K+ channel expression and cell proliferation are regulated by intracellular sodium and membrane depolarization in oligodendrocyte progenitor cells

Abstract

The effects of a variety of antiproliferative agents on voltage-dependent K+ channel function in cortical oligodendrocyte progenitor (O-2A) cells were studied. Previously, we had shown that glutamate receptor activation reversibly inhibited O-2A cell proliferation stimulated by mitogenic factors and prevented lineage progression by attenuating outward K+ currents in O-2A cells. We now show that the antiproliferative actions of glutamate receptor activation are Ca2+-independent and arise from an increase in intracellular Na+ and subsequent block of outward K+ currents. In support of this mechanism, agents that acted to depolarize O-2A cells or increase intracellular sodium similarly had an antiproliferative effect, attributable at least in part to a reduction in voltage-gated K+ currents. Also, these effects were reversible and Ca2+-independent. Chronic treatment with glutamate agonists was without any long-term effect on K+ current function. Cells cultured in elevated K+, however, demonstrated an upregulation of inward rectifier K+ currents, concomitant with an hyperpolarization of the resting membrane potential. This culture condition therefore promoted a current phenotype typical of pro-oligodendroblasts. Finally, cells chronically treated with the mitotic inhibitor retinoic acid displayed a selective downregulation of outward K+ currents. In conclusion, signals that affect O-2A cell proliferation do so by regulating K+ channel function. These data indicate that the regulation of K+ currents in cells of the oligodendrocyte lineage plays an important role in determining their proliferative potential and demonstrate that O-2A cell K+ current phenotype can be modified by long-term depolarization of the cell membrane.

Fig. 1.

Both transient and sustained voltage-dependent outward K⁺ current phenotypes are observed in O-2A progenitor cells. A, Outward currents were activated by test potentials up to +70 mV (10 mV increments, 0.1 Hz,V_hold = −70 mV). A prepulse to either −110 or −40 mV (100 msec duration, see insets) permitted the isolation of both the sustained and transient current components. Currents evoked from −40 mV possessed only a sustained current phenotype (left panel). When a prepulse to −110 mV was included, an additional transient current component was observed in the total current activated (middle trace). Digital subtraction of the sustained current component (left panel) from the total outward current (middle panel) permitted the isolation of the inactivating transient current component (right panel). The sustained current demonstrated modest inactivation (10%) during a 500 msec test pulse. B, The sum of two Boltzmann distributions was required to adequately describe the voltage-dependence of sustained current activation. The mean half-activations of the two current components were −23.7 ± 0.5 mV (k = 5) and 12.9 ± 1.8 mV (k = 13) (n = 25). The two currents represented 37 and 63% of the total current, respectively. This suggests that the total sustained current in O-2A cells reflects the temporal overlap of two current components. C, Transient currents were observed in only 63% of cells. In contrast to the sustained current, a single Boltzmann distribution was sufficient to describe the voltage-dependence of activation. Transient currents had a mean half-activation of −10.4 ± 0.9 mV (n = 24).

Fig. 3.

The block of outward K⁺ currents by GluR activation in O-2A cells is [Na⁺]_i-dependent. A, Addition of kainate (KA, 200 μm) reversibly attenuated the sustained current by 48.5 ± 5.3% (n = 11). Currents were activated by a test pulse to +70 mV (with or without a prepulse to −110 or −40 mV; see Fig. 1). In addition, the isolated transient current was also blocked by GluR activation (41.2 ± 9%, n = 6,E). B, The AMPA-preferring receptor antagonist DNQX (20 μm) blocked the kainate-induced attenuation of K⁺ currents (B,E), confirming a requirement for AMPA receptor activation in K⁺ current attenuation. C, When Na⁺ in the extracellular medium was replaced by NMDG, kainate application no longer reduced O-2A K⁺ currents (94.2 ± 5.3% of control), confirming that an increase in [Na⁺]_i alone was responsible for the K⁺ current block. D, A direct increase in [Na⁺]_i by application of veratridine (50 μm) also attenuated both the transient and sustained current (53.7 ± 3.1% and 22.2 ± 7.1%, n = 10), respectively. E, Summary histogram of the effects shown in A–D for both the isolated sustained and transient current components.

Fig. 4.

An elevation of extracellular K⁺reduces outward and augments inward K⁺ currents in O-2A cells. Elevation of [K⁺]_o from 2.5 to 45 mm reduced outward currents (A) and augmented the inward rectifying current (B). The magnitudes of both the sustained current reduction and the Kir augmentation were as predicted from a simple change in the K⁺ driving force. Aii, The current amplitude was reduced by 52 ± 5% (n = 6), a value close to the calculated value (56%). Bii, Likewise, the observed augmentation of the Cs⁺-sensitive Kir was 440 ± 60% (n = 5), a value close to the predicted value of 469%.

Fig. 2.

