Monitoring calcium in turtle hair cells with a calcium-activated potassium channel

1. An apamin-sensitive Ca2+-activated K+ channel was characterized in turtle hair cells and utilized to monitor submembranous intracellular Ca2+ and to evaluate the concentration of the mobile endogeneous calcium buffer. 2. Isolated hair cells were voltage clamped with whole-cell patch electrodes filled with a Cs+-based intracellular solution to block the large-conductance Ca2+-activated K+ (BK) channel. Ca2+ currents evoked by depolarization were followed by inward tail currents lasting several hundred milliseconds. Both the Ca2+ current and slow tail current were abolished by nifedipine. 3. The tail current was carried by K+ and Cs+ (relative permeabilities P-Cs/P-K = 0.22), and was fully blocked by 0.1 mu M apamin and half blocked by 5 mM external TEA. These properties suggest the tail current flows through a Ca2+-activated K+ channel distinct from the BK channels. 4. Intracellular Ca2+ was imaged with a confocal microscope in hair cells filled with the indicator Calcium Green-5N introduced via the patch pipette. Increases in Ca2+ evoked by depolarization were localized to hotspots on the basolateral surface of the cell. The time course of the tail current closely matched the fast component of the fluorescence monitored at a hotspot. 5. Ca2(+)-ATPase pump inhibitors thapsigargin, 2,4-di-(t-butyl)hydroquinone (BHQ) and vanadate, which are known to influence calcium regulation in turtle hair cells, prolonged the time course of the tail current, supporting the idea that the channel monitors cytoplasmic Ca2+. 6. The mobile endogenous buffer was estimated by combining perforated-patch and whole-cell recordings on a single cell. After recording tail currents with an amphotericin-perforated patch, the patch was ruptured to obtain the whole-cell mode, thus allowing washout of soluble cytoplasmic proteins and exchange with pipette buffers. By varying the concentration of Ca2+ buffer in the pipette, the mobile endogenous buffer was found to be equivalent to about 1 mM BAPTA.