineage cells (5–9). Proliferating OPs possess large IK, whereas postmitotic oligodendrocytes do not express such currents (5–9). However, few studies have attempted to identify the cellular mechanisms responsible for these K⫹ channel changes in OPs. Furthermore, although the functional properties of these channels indicate that they are composed of subunits of the Kv1 subfamily (9, 10), the molecular identity of the K⫹ channel subunits involved in the developmental alterations of K⫹ channel expression in OPs has not been extensively analyzed. We have previously used a cell culture model system that allows OPs to be synchronized in a nonproliferative state (G0) and be induced to enter the cell cycle upon mitogen treatment (11, 12). By using this approach, we have previously shown that mitogen-induced entry of OPs into the cell cycle is accompanied by a marked increase in IK.§ In the present study, we take further advantage of this culture system to: (i) analyze the time course of the mitogen-induced increase of IK in OPs and determine the 2350 –2355 兩 PNAS 兩 February 19, 2002 兩 vol. 99 兩 no. 4
Materials and Methods Materials. Platelet-derived growth factor (PDGF; human, AB,
heterodimer form), basic fibroblast growth factor (bFGF; human), and anti-cyclin-D antibody were obtained from Upstate Biotechnology (Lake Placid, NY). Anti Kv1.1–1.6 antibodies, r-agitoxin and r-margatoxin, were obtained from Alomone Labs (Jerusalem). Anti-NG2 polyclonal antibody was a gift of Bill Stallcup (Burnham Institute, La Jolla, CA). Anti-BrdUrd antibody was obtained from Dako. Rapamycin was obtained from Biomol (Plymouth Meeting, PA). All other reagents were obtained from Sigma. OP cell cultures were prepared as described (12). Brain organotypic slice cultures were prepared from P3 coronal slices, containing the lateral ventricles of Sprague–Dawley rats, as described (13).
Immunocytochemistry. For immunostaining of acutely isolated cells from brain slices, 400-m cerebral coronal sections from P3 rats were dissociated, and total cell suspensions were prepared as described (13). For immunostaining of OP cultures, cells were fixed in 4% (wt兾vol) paraformaldehyde for 15 min and permeabilized in 95% ethanol兾5% acetic acid for 3 min. After incubation with anti-Kv1.3 and anti-Kv1.5 (1:50) overnight at 4°C, and with fluorescein-conjugated goat anti-rabbit IgG for 30 min
Abbreviations: AgTx, r-agitoxin; bFGF, basic fibroblast growth factor; IA, A-type transient outward K⫹ channel current; IK, delayed outward-rectifying K⫹ channel current; MgTx, r-margatoxin; PDGF, platelet-derived growth factor; SVZ, subventricular zone; TEA, tetraethylammonium; OPs, oligodendrocyte precursor cells. *Present address: Mental Retardation Research Center, University of California, Los Angeles, CA 90024-1759. †To
whom reprint requests should be addressed. E-mail:
[email protected].
‡Present
address: Neuroscience Research Center, Center 5, Children’s Research Institute, Children’s Hospital, 111 Michigan Avenue, NW, Washington, DC 20010-2970.
§Heckman,
T., Ghiani, C. A., Armstrong, R., Gallo, V. & McBain, C. J. (1999) Soc. Neurosci. Abstr. 25, no. 83.1.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
www.pnas.org兾cgi兾doi兾10.1073兾pnas.042698399
at room temperature, cells were washed and mounted in Vectashield. Western Blots. Western blot analysis of cyclin-D and Kv channel subunit expression was performed as described (11, 14). AntiKv1.1–anti-Kv1.6 antibodies were used at a dilution of 1:1,000. Cell Proliferation Assays in Purified OP Cultures and in Tissue Slice Cultures. Cultured cells were incubated with BrdUrd (25 M) for
24 h, fixed, permeabilized, and stained with anti-BrdUrd antibodies as described (13). In tissue slice cultures, coronal slices containing the lateral ventricles were incubated with BrdUrd (25 M) for 24 h. After enzymatic dissociation (13), cells were stained with antibodies (NG2, O4, and anti-BrdUrd), and percentages of labeled cells were determined (13). Electrophysiology in Cultured OPs and NG2ⴙ Cells in Acutely Isolated Slices. Whole-cell patch–clamp recordings were performed on
OPs that were synchronized in G0 or were treated with PDGF or bFGF (10 ng兾ml). IK and A-type transient K⫹ channel currents (IA) in cultured OPs were isolated and measured as described (9). Measurements of IK expressed by OPs in SVZ or in developing white matter of acutely isolated brain slices were performed as follows. Sprague-Dawley rats (P6–P10) were killed following National Institutes of Health Animal Welfare Guidelines, and 300 M vibratome sagittal sections were obtained. Chittajallu et al.