Kir currents in O-2A progenitor cells are revealed at negative test potentials. Kir were activated by either one of two protocols. A, A ramp protocol (inset) delivered from −120 mV to +50 mV (60 mV/sec) evoked both inward and outward currents. Addition of Cs⁺ (5 mm) to the extracellular solution selectively blocked the Kir. B, Kir were isolated by digitally subtracting the current obtained in the presence of Cs⁺ from that obtained in control. The reversal potential of Kir was −96 mV, close to the calculated reversal potential for K⁺ (E_Kcalc = −100 mV). At potentials positive to −90 mV, the Cs⁺-sensitive current became outward until strong rectification was observed and no current was observed at the most positive potentials. C, Alternatively, Kir were activated by voltage-clamping cells at −70 mV and delivering test potentials to negative voltage-steps (5 mV increments, 0.1 Hz). Application of Cs⁺ removed all time-dependent currents (middle panel) and permitted the isolation of Kir by digital subtraction (bottom panel). D, The KirE_rev were shifted in a predictable manner when [K⁺]_o was elevated from 2.5 to 45 mm (E_rev =E_Kcalc = −26 mV, D). This confirms that the major permeant ion is K⁺, confirming that the isolated current was Kir and not the hyperpolarizing-activated current I_h.

Fig. 5.

Membrane depolarization and increased [Na⁺]_i inhibit O-2A cell proliferation—[³H]thymidine incorporation assays.A, High extracellular K⁺ inhibits O-2A cell proliferation. B, Veratridine inhibits O-2A cell proliferation. Cells were plated in 24-well plates at a density of 30,000 cells/well and cultured in DME-N1 + 0.5% FBS with PDGF and/or bFGF (both 10 ng/ml). Veratridine was added at a concentration of 10 or 50 μm, whereas the high K⁺ media contained 25 or 45 mm KCl, respectively. [³H]thymidine (0.5 μCi/ml) was added to the cultures 2 hr after plating the cells. After 22 hr, [³H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages of three experiments in triplicate ± SEM are shown.A, *p < 0.001, **p < 0.05 compared with their respective controls (Student’s t test). B–D, Proliferation of cells treated with veratridine (50 and 10 μm) was significantly different from controls (p < 0.05 and p < 0.001, respectively). The antiproliferative effects of high K⁺ and veratridine are reversible. Time course after removal of high K⁺- (C) or veratridine-containing (D) medium. Progenitor cells were cultured in PDGF (10 ng/ml) in the absence (control condition) or presence of 45 mmK⁺- or veratridine-containing (20 μm) medium. After 22 hr, all cells were shifted to fresh culture medium without high K⁺ or veratridine, but containing PDGF (10 ng/ml) and [³H]thymidine (0.5 μCi/ml). At 22 hr, before the shift to low -K⁺ or veratridine-free medium, high K⁺ and veratridine inhibited [³H]thymidine incorporation by 70 and 47%, respectively. Cells were harvested after 6, 12, and 24 hr after shift to low-K⁺ or veratridine-free medium, and [³H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages ± SEM (n = 3) are shown.

Fig. 6.

The inhibitory effects of GluR agonists, agents that increase [Na⁺]_i and membrane depolarization on O-2A cell proliferation, are independent on extracellular Ca²⁺. A, Absence of extracellular Ca²⁺ does not prevent kainate-, veratridine-, and high K⁺-induced inhibition of O-2A cell proliferation. Cells were plated in 24-well plates at a density of 30,000 cells/well and cultured in DME-N1 + 0.5% FBS with PDGF and/or bFGF (both 10 ng/ml), in the presence or absence of extracellular Ca²⁺(see Materials and Methods for media composition). Veratridine was added at a concentration of 25 μm, kainate was 100 μm, whereas the high K⁺ media contained 45 mm KCl. [³H]thymidine (0.5 μCi/ml) was added to the cultures 2 hr after plating the cells. After 22 hr, [³H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages of two experiments in triplicate ± SEM are shown. All treatments (kainate, veratridine, and high K⁺) were significantly different from their respective controls, in both the presence and absence of Ca²⁺ (p < 0.001, Student’s t test). B, Treatment with the Ca²⁺ ionophore A23187 does not modify O-2A cell proliferation. Cells were plated in a DME-N1 medium containing Ca²⁺ with PDGF and/or bFGF (both 10 ng/ml), in the presence or absence of A23187 (1–30 nm) or AMPA (100 μm). [³H]thymidine (0.5 μCi/ml) was added to the cultures 2 hr after plating the cells. After 22 hr, [³H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages of three experiments in triplicate ± SEM are shown.

Fig. 7.

Depolarization with high extracellular K⁺ prevents O-2A lineage progression, as detected by staining with O4 antibody. O-2A progenitor cells were purified and cultured on coverslips in DME-N1 medium with 0.5% FBS with PDGF (10 ng/ml), or bFGF (10 ng/ml), or PDGF + bFGF. K45indicates that cells were cultured in the same medium with the addition of 45 mm K⁺ (see Materials and Methods for media composition). After 48 hr, cells were immunostained with O4 antibody and counted. Averages ± SEM obtained from three experiments (n = 30) are shown. The total number of cells counted for each culture condition ranged from 1020 to 2046; *p < 0.001; **p < 0.005 (Student’s t test).

