Conceived and designed the experiments: GS FW SCS HS JS. Performed the experiments: GS. Analyzed the data: GS FW HS. Wrote the paper: GS FW HS. Revised the article, gave final approval of the version to be published: FW SCS HS JS.
The authors have declared that no competing interests exist.
Voltage-gated potassium (Kv) channels are among the earliest ion channels to appear during brain development, suggesting a functional requirement for progenitor cell proliferation and/or differentiation. We tested this hypothesis, using human neural progenitor cells (hNPCs) as a model system.
In proliferating hNPCs a broad spectrum of Kv channel subtypes was identified using quantitative real-time PCR with a predominant expression of the A-type channel Kv4.2. In whole-cell patch-clamp recordings Kv currents were separated into a large transient component characteristic for fast-inactivating A-type potassium channels (IA) and a small, sustained component produced by delayed-rectifying channels (IK). During differentiation the expression of IA as well as A-type channel transcripts dramatically decreased, while IK producing delayed-rectifiers were upregulated. Both Kv currents were differentially inhibited by selective neurotoxins like phrixotoxin-1 and α-dendrotoxin as well as by antagonists like 4-aminopyridine, ammoniumchloride, tetraethylammonium chloride and quinidine. In viability and proliferation assays chronic inhibition of the A-type currents severely disturbed the cell cycle and precluded proper hNPC proliferation, while the blockade of delayed-rectifiers by α-dendrotoxin increased proliferation.
These findings suggest that A-type potassium currents are essential for proper proliferation of immature multipotent hNPCs.
Human neural progenitor cells (hNPCs) isolated from fetal brain tissue are considered a promising source for cell replacement therapies in neurodegenerative disorders
While immature progenitor cells rarely exhibit sodium currents and cannot generate action potentials
After identification of the four Kv channel genes
In the present study we show that proliferating hNPCs express functional Kv channels, while they do neither exhibit sodium currents nor action potential firing. An overview of the investigated Kv1-4 channels and their published functional characteristics is given in
Human neural progenitor cells (hNPCs) derived from aborted fetal brain tissue 12 weeks post-fertilization were isolated as described previously
Patch pipettes were formed from borosilicate glass (BioMedical Instruments, Zöllnitz, Germany) with a horizontal puller (Sutter Instruments P-97, Novato CA, USA) and fire-polished to final resistances of 2–4 MΩ. The pipette solution contained (mM): 130 KCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 10 EGTA and 2 Mg-ATP, pH adjusted to 7.3 with KOH (260 mOsm). Poly-L-lysine (PLL)-coated culture dishes (∅ 35 mm) with proliferating hNPCs or differentiated cells were used as recording chamber and filled with a bath solution containing (mM): 150 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose and 5 HEPES, pH adjusted to 7.3 with NaOH (280 mOsm). Different antagonists (all from Sigma-Aldrich GmbH if not stated otherwise) were dissolved in this bathing solution: 4-aminopyridine (4-AP, 0.1–10 mM), phrixotoxin-1 (PTX, 1–1000 nM, Alomone Labs, Jerusalem, Israel), ammonium chloride (NH4Cl, 1–100 mM), quinidine (QND, 0.1–100 µM), α-dendrotoxin (DTX, 1–1000 nM), margatoxin (MTX, 0.1–50 nM) and tetraethylammonium chloride (TEA, 1–100 mM). A fast application system with a triple-barrel glass pipette attached to an electromechanical switching device (SF-77B, Warner Instruments, Hamden, CT, USA) was arranged with the external bath solution flowing centrally and the antagonist solutions flowing through the side tubes. Whole-cell patch clamp experiments were performed at 20–22°C under optical control (inverted microscope DMIL, Leica, Bensheim, Germany). Seal resistances ranged from 1–3 GΩ. Whole-cell currents were amplified using an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany), low-pass filtered at 2 kHz, and sampled at 10 kHz. Capacitances were compensated and leak currents were substracted (P/n) using the facilities of the Pulse software (HEKA Elektronik, Lambrecht, Germany). Series resistances (Rs = 14±7 MΩ) and liquid junction potentials (VL = 4.3 mV, calculated with Clampex 9.2, Molecular Devices, Sunnyvale, USA) were not corrected.