Slices were perfused with extracellular solution of the following composition (in mM): 124 NaCl兾3 KCl兾2.5 CaCl 2 兾1.3 MgSO4兾26 NaHCO3兾1.25 NaH2PO4兾15 glucose兾0.001 tetrodotoxin, saturated with 95% O2兾5% CO2. Whole-cell somatic recordings were made in SVZ and developing white matter dorsal to the lateral ventricles. Cells were voltage-clamped at ⫺70 mV, and IK or IA was elicited as described for cultured OPs (9). Intracellular solution used was as described (9) but with the addition of 5 mg兾ml biocytin. All membrane potentials were adjusted for a calculated junction potential of 13.7 mV. In both the culture and slice electrophysiological experiments, currents were recorded using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA), filtered at 5 kHz and digitized at 10 kHz. All offline analysis was performed by using the CLAMPFIT analysis program (Axon Instruments). Biocytin and NG2 Double Immunostaining After Electrophysiological Recording from Acutely Isolated Slices. Following electrophysio-
logical recordings, slices were immediately fixed and processed as described (15). For NG2 staining, sections were incubated with a blocking solution [20% (vol兾vol) goat serum in PBS], followed by anti-NG2 polyclonal antibody overnight at 4°C, and then mounted.
Statistical Analysis. For all experiments described where statistical
analysis was performed, an unpaired Student’s t test was used (*, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001).
Results Time course of IK up-regulation following mitogen treatment of quiescent OPs. Cultured OPs synchronized in G0 phase (i.e., withdrawn from the cell cycle by culturing in serum-free DMEM-N1 for 24 h) displayed small IK (Fig. 1A). On treatment with PDGF or bFGF (10 ng兾ml), an up-regulation of IK was PNAS 兩 February 19, 2002 兩 vol. 99 兩 no. 4 兩 2351
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Fig. 1. IK is up-regulated following 24-h (B) PDGF and (C) bFGF treatment (10 ng兾ml), when compared with IK in OPs in G0 (DME-N1 medium) (A). (D and E) Time course of up-regulation of IK in OPs following PDGF and bFGF (total cells ⫽ 131 and 147, respectively) treatment. (F and G) The PDGF- and bFGFinduced increase is not prevented by the G1兾S transition blockers, rapamycin (30 nM) and aphidicolin (3 M), after 12 or 24 h following mitogen treatment (data represent means of 8 –11 OP cell recordings per condition from at least three different individual cultures ⫾ SEM). In F and G, Ik density in OP cells cultured in N1 medium without PDGF or bFGF was the same as in D and E.
Fig. 2. Western blot analysis of Kv1.3–Kv1.6 expression in cultured OPs in DME-N1 medium and 24 h after PDGF (P) or bFGF (bF) treatment (10 ng兾ml). A selective and significant increase of normalized protein expression (**, P ⬍ 0.01) is apparent following either PDGF or bFGF treatment of Kv1.3 (A) and Kv1.5 (C) but not of Kv1.4 (B) and Kv1.6 (D) (see Insets for single examples of band staining). Data were normalized with respect to cells cultured in DME-N1 and represent means from three to seven individual cultures ⫾ SEM. Equal protein loading was verified by Ponceau S solution reversible staining of the blots.
