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

Single-channel properties of Ca²⁺-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 M internal Ca²⁺. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime 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 longlived 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 Ca²⁺-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 M 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.