[Ca2+]i elevations detected by BK channels during Ca2+ influx and muscarine-mediated release of Ca2+ from intracellular stores in rat chromaffin cells (original) (raw)
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The Journal of Neuroscience, 1996
Submembrane [Ca 2ϩ ] i changes were examined in rat chromaffin cells by monitoring the activity of an endogenous Ca 2ϩdependent 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.
Journal of neurophysiology, 2000
Large-conductance Ca(2+) and voltage-dependent K(+) channels (BK channels) in many tissues require high Ca(2+) concentrations for activation and therefore might be expected to be tightly coupled to Ca(2+) channels. However, in most cases, little is known about the relative organization of the BK channels and the Ca(2+) channels involved in their activation. We probed the nature of the organization of BK and Ca(2+) channels in rat chromaffin cells by manipulating Ca(2+) influx through Ca(2+) channels and by altering cellular Ca(2+) buffering using EGTA and bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA). The results were analyzed to determine the distance between Ca(2+) and BK channels that would be most consistent with the experimental data. Most BK channels are close enough to Ca(2+) channels to be resistant to the buffering action of millimolar of EGTA, but are far enough to be inhibited by BAPTA. Analysis of the EGTA/BAPTA results suggests that BK channels are at...
Biophysical Journal, 1998
The Ca 2ϩ sensitivity of large conductance Ca 2ϩ -and voltage-activated K ϩ channels (BK V,Ca ) has been determined in situ in freshly isolated myocytes from the guinea pig urinary bladder. In this study, in situ denotes that BK V,Ca channel activity was recorded without removing the channels from the cell. By combining patch clamp recording in the cell-attached configuration and microfluorometry of fura-2, we were able to correlate BK V,Ca channel activity with changes in cytoplasmic intracellular [Ca 2ϩ ] ([Ca 2ϩ ] i ). The latter were induced by ionomycin, an electroneutral Ca 2ϩ ionophore. At 0 mV, the Hill coefficient (n H ) and the [Ca 2ϩ ] i to attain half of the maximal BK V,Ca channel activity (Ca 50 ) were 8 and 1 M, respectively. The data suggest that this large Hill number was not a consequence of the difference between the nearmembrane [Ca 2ϩ ] ([Ca 2ϩ ] s ) and the bulk [Ca 2ϩ ] i , indicated by fura-2. High Hill numbers in the activation by Ca 2ϩ of BK V,Ca channels have been seen by different groups (e.g., filled squares in of Silberberg, S. D., A. Lagrutta, J. P. Adelman, and K. L. . Biophys. J. 70:2640 -2651. However, such high n H has always been considered a peculiarity rather than the rule. This work shows that a high Ca 2ϩ cooperativity is the normal situation for BK V,Ca channels in myocytes from guinea pig urinary bladder. Furthermore, the Ca 50 did not display any significant variation among different channels or cells. It was also evident that BK V,Ca channel activity could decrease in elevated [Ca 2ϩ ] i , either partially or completely. This work implies that the complete activation of BK V,Ca channels occurs with a smaller increment in [Ca 2ϩ ] s than previously expected from in vitro characterization of the Ca 2ϩ sensitivity of these channels. Additionally, it appears that the activity of BK V,Ca channels in situ does not strictly follow changes in near-membrane [Ca 2ϩ ].
Journal of neurophysiology, 1999
BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. Here we characterize the Ca2+ currents expressed in these cells and identify the Ca2+ channel subtypes that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33, 21, 12, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 microM nifedipine or Q-type Ca2+ channels by 2 microM Aga IVA reduced BK current activation by 77 and 42%, respecti...
Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells
Pfl�gers Archiv European Journal of Physiology, 1993
Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca2+]~ measurements were used to characterize Ca2+-activated K + currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 gM) induced a rise of [Ca2+]~ concomitantly with an outward current at holding potentials positive to -80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range -80 mV to -40 mV. Increasing the extracellular K + concentration to 20 mM at the holding potential of -78 mV was associated with inwardly directed currents during the [Ca2+]i elevations induced either by histamine (10 gM) or short voltageclamp depolarizations. The current reversal potential was close to the K + equilibrium potential, being a function of external K + concentration. Current fluctuation analysis suggested a unit conductance of 3-5 pS for the channel that underlies this K + current. The current could be blocked by apamin (1 ~tM). Whole-cell current-clamp recordings showed that histamine (10 txM) application caused a transient hyperpolarization, which evolved in parallel with the [Ca2+]~ changes. It is proposed that a small-conductance Ca2+-activated K + channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.
