[Ca2+]iElevations Detected by BK Channels during Ca2+Influx and Muscarine-Mediated Release of Ca2+from Intracellular Stores in Rat Chromaffin Cells

Submembrane [Ca2+]ichanges 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 BK current inactivation and conductance were calibrated first by using defined [Ca2+]isalines. This information was used to examine submembrane [Ca2+]ielevations arising out of Ca2+influx and muscarine-mediated release of Ca2+from intracellular stores. During Ca2+influx, some BK channels are exposed to [Ca2+]iof at least 60 μm. However, the distribution of this [Ca2+]ielevation is highly nonuniform so that the average [Ca2+]idetected when all BK channels are activated is only ∼10 μm. Intracellular dialysis with 1 mmor higher EGTA spares only the BK channels activated by the highest [Ca2+]iduring influx, whereas dialysis with 1 mmor higher BAPTA blocks activation of all BK channels. Submembrane [Ca2+]ielevations fall rapidly after termination of short (5 msec) Ca2+influx steps but persist above 1 μmfor several hundred milliseconds after termination of long (200 msec) influx steps. In contrast to influx, the submembrane [Ca2+]ielevations 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+]ito be activated.