Fig. 3. (A and B) Densitometric measurement of Western blot band (see Insets) intensity illustrates that Kv1.3 and Kv1.5 protein expression is significantly prevented (*P ⬍ 0.05) by ␣-amanitin (⫹A; 20 g兾ml) in the presence of PDGF (P) and bFGF (bF) to levels that are comparable with those seen in OPs in G0 (i.e., cultured in DME-N1). Data were normalized with respect to cells cultured in DME-N1 and represent means from three to five individual cultures ⫾ SEM. (C and D) ␣-Amanitin also significantly prevents (*, P ⬍ 0.05) the mitogen-induced up-regulation (12- or 24-h treatment) of IK density in OPs. Data represent the means of the calculated IK density elicited by a test pulse to 50 mV and are from 15–21 OPs from at least three individual cultures. (E) Inhibition of RNA synthesis does not prevent the PDGF-induced entry of cultured OPs into the cell cycle, as assessed by cyclin D expression. Samples were processed for Western blot analysis with anti-cyclin D antibody at varying time points following PDGF treatment. ctr, purified cyclin D protein. Equal protein loading was verified by Ponceau S solution reversible staining of the blots.
observed (Fig. 1 B and C, respectively). Twenty-four hours after PDGF or bFGF treatment, IK density increased approximately 2-fold (Fig. 1 D and E). The majority of the increase in IK density occurred within approximately 15 h after mitogen treatment. Prolonged exposure to either PDGF or bFGF (48 h) did not lead to any further significant changes in IK density (Fig. 1D). The Mitogen-Induced Up-Regulation of IK Occurs in G1 Phase of the Cell Cycle. Rapamycin and aphidicolin cause cell arrest in the late G1
phase of OP cell cycle (11). In the presence of either agent, an up-regulation of IK density similar to that observed in cells treated with PDGF or bFGF alone was observed, showing that late G1 arrest did not prevent the mitogen-induced up-regulation (Fig. 1 F and G). These observations, coupled with the time course of the increase in IK and estimates of cell cycle duration in cultured OPs (24 h) (16, 17), indicate that IK is up-regulated during G1 phase of the cell cycle. 2352 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.042698399
Fig. 4. (A) Acutely isolated cells were immunostained with oligodendrocyte lineage antibodies and anti-Kv1.3 or anti-Kv1.5 antibodies. A significant decrease in Kv1.3 and Kv1.5 expression was observed in O4⫹ and O1⫹ cells, as compared with NG2⫹ cells (**, P ⬍ 0.01; ***, P ⬍ 0.001). Data shown are means ⫾ SEM obtained from three independent experiments. The number of total cells counted for each antibody ranged between 226 and 700. (B) Acutely isolated cells were immunostained with NG2, O4, O1, and anti-cyclin D antibodies. A significant decrease in cyclin D expression was observed in O4⫹ and O1⫹ cells, as compared with NG2⫹ cells (***, P ⬍ 0.001). The data shown are means ⫾ SEM obtained from of three independent experiments. The total number of cells counted ranged between 456 and 1,042.
Mitogen-Induced Increase in IK Is Mediated by a Selective Up-Regulation of Kv1.3 and Kv1.5 Subunit Protein. Western blot analysis of
Kv subunit proteins was performed in cultured OPs, either in G0 or 24 h after incubation with PDGF or bFGF. Of the Kv subunits tested (Kv1.1 through Kv1.6), no detectable Kv1.1 protein was observed, and only low levels of Kv1.2 were present. Furthermore, PDGF or bFGF (data not shown) did not affect expression of Kv1.2. In contrast, OPs expressed significant levels of Kv1.3, Kv1.4, Kv1.5, and Kv1.6 proteins (Fig. 2). Following PDGF or bFGF treatment (24 h), a selective increase was only apparent in Kv1.3 (Fig. 2 A) and Kv1.5 (Fig. 2C) expression. No significant change in Kv1.4 (Fig. 2B) or Kv1.6 (Fig. 2D) was observed. Up-Regulation of Kv1.3兾Kv1.5 Subunit Protein Expression and IK Are RNA Synthesis-Dependent. The RNA polymerase inhibitor
␣-amanitin (20 g兾ml) (18) prevented the mitogen-stimulated increase in Kv1.3 and Kv1.5 expression (Fig. 3 A and B). Similarly, no up-regulation of IK was observed in the presence of ␣-amanitin (Fig. 3 C and D). RNA synthesis-inhibition in mitogen-treated OPs reduced Kv1.3兾Kv1.5 protein expression and IK density to levels comparable with those of OPs in G0 (DME-N1 medium) (Fig. 3 A–D). ␣-Amanitin caused no change Chittajallu et al.