Voltage-gated currents were activated from a holding potential of −100 mV by depolarizing steps to 100 mV in 10 mV increments (300 ms). Steady-state inactivation of Kv currents was determined via hyperpolarizing prepulses increasing in 10 mV increments from −130 mV to 50 mV (500 ms) followed by a test pulse to 50 mV (300 ms). Current amplitudes were measured between 0 and 20 (transient component, t.c.) and between 280 and 300 ms (sustained component, s.c.) of each depolarizing voltage pulse. Biophysical separation of a delayed-rectifier current (IK) was obtained in activation protocols by a depolarizing prepulse to −40 mV (500 ms), which inactivated the transient A-type current (IA). IA could be isolated in inactivation protocols by a test pulse to 0 mV, because it activated at slightly more negative potentials than IK. Both current components were additionally separated pharmacologically by application of 10 mM 4-AP to proliferating hNPCs, with IK being identified as the 4-AP-insensitive component measured in activation protocols and IA was isolated by subtracting the 4-AP-insensitive component of steady-state inactivation currents from control currents (
(A): In whole-cell patch-clamp recordings human neural progenitor cells (hNPCs) expressed inactivating A-type (IA) and non-inactivating delayed-rectifier-like potassium currents in activation (i) and inactivation protocols (ii, insets). (B): Pharmacological separation of current components was performed by application of 10 mM 4-aminopyridine (4-AP). IK was defined as 4-AP-insensitive component and IA as 4-AP-sensitive component. (C): Biophysical separation of IK was observed in activation protocols by a depolarizing prepulse to −40 mV (500 ms), which caused inactivation of IA. In inactivation protocols IA was revealed by a test pulse to 0 mV only since it activated at slightly more negative potentials than IK. During each voltage step peak values of the transient component were measured between 0 and 20 ms and sustained currents were determined between 280 and 300 ms. Chord conductances and current values respectively were normalized to their peak amplitudes and fitted to a Boltzmann distribution and current-voltage-relationships of control currents (A), pharmacologically (B) as well as biophysically (C) separated currents were calculated (iii, see
For dose-response relationships the inhibition of biophysically separated peak currents was determined during a single depolarizing voltage step from −100 mV to 100 mV (−40 mV prepulse, for IK) or to 0 mV (−130 mV prepulse, for IA). At the same time antagonists were applied starting 30 s prior to the test pulses. Values were normalized to peak amplitudes recorded in the absence of antagonists and fitted with the Hill equation using Origin 6.1:
Total RNA was isolated from proliferating hNPCs as well as from differentiated cells (4 tissue preparations each) grown in 75 cm2 PLO/FN-precoated culture flasks using the RNeasy mini kit (QIAGEN Sciences, Germantown MD, USA) according to the manufacturer's protocol. First-strand cDNA was prepared from total RNA using the RevertAid first strand cDNA synthesis kit (Fermentas International Inc., Burlington, Canada). 30 µl samples of total RNA were transcribed to cDNA with 600 U of reverse transcriptase. The reaction mixture of 60 µl further contained 5 µM oligo(dT)18 primer, 0.5 mM nucleotide triphosphates (dNTPs), 50 mM KCl, 4 mM MgCl2, 10 mM dithiothreitol (DTT) and 50 mM Tris-HCl (pH 8.3). Oligonucleotide primers for subtypes of the Kv channel families 1–4 (see
