Dataset Information

Characterization of voltage-gated potassium channels in human neural progenitor cells.

ABSTRACT:

Background

Voltage-gated potassium (K(v)) 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 findings

In proliferating hNPCs a broad spectrum of K(v) channel subtypes was identified using quantitative real-time PCR with a predominant expression of the A-type channel K(v)4.2. In whole-cell patch-clamp recordings K(v) currents were separated into a large transient component characteristic for fast-inactivating A-type potassium channels (I(A)) and a small, sustained component produced by delayed-rectifying channels (I(K)). During differentiation the expression of I(A) as well as A-type channel transcripts dramatically decreased, while I(K) producing delayed-rectifiers were upregulated. Both K(v) currents were differentially inhibited by selective neurotoxins like phrixotoxin-1 and alpha-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 alpha-dendrotoxin increased proliferation.

Conclusions/significance

These findings suggest that A-type potassium currents are essential for proper proliferation of immature multipotent hNPCs.

SUBMITTER: Schaarschmidt G

PROVIDER: S-EPMC2702754 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Publications

Characterization of voltage-gated potassium channels in human neural progenitor cells.

Schaarschmidt Grit G Wegner Florian F Schwarz Sigrid C SC Schmidt Hartmut H Schwarz Johannes J

PloS one 20090708 7

<h4>Background</h4>Voltage-gated potassium (K(v)) 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.<h4>Methodology/principal findings</h4>In proliferating hNPCs a broad spectrum of K(v) channel subtypes was identified using quantitative real-time PCR with a predominant expression of t ...[more]

PMID: 19584922

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Project description:Voltage-gated ion channels play a central role in the generation of action potentials in the nervous system. They are selective for one type of ion - sodium, calcium, or potassium. Voltage-gated ion channels are composed of a central pore that allows ions to pass through the membrane and four peripheral voltage sensing domains that respond to changes in the membrane potential. Upon depolarization, voltage sensors in voltage-gated potassium channels (Kv) undergo conformational changes driven by positive charges in the S4 segment and aided by pairwise electrostatic interactions with the surrounding voltage sensor. Structure-function relations of Kv channels have been investigated in detail, and the resulting models on the movement of the voltage sensors now converge to a consensus; the S4 segment undergoes a combined movement of rotation, tilt, and vertical displacement in order to bring 3-4e(+) each through the electric field focused in this region. Nevertheless, the mechanism by which the voltage sensor movement leads to pore opening, the electromechanical coupling, is still not fully understood. Thus, recently, electromechanical coupling in different Kv channels has been investigated with a multitude of techniques including electrophysiology, 3D crystal structures, fluorescence spectroscopy, and molecular dynamics simulations. Evidently, the S4-S5 linker, the covalent link between the voltage sensor and pore, plays a crucial role. The linker transfers the energy from the voltage sensor movement to the pore domain via an interaction with the S6 C-termini, which are pulled open during gating. In addition, other contact regions have been proposed. This review aims to provide (i) an in-depth comparison of the molecular mechanisms of electromechanical coupling in different Kv channels; (ii) insight as to how the voltage sensor and pore domain influence one another; and (iii) theoretical predictions on the movement of the cytosolic face of the Kv channels during gating.

Project description:Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) regulates activities of numerous ion channels including inwardly rectifying potassium (K(ir)) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K(V)) channels might be regulated by PI(4,5)P(2). Wide expression of K(V) channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K(V) channels by PI(4,5)P(2), we have coexpressed several of them in tsA-201 cells with a G protein-coupled receptor (M(1)R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P(2) with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P(2) at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K(V)7.1, K(V)7.2/7.3, and K(ir)2.1 channel current by 90-95%. Activation of M(1)R inhibited K(V)7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P(2) regulation of activity of K(V)1.1/K(V)β1.1, K(V)1.3, K(V)1.4, and K(V)1.5/K(V)β1.3, K(V)2.1, K(V)3.4, K(V)4.2, K(V)4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K(V)1.1/K(V)β1.1 and K(V)3.4, resulting in up-regulation of current density upon activation of M(1)R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P(2) at the plasma membrane by enzymes does not seem to influence activity of most tested K(V) channels, whereas it does strongly inhibit members of the K(V)7 and K(ir) families.

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Dataset Information

Characterization of voltage-gated potassium channels in human neural progenitor cells.

Background

Methodology/principal findings

Conclusions/significance

Publications

Characterization of voltage-gated potassium channels in human neural progenitor cells.

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