Characterization of ionic currents in human neural stem cells

Abstract

The profile of membrane currents was investigated in differentiated neuronal cells derived from human neural stem cells (hNSCs) that were obtained from aborted fetal cortex. Whole-cell voltage clamp recording revealed at least 4 different currents: a tetrodotoxin (TTX)-sensitive Na(+) current, a hyperpolarization-activated inward current, and A-type and delayed rectifier-type K(+) outward currents. Both types of K(+) outward currents were blocked by either 5 mM tetraethylammonium (TEA) or 5 mM 4-aminopyridine (4-AP). The hyperpolarization-activated current resembled the classical K(+) inward current in that it exhibited a voltage-dependent block in the presence of external Ba(2+) (30microM) or Cs(+) (3microM). However, the reversal potentials did not match well with the predicted K(+) equilibrium potentials, suggesting that it was not a classical K(+) inward rectifier current. The other Na(+) inward current resembled the classical Na(+) current observed in pharmacological studies. The expression of these channels may contribute to generation and repolarization of action potential and might be regarded as functional markers for hNSCs-derived neurons.

Keywords: A-type; Human neural stem cells; TTX-sensitive Na+ current; delayed rectifier; hyperpolarization-activated inward current.

Fig. 1

Immunocytochemistry of differentiated human neural stem cells. Cultured hNSCs were differentiated into neural lineage cells by removing the growth factors from the culture medium. 9 days after neural induction, the cells were immunostained with (D~F) anti-β-tubulin III antibody (Tuj-1), and (G~I) anti-GFAP antibody. Immunostained cells were counterstained with DAPI. Phase contrast images of differentiated hNSCs at 100X, 200X, and 400X magnification are shown in (A~C).

Fig. 2

Voltage-dependent Na⁺ current in hNSCs. Membrane depolarization from -90 to 40 mV evoked inward currents (A), which were not observed when external Na⁺ was replaced with an equimolar concentration of NMG (B). The I~V relationships of (A) and (B) are shown in (C). Inward currents elicited by the same protocol are blocked completely by 500 nM TTX (D, E). The I~V relationships of (D) and (E) are shown in (F). Hollow circles indicate the amplitudes of peak inward currents of given commanding potentials in the control, and filled circles indicate the current amplitudes remaining after NMG substitution (C) or TTX application (F).

Fig. 3

Voltage-dependent K⁺ currents in hNSCs. Depolarizing pulses from -90 to 40 mV elicited outward K⁺ currents (A, B, D, E). The symbols above the currents traces indicate the measuring points. Hollow symbols are used in the control current traces, and filled symbols are used in the current traces remaining after the drug applications. I~V relationships before and after the drug applications are shown in (C, F). Hollow circles and squares in (C, F) indicate A-type and delayed rectifier type current amplitudes of each commanding potentials before drug application, while the filled symbols in (C, F) indicate amplitudes of A-type and delayed rectifier type currents after drug application.

Fig. 4

Hyperpolarization-activated inward currents in hNSCs. (A~C) Hyperpolarizing pulses from 0 to -130 mV elicited inward currents, which exhibited increases in amplitude with increases in the external concentration of K⁺ ([K⁺]_ext). The I~V relationships of hyperpolarization-activated inward currents on external K⁺ concentrations are shown in (D : hollow circle - control, filled circle -30 mM [K⁺]_ext, filled triangle -60 mM [K⁺]_ext). The cesium (Cs) or barium (Ba) reversibly blocked the hyperpolarization-activated inward currents (E, F: hollow circle - control, filled circle - Cs or Ba, filled triangle - wash out).