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The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K+ channels is enhanced by cytosolic calcium (Ca2+). Activation is sensitive to a range of [Ca2+] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 α subunits at [Ca2+] and [Mg2+] up to 100 mM. The half-activation voltage (V0.5) is steeply dependent on [Ca2+] in the micromolar range, but shows a tendency towards saturation over the range of 60–300 μM Ca2+. As [Ca2+] is increased to millimolar levels, the V0.5 is strongly shifted again to more negative potentials. When channels are activated by 300 μM Ca2+, further addition of either mM Ca2+ or mM Mg2+ produces similar negative shifts in steady-state activation. Millimolar Mg2+ also produces shifts of similar magnitude in the complete absence of Ca2+. The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca2+] increase activation rates, addition of 10 mM Mg2+ to 0 Ca2+ produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg2+ does not participate in Ca2+-dependent steps that influence current activation rate. We conclude that millimolar Mg2+ and Ca2+ concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca2+-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca2+ binding steps, four voltage sensors, and four independent lower affinity Ca2+/Mg2+ binding steps describes well the behavior of G-V curves over a range of Ca2+ and Mg2+. The ability of a broad range of [Ca2+] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.

Most calcium-activated potassium channels couple changes in intracellular calcium to membrane excitability by conducting a current with a probability that depends directly on submembrane calcium concentration. In rat adrenal chromaffin cells, however, a large conductance, voltage- and calcium-activated potassium channel (BK) undergoes rapid inactivation, suggesting that this channel has a physiological role different than that of other BK channels. The inactivation of the BK channel, like that of the voltage-gated Shaker B potassium channel, is removed by trypsin digestion and channels are blocked by the Shaker B amino-terminal inactivating domain. Thus, this BK channel shares functional and possibly structural homologies with other inactivating voltage-gated potassium channels.

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.

ROMK channels are responsible for K؉ secretion in kidney. The activity of ROMK is regulated by intracellular pH (pH i ) with acidification causing channel closure (effective pK a ϳ6.9). Recently, we and others reported that a direct interaction of the channels with phosphatidyl-4,5-bisphosphate (PIP 2 ) is critical for opening of the inwardly rectifying K ؉ channels. Here, we investigate the relationship between the mechanisms for regulation of ROMK by PIP 2 and by pH i . We find that disruption of PIP 2 -ROMK1 interaction not only decreases single-channel open probability (P o ) but gives rise to a ROMK1 subconductance state. This state has an increased sensitivity to intracellular protons (effective pK a shifted to pH ϳ7.8), such that the subconductance channels are relatively quiescent at physiological pH i . Open probability for the subconductance channels can then be increased by intracellular alkalinization to supra-physiological pH. This increase in P o for the subconductance channels by alkalinization is not associated with an increase in PIP 2 -channel interaction. Thus, direct interaction with PIP 2 is critical for ROMK1 to open at full conductance. Disruption of this interaction increases pH i sensitivity for the channels via emergence of the subconductance state. The control of open probability of ROMK1 by pH i occurs via a mechanism distinct from the regulation by PIP 2 .Potassium channels play important roles in the regulation of potassium transport in kidney (1). Recently, cDNAs for the renal K ϩ channels and splice isoforms, ROMK1, -2, and -3, have been isolated (2-4). ROMKs belong to a large family of inward rectifier K ϩ channels, which also includes the strongly rectifying IRK1, the G protein-gated GIRK1, and the pancreatic ␤-cell inward rectifier (5). These cDNAs encode polypeptides of ϳ300 -500 amino acids, which share ϳ40% or more amino acid identity and have the common structure of a cytoplasmic N terminus, two hydrophobic segments that span the membrane as ␣-helices, one pore-forming partial membranespanning region, and a long cytoplasmic C terminus.Opening of the G protein-gated GIRK channels requires G protein ␤␥ subunits. Other inward rectifier K ϩ channels, such as ROMK1 and IRK1, are constitutively open (5). Inward rectifier K ϩ channels run down (close) when inside-out membrane patches are excised into ATP-free, Mg 2ϩ -containing solution. We and others (6 -9) recently found that direct interaction of inward rectifier channels with the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 is critical for channel opening. Reduction of membrane PIP 2 via activation of the Mg 2ϩ -dependent lipid phosphatases causes channel run-down. Direct application of PIP 2 -containing liposomes to membrane patches reactivates run-down channels (6 -9). Furthermore, PIP 2 is important for regulation of the G protein-gated channels by G␤␥ and by intracellular Na ϩ (8 -11) and modulates ATP sensitivity of K ATP channels (12, 13).ROMK1 channels are also regulated by cAMP-dependent protein kina...

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Most BK-type voltage- and Ca(2+)-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore-blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 microM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.

1. The mechanism by which muscarine, ionomycin or caffeine results in suppression of Caand voltage-dependent outward current in rat adrenal chromaffin cells was evaluated using both whole-cell voltage clamp and single channel recording. 2. The whole-cell current activated following the elevation of the cytosolic calcium concentration ([Ca2+]i) by muscarine inactivates with a time course comparable to that of single Ca2+-and voltage-dependent potassium (BK) channels.3. The whole-cell inactivating current is pharmacologically similar to BK current. 4. The voltage dependence of inactivation and rate of recovery from inactivation are qualitatively similar for both whole-cell current and ensemble averages of single BK channels. Furthermore, changes in the rate of whole-cell current inactivation track expected changes in submembrane [Ca2+]. 5. The suppression of outward current can be accounted for solely by inactivation of BK channels and does not depend on the means by which [Ca2+]i is elevated.6. Muscarinic acetylcholine receptor (mAChR) activation, changes in holding potential (-50 to -20 mV), and step depolarizations of different amplitude and duration were tested for their ability to elevate [Ca2+]i and thereby regulate the availability of BK current for activation. 7. Following muscarine-induced elevation of [Ca2+]i at holding potentials positive to -40 mV, the availability of BK current for activation was typically reduced by more than 50 %. 8. Holding potentials in the range of -50 to -20 mV produced only slight alterations in the availability of BK current for activation. 9.Step depolarizations that cause maximal rates of Ca2+ influx (0 to +10 mV) must exceed 200 ms to reduce the availability of BK current by approximately 50 %.10. The results show that the muscarine-induced elevation of [Ca2+]i produces a profound reduction in the availability of BK channels for activation at membrane potentials likely to be physiologically meaningful. Although depolarization-induced Ca2P influx can inactivate BK current, we propose that short duration depolarizations that occur during normal electrical activity will not significantly alter BK channel availability.