Quantitative real-time PCR was performed using 300 ng cDNA from total RNA, 600 nM forward and reverse primers, Platinum-SYBR Green qPCR Supermix® (SYBR Green I, 0.375 U Platinum Taq DNA polymerase, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, dNTPs 200 µM each, 0.25 U UDG) and 100 nM 6-carboxy-X-rhodamine (both from Invitrogen) using the following protocol in an MX 3000P instrument (Stratagene, La Jolla, CA, USA): 2 min 50°C, 2 min 95°C and 50 cycles of 15 s 95°C, 30 s 60°C. To confirm a single amplicon a product melting curve was recorded. Threshold cycle (Ct) values were placed within the exponential phase of the PCR as described previously by Engemaier et al. (2006). Ct values of 4–12 independent experiments, each performed in duplicate, were normalized to ribosomal protein L22 (Ct−Ct RPL22 = ΔCt)
Evaluation of cell viability was performed by a tetrazolium salt assay using the reagent 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich GmbH). In viable cells MTT is converted by the mitochondrial dehydrogenase to a blue formazan product
According to this, a flow cytometric analysis was performed to substantiate the effects on cell cycle (see
Progenitor cell proliferation was quantified by a colorimetric immunoassay based on the measurement of 5-bromo-2-deoxyuridine (BrdU) incorporation during DNA synthesis
Data were expressed as mean±standard error (SEM). Statistical differences were calculated with Students's t-test (two-tailed, unpaired) using Origin 6.1 (OriginLab Corporation, Northampton MA, USA) or one-way ANOVA, followed by Tukey's post-hoc test using GraphPad Prim 3 (GraphPad Software Inc., La Jolla, USA); p values≤0.05 were considered significant.
To characterize the voltage-dependency of voltage-gated potassium (Kv) currents in proliferating human neural progenitor cells (hNPCs) outward currents were elicited in whole-cell voltage-clamp recordings either in activation protocols or steady-state inactivation protocols (
IA and IK were pharmacologically separated by application of 10 mM 4-aminopyridine (4-AP). IK was classified as 4-AP-insensitive current in activation protocols (30±5 pA/pF, n = 10–13) and contributed 10% to the transient and 47% to the sustained whole-cell current. IA was isolated as 4-AP-sensitive component during steady-state inactivation (207±66 pA/pF, n = 6–7) and constituted 90% of the transient and 53% of the sustained component of Kv outward currents (
Protocol | activation | inactivation | ||||||||||
t.c./IA | s.c./IK | t.c./IA | s.c./IK | |||||||||
Parameter | V1/2 (mV) | dV (mV/e) | n | V1/2 (mV) | dV (mV/e) | n | V1/2 (mV) | dV (mV/e) | n | V1/2 (mV) | dV (mV/e) | n |
control | −16.7±1.1 | 15.0±1.0 | 36–38 | 10.0±3.6 | 8.2±0.5 | 22–36 | −68.6±0.6 | 23.2±3.1 | 38 | −33.1±3.9 | 52.7±5.2 | 14–35 |
pharma-cological separation | −14.5±1.6 | 18.8±1.5 | 4–13 | 29.2±1.5 | 27.4±1.5 | 9–10 | −62.1±1.7 | 13.6±1.5 | 5–7 | −60.6±2.7 | 24.5±2.7 | 3–7 |
biophysical separation | 9.4±1.5 | 22.1±1.4 | 21–37 | −71.7±0.5 | 7.3±0.4 | 36 | ||||||
control | −15.5±1.9 | 16.6±1.7 | 26–27 | −0.6±1.3 | 12.9±1.2 | 20–27 | −73.2±1.4 | 7.9±1.0 | 27 | −4.9±2.4 | 12.0±1.9 | 9–27 |
biophysical separation | 5.7±1.4 | 11.3±1.2 | 5–25 | −76.7±0.9 | 7.8±0.8 | 13–22 |
Parameters of I–V curves fitted to the Boltzmann distribution with V1/2 being the half maximal activation/inactivation, and dV the slope of the voltage dependency. Control inactivation data of the sustained current (s.c.) best fit with a sum of two Boltzmann equations, and because the first component had values similar to the transient current (t.c., IA), only the values of the more depolarized component, assumed to represent IK, are shown. All data presented as mean±SD.
