Ca2+ dependence of small Ca(2+)-activated K+ channels in cultured N1E-115 mouse neuroblastoma cells

Abstract

Single-channel properties of Ca(2+)-activated K+ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K+ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 microM internal Ca2+. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Open-time distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca2+]i, whereas the mean long open time is independent of [Ca2+]i. At low [Ca2+]i the short-lived open state predominates. At saturating [Ca2+]i the number of long-lived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of -16 to +40 mV. The results indicate that activation of K+ current through SK channels is mainly determined by the Ca(2+)-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5-21 microM internal Ca2+. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca2+]i, the Ca2+ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca2+ dependence suggest that BK channels are activated by a local high [Ca2+]i associated with Ca2+ influx, whereas SK channels may be activated by Ca2+ released from internal stores as well.