Large conductance Ca 2-activated K (BK) channel: Activation by Ca 2 and voltage
Biol. Res, 2006
Large conductance Ca 2+ -activated K + (BK) channels belong to the S4 superfamily of K + channels that include voltage-dependent K + (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH 2 -terminus. The BK channel is activated by internal Ca 2+ , and using chimeric channels and mutagenesis, three distinct Ca 2+ -dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca 2+ -binding domains activate the BK channel when cytoplasmic Ca 2+ reaches micromolar concentrations, and a low Ca 2+ affinity mechanism may be involved in the physiological regulation by Mg 2+ . The presence in the BK channel of multiple Ca 2+ -binding sites explains the huge Ca 2+ concentration range (0.1 μM-100 μM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca 2+ . Therefore, Ca 2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.
The Journal of Physiology, 1993
1. To study Ca2"-activated K' currents in dimethyl sulphoxide (DMSO)differentiated HL-60 cells (HL-60 granulocytes), we have combined the patch clamp technique with microfluorimetric measurements of the cytosolic free Ca2+ concentration ([Ca2+]i). 2. Elevations of [Ca2+]i induced by the receptor agonist N-formyl-L-methionyl-Lphenylalanine (f-MLP), by cellular spreading or by the Ca2+ ionophore ionomycin, activated whole-cell currents. The kinetics of the current elevations closely paralleled the kinetics of the elevations in [Ca2+]i. Cellular spreading induced oscillations in [Ca2 ]i and parallel oscillatory changes in the amplitude of the recorded currents. 3. The reversal potential of the Ca2+-activated current was a function of the extracellular K+ concentration (56-1 mV per log [K+]), demonstrating that the underlying conductance was selective for K+. 4. The current was blocked by charybdotoxin, but insensitive to apamin. 5. The whole-cell current was inwardly rectifying. No time-dependent activation or inactivation of the current could be observed within the range of voltages tested (-100 to + 100 mV). 6. The dependence of the current amplitude on the measured [Ca2+]i revealed a half-maximal activation at approximately 350 nm [Ca2+]i, and a highly cooperative activation by [Ca2+]i with an apparent Hill coefficient of approximately 8. Neither the half-maximal activation by [Ca2+]i nor the apparent Hill coefficient depended on the voltage, and they were identical for Ca2+ elevations caused by the ionophore and the receptor agonist. 7. Analysis of Ca2+-activated single-channel events in cell-attached recordings revealed an inwardly rectifying K+ channel with a slope conductance of 35 pS. Fluctuation analysis of the Ca2+-activated whole-cell current suggested an underlying single-channel conductance of a similar size (28 pS).
Pflugers Archiv-european Journal of Physiology
BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca2+ during action potentials. Indeed, Ca2+-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K+ currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca2+) Ca2+ concentrations. Despite their low open probability under these conditions (V50 of +146.8 mV), BK channels were responsible for more than 25% of the total K+ efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K+ channels. The other main source of fast voltage-dependent K+ efflux at such a low Ca2+ was a transient K+ (IA-type) current activating with V 50 = −14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca2+. Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca2+ concentration, and suggest that they could do so also in the presence of extracellular Ca2+, before Ca2+ entering the cell facilitates their activity.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1995
The properties of Ca(2+)- and voltage-dependent K+ currents and their role in defining membrane potential were studied in cultured rat chromaffin cells. Two variants of large-conductance, Ca2+ and voltage-dependent BK channels, one noninactivating and one inactivating, were largely segregated among patches. Whole-cell noninactivating and inactivating currents resulting from each of these channels were segregated among different chromaffin cells. Cell-to-cell variation in the rate and extent of whole-cell current decay was not explained by differences in cytosolic [Ca2+] regulation among cells; rather, variation was due to differences in the intrinsic properties of the underlying BK channels. About 75% of rat chromaffin cells and patches express inactivating BK current (termed BKi) while the remainder express noninactivating BK current (termed BKs). The activation time course of both currents is similar, as is the dependence of activation on [Ca2+] and membrane potential. However, de...
Channelopathy of small- and intermediate-conductance Ca2+-activated K+ channels
Acta Pharmacologica Sinica
Part of the Medical Genetics Commons, and the Medicinal and Pharmaceutical Chemistry Commons Channelopathy of Small-and Intermediate-Conductance Ca Channelopathy of Small-and Intermediate-Conductance Ca 2+ 2+-activated K-activated K + + Channels Channels Comments Comments This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Acta Pharmacologica Sinica in 2022 following peer review. The final publication may differ and is available at Springer via