in IK in G0 cells (data not shown), suggesting that under quiescent conditions the tonic levels of K⫹ channel expression were not dependent on RNA synthesis. The effects of ␣-amanitin on Kv1.3兾Kv1.5 and IK up-regulation were not because of a failure of OPs to enter G1 phase, as demonstrated by the increase in the levels of the G1 phase marker, cyclin D (11, 19) (Fig. 3E). In OPs cultured with PDGF alone, cyclin D levels decreased between 18 and 24 h, whereas in OPs treated with ␣-amanitin cyclin D levels remained constant between 18 and 24 h (Fig. 3E), indicating that preventing Kv1.3 and Kv1.5 up-regulation caused cell cycle arrest in G1 phase (11, 12). Kv1.3 and Kv1.5 Are Developmentally Regulated with Progression Through the Oligodendrocyte Lineage. Immunocytochemical stain-
ing of oligodendrocyte lineage cells acutely isolated from tissue Chittajallu et al.
slices demonstrated that expression of Kv1.3 and Kv1.5 subunits was higher in NG2⫹ and O4⫹ progenitors than in mature O1⫹ oligodendrocytes (Fig. 4A). Furthermore, expression of the same subunits within the NG2⫹, O4⫹, and O1⫹ populations paralleled that of cyclin D (Fig. 4B). Immunoreactivity for Kv1.4 and Kv1.6 was low both in NG2⫹ and O4⫹ cells (data not shown). These data demonstrate that both Kv1.3 and Kv1.5 expression is down-regulated as OPs progress through the lineage to mature oligodendrocytes and is correlated with the proliferative potential of these cells. OPs in Acutely Isolated Slices Display IK That Are Partially Sensitive to Kv1.3 Block. Whole-cell voltage–clamp recordings were per-
formed from OPs located in the SVZ and in developing white matter of acutely isolated brain slices in rats aged P6–P11. To
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Fig. 5. (A) A representative example of a biocytin-filled cell from which electrophysiological recordings were made. (B) Illustration that this cell (see arrow) was immunoreactive for NG2 (scale bars ⫽ 20 M). (C) All cells expressed IK (Middle) and, in some cases, IA (64% of cells; n ⫽ 25, Bottom). Insets in top and middle panels illustrate voltage protocols used. (D) Activation curves for IK illustrate the presence of two current components; data yielded a significantly better fit when described by the sum of two Boltzmann distributions (single Boltzmann vs. double Boltzmann fit; 2 values ⫽ 0.0013 ⫾ 0.0001 vs. 0.0006 ⫾ 0.00007; P ⬍ 0.001; n ⫽ 16). Data for the activation plots are expressed as means ⫾ SEM from 25 cells. IK was partially inhibited by either AgTx (50 nM) (E) or MgTx (50 nM) (F). Subsequent addition of TEA (10 mM) caused a further inhibition. Data are means ⫾ SEM obtained from five to six cells. The traces in E and F are individual examples of IK taken at the times indicated by the numbers.
