Voltage‐gated K⁺ channels play a role in cAMP‐stimulated neuritogenesis in mouse neuroblastoma N2A cells

Abstract

Neuritogenesis is essential in establishing the neuronal circuitry. An important intracellular signal causing neuritogenesis is cAMP. In this report, we showed that an increase in intracellular cAMP stimulated neuritogenesis in neuroblastoma N2A cells via a PKA-dependent pathway. Two voltage-gated K(+) (Kv) channel blockers, 4-aminopyridine (4-AP) and tetraethylammonium (TEA), inhibited cAMP-stimulated neuritogenesis in N2A cells in a concentration-dependent manner that remarkably matched their ability to inhibit Kv currents in these cells. Consistently, siRNA knock down of Kv1.1, Kv1.4, and Kv2.1 expression reduced Kv currents and inhibited cAMP-stimulated neuritogenesis. Kv1.1, Kv1.4, and Kv2.1 channels were expressed in the cell bodies and neurites as shown by immunohistochemistry. Microfluorimetric imaging of intracellular [K(+)] demonstrated that [K(+)] in neurites was lower than that in the cell body. We also showed that cAMP-stimulated neuritogenesis may not involve voltage-gated Ca(2+) or Na(+) channels. Taken together, the results suggest a role of Kv channels and enhanced K(+) efflux in cAMP/PKA-stimulated neuritogenesis in N2A cells.

Inhibition of voltage-gated K+ channels and Ca2+ channels by diphenidol

“…We could not detect VGCC currents in N2A cells by patch clamp recording [8]. Consistently, microfluorimetric measurements using fura-2 demonstrated very small KCl depolarizationevoked [Ca 2+ ] i responses in both undifferentiated and differentiated N2A cells, indicating very low densities of VGCC [7]. The absence of VGCC currents in N2A cells may be due to the fact that the very small VGCC currents run down rapidly.…”

“…In the present work we examined if diphenidol could also affect Kv channels in N2A cells. Kv channels of N2A cells are of the delayed rectifier type [3], characteristically sensitive to tetraethylammonium and 4-aminopyridine, and composed mainly of subfamily members Kv1.1, Kv1.4 and Kv2.1 [7]. Figure 1 shows that diphenidol inhibited Kv channels in a concentration-dependent manner, with IC 50 of 28.2 µM.…”

“…The absence of VGCC currents in N2A cells may be due to the fact that the very small VGCC currents run down rapidly. The limitation of the N2A cell model prompted us to employ the NG108-15 cell model, which after differentiation, would exhibit larger [Ca 2+ ] i rises in response to KCl-elicited depolarization [7]. Nonetheless, electrophysiological recording of differentiated NG108-15 cells still detected very small VGCC currents, which might have run down very rapidly.…”

“…Electrophysiological experiments were performed as previously reported [7]. N2A cells were voltageclamped in the whole-cell configuration.…”

“…Microfluorimetric measurement of cytosolic Ca 2+ concentration ([Ca 2+ ] i ) was performed using fura-2 AM (Invitrogen, Carlsbad, CA, USA) as a Ca 2+ -sensitive fluorescent dye [7]. Briefly, differentiated NG108-15 cells or RBA-1 astrocytes were incubated with 5 µM fura-2 AM for 1 h at 37°C and then washed and bathed in the bath solution mentioned above.…”