Single channel study of a Ca(2+)‐activated K+ current associated with ras‐induced cell transformation.

Abstract

1. Ras-transformed fibroblasts have a whole-cell Ca(2+)-activated K+ current which is either absent or unavailable for activation in their non-transformed counterparts. To better understand the physiological significance of this K+ current the single channel basis for the current was characterized in ras-transformed cells. 2. More than 90% of inside-out patches from ras-transformed balb 3T3 cells had a channel type which was Ca(2+)-activated (threshold < 0.2 microM internal Ca2+), K(+)-selective (permeability ratio PNa:PK < 0.02), and inwardly rectifying in symmetric 150 mM KCl solutions (conductances at -60 and 60 mV of 33 +/- 1 and 17 +/- 1 pS respectively). Channel opening probability increased 25-50% between -60 and 60 mV due to an increase in the frequency of opening. Single K+ channels in outside-out patches were blocked by externally applied 10 mM TEA or 100 nM charybdotoxin, as were whole-cell Ca(2+)-activated K+ currents. The properties of this class of K+ channel are sufficient to account for the whole-cell Ca(2+)-activated current in ras-transformed cells. 3. Inside-out patches from C3H10T1/2 and NIH 3T3 fibroblasts transformed by the H-ras oncogene had Ca(2+)-activated K+ channels identical to those observed in K-ras-transformed balb 3T3 cells. 4. As predicted from whole-cell experiments Ca(2+)-activated K+ channels were not observed in inside-out patches from non-transformed balb 3T3 cells. The purpose of the excised patch recordings was, instead, to rule out potential technical complications with the whole-cell experiments. For instance A23187, which evoked whole-cell K+ currents in transformed cells, may not have elevated Ca2+ sufficiently to allow K+ channel activation in non-transformed cells. Another possibility was that trypsin pretreatment used to round-up cells for whole-cell recording may have preferentially disabled channels in non-transformed cells. The first problem was addressed by exposing patches from non-transformed cells to 100-1000 microM Ca2+. Excised patches were also taken from non-transformed cells which had not been exposed to trypsin. K+ channel activity was not observed under either condition. 5. Patches from both ras-transformed and non-transformed cells had a type of non-specific cation channel which was activated at internal Ca2+ concentrations > or = 100 microM. This channel was sensitive to membrane voltage, mean open time increasing from 12 to 72 ms between -90 and 90 mV.