Abstract
BackgroundVoltage-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.Methodology/Principal FindingsIn 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.Conclusions/SignificanceThese findings suggest that A-type potassium currents are essential for proper proliferation of immature multipotent hNPCs.
Highlights
Human neural progenitor cells isolated from fetal brain tissue are considered a promising source for cell replacement therapies in neurodegenerative disorders [1]
Expansion of human neural progenitor cells (hNPCs) was performed in serumfree proliferation medium (PM) based on Dulbecco’s modified Eagle medium and Ham’s F12 containing the supplements N2 or B27 [2,42,43,44], the antibiotics Penicillin/Streptomycin, the mitogens epidermal growth factor (EGF) and fibroblast growth factor (FGF2; 20 ng/ml each; both from PAN Biotech GmbH, Aidenbach, Germany)
Voltage-gated potassium currents in proliferating hNPCs To characterize the voltage-dependency of voltage-gated potassium (Kv) currents in proliferating human neural progenitor cells outward currents were elicited in whole-cell voltageclamp recordings either in activation protocols or steady-state inactivation protocols (Fig. 1A)
Summary
Human neural progenitor cells (hNPCs) isolated from fetal brain tissue are considered a promising source for cell replacement therapies in neurodegenerative disorders [1] They bear an immense potential to proliferate and represent an appropriate in vitro model for investigating mechanisms of early human brain development [2] including ion channel function. 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
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