Fig. 8.

Chronic exposure of O-2A cells to 45 mm [K⁺]_o upregulates the Kir current. Cells cultured in 45 mm[K⁺]_o for 48 hr possessed properties distinct from those observed in control cultured O-2A cells. After removal of the elevated K⁺ culturing medium, cells were perfused with a control solution (i.e., 2.5 mm K⁺) for electrophysiological recordings. The resting membrane potential of cells cultured in 45 mm [K⁺]_o was significantly more negative than that seen in control (Table 3).A, An increase in the sustained current density was observed after culture in 45 mm[K⁺]_o for 48 hr (see also Table 1).Ai, Representative current traces obtained from two different cells cultured under control and 45 mm[K⁺]_o conditions demonstrate the upregulation of the sustained current component after chronic exposure to 45 mm [K⁺]_o. Aii, Summary histograms of data obtained at a test pulse of +70 mV reveals an upregulation of both the current amplitude and the current density when normalized for any changes in membrane capacitance.Bi, Similarly, an upregulation of the Kir current density was also observed after chronic exposure to 45 mm[K⁺]_o culturing conditions (Bi, 1, 3,4) (see also Table 3). Bi,2, Normalization of the Kir currents obtained in control (n = 8) versus the 45 mm[K⁺]_o (n = 6) revealed no change in the current–voltage relationship of the Kir after chronic 45 mm [K⁺]_o culture.Aii, Bii, Summary histograms of sustained and Kir current data obtained from both control (2.5 mm[K⁺]_o) and 45 mm[K⁺]_o culturing conditions reveal an upregulation of both the total current and the current density in 45 mm [K⁺]_o culturing conditions. Data shown in the histogram was obtained from a current step to −120 mV.

Fig. 9.

RA reversibly inhibits O-2A cell proliferation and prevents lineage progression. A, RA inhibits O-2A cell proliferation. Cells were plated in 24-well plates. After 2 hr, PDGF and/or bFGF (both 10 ng/ml), as well as RA (0.1–3 μm), were added to the cultures, together with [³H]thymidine (0.5 μCi/ml). After 22 hr, [³H]thymidine incorporation was measured by trichloroacetic acid precipitation and scintillation counting. Averages ± SEM obtained from three to six experiments run in triplicate are shown. All concentrations of RA significantly (p < 0.05) inhibited O-2A cell proliferation, except for 0.01 μm in bFGF and P + F (Student’st test). B, The antiproliferative effects of RA are reversible. Progenitor cells were cultured in PDGF (10 ng/ml) in the absence or presence of RA (1 μm). After 22 hr, all cells were shifted to fresh culture medium, without RA, containing PDGF (10 ng/ml) and [³H]thymidine (0.5 μCi/ml). Cells were harvested after 6, 12, and 24 hr, and [³H]thymidine incorporation was determined by trichloroacetic acid precipitation and scintillation counting. Averages ± SEM (n = 3) are shown. At 22 hr, before the shift to RA-free medium, RA inhibited [³H]thymidine incorporation by 47%.C, Treatment with RA prevents O-2A lineage progression, as detected by staining with O4 antibody. O-2A progenitor cells were purified and cultured on coverslips in DME-N1 medium with 0.5% FBS with PDGF (10 ng/ml), or bFGF (10 ng/ml), or PDGF + bFGF. RA (1 μm) was added to the culture medium 2 hr after plating. After 48 hr, cells were immunostained with O4 antibody and counted. Averages ± SEM obtained from three experiments (n = 20) are shown. The total number of cells counted for each culture condition ranged from 683 to 2176; *p < 0.001 (Student’s ttest).

Fig. 10.

Long-term treatment with RA downregulates both the transient and sustained outward K⁺ currents in O-2A cells. A, B, Cells cultured in RA-containing (1 μm) media for 48 hr displayed a downregulation of both the isolated sustained current (Ai) and transient current density (Aii) (see also Table 3). Representative traces from two different cells in both control and RA culture conditions are depicted inAi and Bi to illustrate the downregulation of both the transient and sustained currents in RA culturing conditions. Aii, Bii, Summary histograms of the effects of chronic exposure to RA on both the isolated sustained and transient current components. Both the transient and the sustained current densities were significantly reduced after chronic exposure to RA. Aiii, Biii, RA had no effect on the voltage-dependent properties of activation of either the sustained or the transient current components.Aiii, Two sustained current components could be resolved, possessing half-activations of −22.4 ± 0.7 (k = 6; n = 13) and 16.1 ± 1.6 mV (k = 7); values similar to those seen in control. The relative contribution of each current component was unchanged from control untreated cells. Biii, Despite a 60% attenuation of current density, the transient current component possessed voltage-dependent activation properties (−6 ± 1.3 mV,n = 13) identical to that seen in control treated cells.