Gamma-Dendrotoxin blocks large conductance Ca 2+-activated K + channels in neuroblastoma cells

Abstract

In N1E 115 neuroblastoma cells, γ-dendrotoxin (DTX, 200 nM) blocked the outward K + current by 31.1 ± 3.5% (n = 4) with ∼500 nM Ca 2+ in the pipet solution, but had no effect on the outward K + current when internal Ca 2+ was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM γ-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca 2+ concentration was ∼500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and γ-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM γ-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. γ-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC 50 of 8.5 nM. In GH 3 cells γ-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K + currents. These results suggest that γ-DTX blocks a large conductance Ca 2+ activated K + current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca 2+ activated K + channels.