In proliferating hNPCs half-maximal activation of IK was determined at 10 to 30 mV by fitting activation curves of normalized chord conductances to the Boltzmann distribution. Fitted inactivation curves of current values showed half-maximal inactivation of IA at −60 to −70 mV (
To investigate the development of Kv currents during differentiation, hNPCs were exposed to a differentiation medium (DM) for 14 days prior to the recording (
Potassium outward currents evoked in hNPCs, which were differentiated for 14 days in differentiation medium (DM). (A): Transient (t.c.) and sustained (s.c.) whole-cell Kv currents elicited via activation (i) and inactivation protocol (ii, insets) were measured between 0 and 20 ms and between 280 and 300 ms, respectively, of each depolarizing voltage pulse. Chord conductances and current values were normalized to their peak amplitudes and fitted to a Boltzmann distribution (iii, see
The biophysically separated IK showed similar half-maximal activation (6 mV in DM vs. 91 mV in PM), but lower voltage dependency (11 mV/e-fold in DM vs. 22 mV/efold in PM). Current-voltage relationships of the transient IA were comparable - half-maximal inactivation at −72 mV in DM vs. −77 mV in PM, voltage dependency 8 mV/e-fold in DM vs. 7 mV/e-fold in PM (
Protocol | activation | inactivation | ||||||
IK | IA | |||||||
Parameter | IC50 | IC80 | dc | n | IC50 | IC80 | dc | n |
Inhibitor | ||||||||
0.5±0.1 | - | 2.5±0.1 | 3–9 | 1.7±0.3 | 4.6 | 1.4±0.2 | 7–9 | |
- | - | - | - | 1.8±0.7 | 28.4 | 0.5±0.1 | 3–8 | |
255.6±7.7 | 811.5 | 1.2±0.1 | 6–8 | 35.5±2.4 | 159.6 | 0.9±0.1 | 6–8 | |
3.4±0.3 | 18.3 | 0.8±0.1 | 8–9 | 42.0±10.5 | 531.4 | 0.5±0.1 | 9 | |
163.9±20.9 | 2622.1 | 0.7±0.1 | 14 | - | - | - | - | |
18.4±5.9 | 293.9 | 0.5±0.1 | 6–8 | 48.7±6.0 | 164.0 | 1.1±0.2 | 8 |
Parameters of dose-response relationships fitted with the Hill equation, where IC50 is the half maximal, IC80 the 80 percent inhibitory concentration and dc the Hill coefficient determining the slope of the concentration dependency. All data presented as mean±SD.
Furthermore, in differentiated cells the mean current density of IK was significantly increased (45±6 pA/pF in DM vs. 29±3 pA/pF in PM, n = 23–36), while IA amplitudes decreased (54±12 pA/pF vs. 96±8 pA/pF in PM, n = 22–36;
Kv channel subtypes in proliferating hNPCs as well as in differentiated cells were identified using reverse transcription polymerase chain reaction (RT-PCR) analysis based on mRNA expression. Specific primers for several Kv channel subtypes were designed and tested by means of conventional PCR (see
(A): Identification of Kv channels was performed via reverse transcription PCR analysis in proliferating hNPCs (PM) as well as in differentiated cells (DM) after isolation of total mRNA using specific primers for Kv1–4 subtypes given in
The expression of several Kv channel transcripts was quantified by real-time PCR analysis (
There is a broad spectrum of specific and less specific Kv antagonists
Biophysically separated A-type (IA) and delayed-rectifying (IK) Kv currents in proliferating hNPCs were differentially inhibited by the 4-aminopyridine (4-AP, i), phrixotoxin-1 (PTX, ii), ammonium chloride (NH4Cl, iii), tetraethylammonium chloride (TEA, iv), quinidine (QND, v) and α-dendrotoxin (DTX, vi). (A): Peak amplitudes of IA were measured during a depolarizing voltage step from −130 mV to 0 mV between 0 and 20 ms (inset). (B): IK was determined between 280 and 300 ms of a 100 mV depolarization step following a −40 mV prepulse during the application of different antagonist concentrations (insets). (C): Both current values were normalized to the non-inhibited peak amplitudes. Dose-response relationships were fitted with the Hill equation and IC50 values were determined (see
Furthermore, the classical potassium channel antagonist TEA, which is typically used to block IK, but with moderate specificity, was applied to the cells
Taken together, 4-AP and NH4Cl preferentially and PTX specifically blocked IA, while QND stronger and DTX selectively inhibited IK. TEA acted as a non-specific Kv channel blocker in hNPCs.