⫺23 ⫾ 1.4 mV and 19 ⫾ 2.4 mV, respectively (Fig. 5D), similar to those found in cultured OPs (9). The relative contribution of the two current components to the total K⫹ current was 40兾60% in NG2⫹ cells in brain slices and 37兾63% in cultured OPs (9). These data illustrate the striking similarity between the functional properties of IK in NG2⫹ cells in brain slices and in cultured OPs. To examine whether IK expressed by NG2⫹ OPs in the SVZ兾developing white matter contain or comprise Kv1.3 subunits, we studied the effects of the Kv1.3 channel blockers, r-agitoxin (AgTx) and r-margatoxin (MgTx), at concentrations shown to be selective for Kv1.3-containing channels (21–24). Both of these toxins only partially inhibited IK in these cells (Fig. 5 E and F). Subsequent addition of tetraethylammonium (TEA) caused a further inhibition of IK (Fig. 5 E and F), suggesting that not all IK is attributable to channels containing or comprising Kv1.3. Kv1.3 Block Inhibits Proliferation of NG2ⴙ Cells in Tissue Slice Cultures and Prevent G1兾S Transition in Cultured OPs. In brain tissue slice
cultures, AgTx and MgTx significantly reduced proliferation of NG2⫹ OPs and O4⫹ preoligodendrocytes, as assessed by BrdUrd staining (Fig. 6A). Because it is not possible to easily determine the phase of the cell cycle at which Kv1.3 block affects cell proliferation in tissue slice cultures, we examined the effects of AgTx and MgTx on PDGF-induced proliferation of cultured OPs (Fig. 6B). These toxins also inhibited proliferation of cultured OPs (Fig. 6B) but did not affect PDGF-induced G0兾G1 transition, as measured by cyclin D expression (Fig. 6C). These data suggest that blockade of currents through Kv1.3-containing channels inhibited cell cycle progression in the G1 phase.
Fig. 6. (A) Cortical coronal slices were cultured for 48 h in the absence or presence of TEA (10 mM), r-agitoxin (AgTx; 50 nM), or r-margatoxin (MgTx; 50 nM) and incubated with BrdUrd for the last 24 h. Acutely isolated cell suspensions from these slices were immunostained with NG2 or O4 antibodies, together with anti-BrdUrd antibodies. All K⫹ channel blockers significantly inhibited cell proliferation in the NG2⫹ and O4⫹ cell populations (**, P ⬍ 0.01; ***, P ⬍ 0.001). Data shown are means ⫾ SEM obtained from three to four experiments. Total number of NG2⫹ and O4⫹ cells counted for the different treatments ranged between 654 and 958. (B) Treatment of cultured OPs with AgTx or MgTx inhibits cell proliferation to the same extent as TEA. Data are means ⫾ SEM of four to five independent experiments run in duplicate. The total number of cells counted for each treatment ranged between 2,843 and 3,616. (C) Time course of cyclin D expression following PDGF treatment of OPs in the absence or presence of r-agitoxin (AgTx; 5 and 50 nM) or r-margatoxin (MgTx; 5 and 50 nM).
unequivocally identify these cells as OPs, both immunocytochemical and electrophysiological characterizations were performed. IK was detected in NG2⫹ OPs (Fig. 5 A–C). These cells had a mean resting membrane potential of ⫺42 ⫾ 3 mV (cf. ⫺50 ⫾ 3 mV in cultured OPs) (9). In addition, 64% of the cells tested expressed IA (Fig. 5C Bottom). This proportion was comparable to that seen previously in cultured OPs (6, 9, 20). The voltage dependence of IK in OPs of the SVZ was composed of an overlap of two separate current components (Fig. 5D). The two current components displayed V1/2 activation values of 2354 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.042698399
Discussion Here, we show that Kv1.3–Kv1.6 subunit protein expression is detectable in quiescent and proliferating OPs, consistent with previous observations demonstrating that OPs and mature oligodendrocytes express Kv1.3–Kv1.6 mRNA transcripts (20, 25). We demonstrate that transition of quiescent OPs into G1 phase of the cell cycle is accompanied by a selective increase in Kv1.3 and Kv1.5 protein expression. This process is RNA synthesisdependent and is accompanied by an up-regulation of IK. Therefore, the insertion of newly transcribed and translated subunit proteins into the cell membrane produces the increase in IK. Changes in the expression levels of various channel proteins following alterations in the rate of mRNA translation are mainly governed by the protein turnover rate. The half-lives of other voltage-gated channels (Na⫹ and Ca2⫹) have been estimated to be 20–48 h (26, 27). Such long half-lives would not allow for the rapid changes in the mRNA synthesis-dependent up-regulation of K⫹ channel expression noted in this study. However, unlike voltage-gated Na⫹ and Ca⫹ channels, previous studies have indicated mechanisms that produce a dynamic regulation of Kv transcript levels (28). These temporal differences have been attributed to the relatively short half-lives of Kv mRNA and protein (35 min and 4 h, respectively) (29, 30). Importantly, these changes translated to a corresponding rapid increase in the functional expression of the voltage-gated K⫹ current (29, 30). Thus, it is clear that under certain conditions, K⫹ channel activity can be rapidly regulated under the control of mRNA transcription, and the time course of up-regulation noted in the present study is in agreement with previous observations. A correlation between the cyclin E兾cdk2-associated kinase activity and OP cell proliferation has been observed (14). Thus, cdk2-associated kinase activity is a reliable marker of the proliferative state of OPs. Here, we found that mitogen-induced alterations of IK in OPs and cdk2 activity (14) follow a similar time course, strengthening the link between K⫹ channel expresChittajallu et al.
