[Ca²⁺](i) elevations detected by BK channels during Ca²⁺ influx and muscarine-mediated release of Ca²⁺ from intracellular stores in rat chromaffin cells

Submembrane [Ca2²⁺](i) changes were examined in rat chromaffin cells by monitoring the activity of an endogenous Ca²⁺dependent protein: the large conductance Ca²⁺- and voltage-activated K⁺ channel (also known as the BK channel). The Ca²⁺ and voltage dependence of BE current inactivation and conductance were calibrated first by using defined [Ca²⁺](i) salines. This information was used to examine submembrane [Ca²⁺](i) elevations arising out of Ca²⁺ influx and muscarine-mediated release of Ca²⁺ from intracellular stores. During Ca²⁺ influx, some BK channels are exposed to [Ca²⁺](i) of at least 60 μM. However, the distribution of this [Ca²⁺](i) elevation is highly nonuniform so that the average [Ca²⁺](i) detected when all BK channels are activated is only ~10 μM. Intracellular dialysis with 1 mM or higher EGTA spares only the BK channels activated by the highest [Ca²⁺](i) during influx, whereas dialysis with 1 mM or higher BAPTA blocks activation of all BK channels. Submembrane [Ca²⁺](i) elevations fall rapidly after termination of short (5 msec) Ca²⁺ influx steps but persist above 1 μM for several hundred milliseconds after termination of long (200 msec) influx steps. In contrast to influx, the submembrane [Ca²⁺](i) elevations produced by release of intracellular Ca²⁺ by muscarinic actetylcholine receptor (mAChR) activation are much more uniform and reach peak levels of 3-5 μM. Our results suggest that during normal action potential activity only 10-20% of BK channels in each chromaffin cell see sufficient [Ca²⁺](i) to be activated.