We further investigated whether Kv channels play a role in cell survival. Towards this end, we applied various concentrations of the Kv antagonists for 3 days prior to analysis by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which colorimetrically measured the production of MTT formazan in viable cells (
Determination of cell viability in proliferating hNPCs via 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium salt (MTT) assay. (A): Cell viability was measured colorimetrically after 72 h of Kv channel inhibition with different concentrations of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH4Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) and normalized to control values without addition of inhibitor. (B): Viability of hNPCs was significantly reduced by electrophysiologically determined inhibitory doses (IC50/IC80) of 4-AP, PTX and NH4Cl, which specifically blocked IA, as well as by TEA and higher doses of QND, which inhibited both current components (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, ***p<0.001).
A significant reduction of cell viability was observed after blocking IA with electrophysiologically determined inhibitory doses (IC50/IC80) of 4-AP, PTX and NH4Cl as well as after treatment with TEA and QND, which blocked all Kv currents. On the contrary, low doses of QND and DTX, which specifically inhibited IK, did not affect cell viability (
To unravel the contribution of Kv channels to progenitor cell proliferation we performed a BrdU assay after 3 days of potassium channel blockade according to MTT assay. The inhibition of A-type Kv channels by 4-AP, PTX and NH4Cl significantly impaired cell proliferation. Blocking all Kv currents by TEA and QND had similar effects. In contrast, specific inhibition of delayed-rectifier channels by low doses of QND did not affect proliferation, while the application of DTX even increased proliferation of hNPCs (
Proliferation of hNPCs was analyzed via BrdU incorporation assay. (A): Progenitor cell proliferation was measured colorimetrically after 72 h of Kv channel inhibition and normalized to control values without addition of inhibitor. Electrophysiologically determined inhibitory doses (IC50/IC80) of 4-aminopyridine (4-AP), phrixotoxin-1 (PTX), ammonium chloride (NH4Cl), tetraethylammonium chloride (TEA), quinidine (QND) and α-dendrotoxin (DTX) were applied. Progenitor cell proliferation was significantly reduced by inhibition of IA with 4-AP, PTX, NH4Cl as well as by unspecific blockers like TEA and higher doses of QND. In contrast, the IK antagonist DTX increased proliferation of hNPCs (n≥4, 3 tissue preparations; one-way ANOVA, followed by Tukey's post-hoc test, *p<0.05, **p<0.01, ***p<0.001).
Taken together, these results demonstrate a substantial effect of Kv currents on cell survival and proliferation, mainly mediated by IA.
We tested the hypothesis that voltage-gated potassium (Kv) channels play a functional role in the development of human neural progenitor cells (hNPCs).
