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

Abstract

Submembrane [Ca 2ϩ ] i changes were examined in rat chromaffin cells by monitoring the activity of an endogenous Ca 2ϩ -dependent protein: the large conductance Ca 2ϩ -and voltageactivated K ϩ channel (also known as the BK channel). The Ca 2ϩ and voltage dependence of BK current inactivation and conductance were calibrated first by using defined [Ca 2ϩ ] i salines. This information was used to examine submembrane [Ca 2ϩ ] i elevations arising out of Ca 2ϩ influx and muscarine-mediated release of Ca 2ϩ from intracellular stores. During Ca 2ϩ influx, some BK channels are exposed to [Ca 2ϩ ] i of at least 60 M. However, the distribution of this [Ca 2ϩ ] i elevation is highly nonuniform so that the average [Ca 2ϩ ] i detected when all BK channels are activated is only ϳ10 M. Intracellular dialysis with 1 mM or higher EGTA spares only the BK channels activated by the highest [Ca 2ϩ ] i during influx, whereas dialysis with 1 mM or higher BAPTA blocks activation of all BK channels. Submembrane [Ca 2ϩ ] i elevations fall rapidly after termination of short (5 msec) Ca 2ϩ influx steps but persist above 1 M for several hundred milliseconds after termination of long (200 msec) influx steps. In contrast to influx, the submembrane [Ca 2ϩ ] i elevations produced by release of intracellular Ca 2ϩ by muscarinic actetylcholine receptor (mAChR) activation are much more uniform and reach peak levels of 3-5 M. Our results suggest that during normal action potential activity only 10 -20% of BK channels in each chromaffin cell see sufficient [Ca 2ϩ ] i to be activated.

Ca2+‐Dependent K+ Currents Induced by Muscarinic Receptor Activation in Guinea Pig Adrenal Chromaffin Cells

“…Therefore, it is likely that the spatial Ca2~]gradient is formed from internal Ca2~stores to the plasma membrane by the muscarine-induced Ca2r elease, and vice versa by the depolarization-induced Ca2~influx. An estimation of submembrane [Ca2~]m ade by monitoring the K~channel activation suggests that the increment of submembrane [Ca2~]~induced by Ca2~entry is higher than that by Ca2~release in rat chromaffin cells (Prakriya et al, 1996). This idea was supported by the present finding that the increases in the K~current evoked by the Ca2~release from the stores were much smaller than those evoked by the Ca2~influx, although the~Ca2~], increase was at almost the same level regardless of the Ca2~source.…”

“…It is therefore of interest to examine whether muscarine activates Ca 2~-dependentK~channels by releasing Ca2~from internal stores in guinea pig chromaffin cells. Ca2~-dependent K~currents are used as a Ca 2~indicator for monitoring just beneath the plasma membrane (Prakriya et al, 1996). The [Ca2~] 1monitored with fura-2 is considered to reflect the average or mean cytosolic Ca 2~concentration in the cells .…”

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

“…Following the same approach and using a double-pulse protocol to quantify the amount and type of BK currents, Chris Lingle and coworkers could formulate a quite realistic picture of how L-type and BK channels are coupled in RCCs. 61,62 The experimental data are consistent with a model in which BK channels are located between 160 and 50 nm from the Ca 2+ channels that fuel them. 63 More precisely, 30 to 40% of fast inactivating BK channels in RCCs are insensitive to EGTA buffers and are therefore positioned sufficiently close to LTCCs (between 50 and 160 nm) to be influenced by the Ca 2+ influx through these channels during brief depolarization steps.…”

Molecular Basis for the Inactivation of Ca2+- and Voltage-Dependent BK Channels in Adrenal Chromaffin Cells and Rat Insulinoma Tumor Cells

“…Specifically, the voltage of halfactivation at 10 M Ca 2ϩ for Slo␤3 channels is approximately Ϫ20 to Ϫ30 mV. This is negative to that reported for the activation of BK i currents in native chromaffin cells by 10 M Ca 2ϩ , which was ϳ3 mV (Prakriya et al, 1996). Although a difference in methodology might account for these differences, two other factors may contribute to this difference.…”

“…In chromaffin cells this value is in the range of 25-40 msec (Solaro and Lingle, 1992;Prakriya et al, 1996). The variability in the chromaffin cells probably arises from the possibility that the BK Figure 5.…”