sion and the proliferative state of OPs. Because K⫹ channel blockers inhibit cyclin E兾cdk2 activity (14), one possible function of outward K⫹ channels is to sustain cdk2 activity in OPs, thus regulating G1兾S progression through a mechanism yet to be determined. Nonselective block of K⫹ channels with TEA causes a failure in G1兾S transition (11). Here, we show that selective block of Kv1.3-containing channels alone with AgTx and MgTx is sufficient to also elicit G1 arrest. Furthermore, the increase in both Kv1.3 subunit protein and IK occurs during G1 phase. These data illustrate that Kv1.3 up-regulation during G1 is a prerequisite for G1兾S transition. In fact, Kv1.3 has been implicated in the proliferation of other cell types (24, 31, 32). To extend these observations to a scenario that is closer to an in vivo situation, we demonstrate that IK expressed by OPs in acutely isolated slices is partially sensitive to Kv1.3 block. These data show that OPs in an intact system express Kv1.3, and this subunit functionally contributes to IK. Kv1.3 block with AgTx and MgTx also prevents OP cell proliferation in tissue slice cultures to a similar extent as TEA. This is observed despite a TEA-sensitive component of IK in the presence of the Kv1.3selective toxins in OPs from acutely isolated slices. Furthermore, in acutely isolated cells, a decrease in Kv1.3 expression occurs as OPs progress through the lineage and become less proliferative. These latter experiments strengthen the initial observations in cultured OPs and illustrate that the observations in this study are relevant to OPs in situ. Unfortunately, we could not assess the role of Kv1.5 in G1兾S transition, as no selective pharmacological agents are available. In a previous study, treatment of OPs with Kv1.5 antisense oligonucleotides caused a reduction in Kv1.5 protein expression
and IK density (20), but no effect on proliferation was observed (20). Here, we show that Kv1.3 block prevents OP proliferation to the same extent as TEA, suggesting that this subunit exerts a functionally dominant role in G1兾S cell cycle progression. Furthermore, the concentration of TEA used in the cell proliferation assays was close to the IC50 for Kv1.3-containing channels (10) and blocked 50% of outward K⫹ currents in the electrophysiological experiments. In contrast, channels formed by Kv1.5 are insensitive to block by TEA (10). Therefore, the concentration of TEA used in the OP proliferation assays (10 mM) would be expected to exert no effects on Kv1.5-containing channels. Thus, although Kv1.5 subunit is up-regulated during G1 phase of the cell cycle, it is conceivable that this subunit plays a relatively minor role when compared with Kv1.3 in OP G1兾S cell cycle progression (20). Conversely, treatment of astrocytes with Kv1.5 antisense oligonucleotides does lead to an inhibition of their proliferation (33). These data indicate that differing Kv subunits are involved in the proliferation of distinct glial cell types. In conclusion, functional K⫹ channels comprising Kv1.3 are expressed in OPs in culture and in vivo. These channels are crucial for G1兾S transition and proliferation of OPs and thus play an important role in the processes leading to the generation of mature oligodendrocytes. Future studies are required to determine the intracellular targets that K⫹ channel expression impinges upon to modulate cell cycle progression.
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NEUROBIOLOGY
We thank Dr. Bill Stallcup for the gift of the monoclonal NG2 antibody. We thank Shibeshih Belachew, Douglas Fields, and Beth Stevens for critically reading the manuscript. R.C. is a Wellcome Trust Traveling Fellow.
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PNAS 兩 February 19, 2002 兩 vol. 99 兩 no. 4 兩 2355