In whole-cell patch clamp recordings the biophysical separation of two Kv currents, IA and IK, was obtained by different voltage protocols. The transient current IA was determined in steady-state inactivation protocols by a test pulse to 0 mV, because it is activated at slightly more negative potentials than IK. IK was measured as the sustained outward current in activation protocols following a prepulse to −40 mV, which inactivated IA. Two types of delayed rectifier currents have been described previously: IDR and ID. While IDR is slowly activated with a time to peak of 50–100 ms and does not show pronounced steady-state inactivation, the delay current ID is rapidly activated and slowly inactivated
During differentiation IK amplitudes increased, while IA decreased without considerable changes in current-voltage dependencies. An increase in voltage-activated Kv currents during development was observed before in several other cell types, for example in rat retinal ganglion cells
In hNPCs a broad pattern of Kv channel subtypes was detected with almost all Kv1–4 channels being expressed except Kv1.4, 3.2 and 3.3. The A-type channel transcript Kv4.2 showed predominant expression levels and, thus, seems to have a critical impact on the physiological characteristics of immature progenitor cells. However, expression of Kv channel mRNA and electrophysiological or pharmacological Kv properties are quite distinct
Pharmacological investigations revealed different sensitivities of IA and IK to the applied Kv antagonists. PTX selectively blocked Kv4.2 and 4.3
Potassium channel function is assumed to be a key requirement for proper progenitor cell proliferation and also essential for functional neuronal differentiation
Furthermore, by using the snake toxin DTX we were able to selectively block IK. DTX did not cause accelerated cell death, but slightly increased proliferation of hNPCs. If we vice versa disrupted proliferation and induced differentiation, functional delayed-rectifier channels were upregulated. An increase in proliferation was also described in rat midbrain-derived NPCs after selective blockade of the DR channels Kv1.3 and 3.1. Two explanations were described: First, a Ca2+ independent regulation via cell cycle mechanisms. Second, the mediation by a higher open probability of voltage-gated Ca2+ channels in response to the depolarizing effect caused by the Kv channel block and an increase of intracellular Ca2+
In summary, hNPCs generated Kv currents that consist to 90% of A-type currents predominantly produced by Kv4.2 channels. Whereas delayed-rectifying currents mainly generated by Kv1.1 and 1.6 were small. Inhibiting IA function caused a dramatic decrease in proliferation and extensive cell death and, vice versa, disrupting proliferation reduced A-type current formation. These findings emphasize that even A-type potassium channels may play a key role in proliferation and survival of immature progenitor cells. On the other hand, the inhibition of IK was less toxic and in case of DTX even increased progenitor cell proliferation. This is in line with the finding that non-proliferating, differentiating cells upregulated these channels.
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Pharmacological inhibition of Kv currents in hNPCs by MTX. Delayed-rectifying (IK) Kv currents in proliferating hNPCs were inhibited by margatoxin (MTX), while A-type currents (IA) were not affected. (A): Peak amplitudes of IA were measured during a depolarizing voltage step from 130 mV to 0 mV between 0 and 20 ms (inset). (B): IK was determined between 280 and 300 ms of a 100 mV depolarization step following a −40 mV prepulse during the application of different antagonist concentrations (insets). (C): Both current values were normalized for the non-inhibited peak amplitudes. Dose-response relationships were fitted with the Hill equation and following parameters were obtained: IC50 = 180.7±46.9 nM, IC80 = 2.9 µM and a Hill coefficient of 0.5±0.1 (n = 4–8, mean±SD).
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Cell cycle analysis after inhibition of voltage-gated potassium (Kv) channels. Analysis of cell cycle phases in proliferating hNPCs was performed by means of flow cytometry using propidium iodide as an intercalating agent for DNA staining. (A): Cell cycle phases were determined after 72 h of Kv channel inhibition with 100 mM TEA, 2 mM 4-AP, 50 µM QND, 0.5 µM DT\and 0.1 µM MTX and their distribution was calculated by dividing through the total cell number. (B): Cell cycle rates were normalized to controls without addition of an inhibitor. The application of TEA and 4-AP increased cell death about 7 times, while G1/G0, G2/M and S phase were decreased compared to control. QND, DTX and MTX were less toxic (n = 10,000, 4 experiments; one-way ANOVA, followed by Tukey's post-hoc test, *p<0.05, ***p<0.001).
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The authors thank Ute Römuβ for primary cell culturing and Annett Brandt for testing primers (Department of Neurology, University of Leipzig, Germany). Thanks also to Javorina Milosevic (Translational Centre for Regenerative Medicine, University of Leipzig, Germany) for introduction in flow cytometry.