Membrane-delimited inhibition of maxi-K channel activity by the intermediate conductance Ca2+-activated K channel

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.     

Activation of Ca2+-activated K+ (maxi-K+) channel by angiotensin II in myocytes of the guinea pig ileum

We investigatedthe regulation of theCa²⁺-activatedK⁺(maxi-K⁺) channel by angiotensinII (ANG II) and its synthetic analog, [Lys²]ANG II, infreshly dispersed intestinal myocytes. We identified amaxi-K⁺ channel population in theinside-out patch configuration on the basis of its conductance (257 ± 4 pS in symmetrical 150 mM KCl solution), voltage andCa²⁺ dependence of channelopening, lowNa⁺-to-K⁺andCl-to-K⁺permeability ratios, and blockade by externalCs⁺ and tetraethylammoniumchloride. ANG II and[Lys²]ANG II caused anindirect, reversible, Ca²⁺- anddose-dependent activation ofmaxi-K⁺ channels in cell-attachedexperiments when cells were bathed inhigh-K⁺ solution. This effect wasreversibly blocked by DUP-753, being that it is mediated by theAT₁ receptor.Evidences that activation of themaxi-K⁺ channel by ANG II requiresa rise in intracellular Ca²⁺concentration([Ca²⁺]_i)as an intermediate step were the shift of the open probability of thechannel-membrane potential relationship to less positive membranepotentials and the sustained increase in[Ca²⁺]_iin fura 2-loaded myocytes. The preservation of the pharmacomechanical coupling of ANG II in these cells provides a good model for the studyof transmembrane signaling responses to ANG II and analogs in thistissue.

     

A lineage-specific Ca(2+)-activated K+ conductance in HL-60 cells.

Cells of the human promyelocytic cell line HL-60 can be controllably induced to terminally differentiate into either granulocytes or monocyte/macrophages. HL-60 promyelocytes and terminally differentiated macrophages express a K(+)-selective ion channel which is activated by intracellular free Ca2+ concentrations above 10(-7) M. Because of its voltage independence, this channel can be distinguished from the voltage- and Ca(2+)-activated family of outward-rectifying channels. The channel is selective for K+ against Na+ and is blocked by Ba2+, thus it may be similar to the Ca(2+)-activated K+ channel previously described in human macrophages. In its sensitivity to block by charybdotoxin, this channel also resembles a Ca(2+)-activated K+ channel of lymphocytes, which plays a role in activation-dependent hyperpolarization. In contrast to promyelocytes and macrophages, functional expression of the Ca(2+)-activated K+ channel is suppressed to nearly undetectable levels in granulocytes derived from HL-60 cells by retinoic acid-induced differentiation. These data suggest that signals which produce elevation of intracellular Ca2+ will hyperpolarize promyelocytes and differentiated macrophages by activating this conductance; however, signals which elevate free Ca2+ in granulocytes must act on other effectors, which may produce a different final influence on membrane potential.     

Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.     

Ion effects on gating of the Ca(2+)-activated K+ channel correlate with occupancy of the pore.

We studied the effects of permeant ions on the gating of the large conductance Ca(2+)-activated K+ channel from rat skeletal muscle. Rb+ blockade of inward K+ current caused an increase in the open probability as though Rb+ occupancy of the pore interferes with channel closing. In support of this hypothesis, we directly measured the occupancy of the pore by the impermeant ion Cs+ and found that it strongly correlates with its effect on gating. This is consistent with the "foot-in-the-door" model of gating, which states that channels cannot close with an ion in the pore. However, because Rb+ and Cs+ not only slow the closing rate (as predicted by the model), but also speed the opening rate, our results are more consistent with a modified version of the model in which the channel can indeed close while occupied, but the occupancy destabilizes the closed state. Increasing the occupancy of the pore by the addition of other permeant (K+ and Tl+) and impermeant (tetraethylammonium) ions did not affect the open probability. To account for this disparity, we used a two-site permeation model in which only one of the sites influenced gating. Occupancy of this "gating site" interferes with channel closing and hastens opening. Ions that directly or indirectly increase the occupancy of this site will increase the open probability.     

Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel.

Iberiotoxin, a toxin purified from the scorpion Buthus tamulus is a 37 amino acid peptide having 68% homology with charybdotoxin. Charybdotoxin blocks large conductance Ca(2+)-activated K+ channels at nanomolar concentrations from the external side only (Miller, C., E. Moczydlowski, R. Latorre, and M. Phillips. 1985. Nature (Lond.). 313:316-318). Like charybdotoxin, iberiotoxin is only able to block the skeletal muscle membrane Ca(2+)-activated K+ channel incorporated into neutral-planar bilayers when applied to the external side. In the presence of iberiotoxin, channel activity is interrupted by quiescent periods that can last for several minutes. From single-channel records it was possible to determine that iberiotoxin binds to Ca(2+)-activate K+ channel in a bimolecular reaction. When the solution bathing the membrane are 300 mM K+ internal and 300 mM Na+ external the toxin second order association rate constant is 3.3 x 10(6) s-1 M-1 and the first order dissociation rate constant is 3.8 x 10(-3) s-1, yielding an apparent equilibrium dissociation constant of 1.16 nM. This constant is 10-fold lower than that of charybdotoxin, and the values for the rate constants showed above indicate that this is mainly due to the very low dissociation rate constant; mean blocked time approximately 5 min. The fact that tetraethylammonium competitively inhibits the iberiotoxin binding to the channel is a strong suggestion that this toxin binds to the channel external vestibule. Increasing the external K+ concentration makes the association rate constant to decrease with no effect on the dissociation reaction indicating that the surface charges located in the external channel vestibule play an important role in modulating toxin binding.     

Ca(2+)-activated K+ channel inhibition by reactive oxygen species

We studied the effect of H(2)O(2) on the gating behavior of large-conductance Ca(2+)-sensitive voltage-dependent K(+) (K(V,Ca)) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevis oocytes membranes expressing the human Slowpoke (hSlo) alpha-subunit. Exposure of the intracellular side of K(V,Ca) channels to H(2)O(2) (4-23 mM) leads to a time-dependent decrease of the open probability (P(o)) without affecting the unitary conductance. H(2)O(2) did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H(2)O(2) action. Desferrioxamine (60 microM) and cysteine (1 mM) completely inhibited the effect of H(2)O(2), indicating that the decrease in P(o) was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H(2)O(2). However, DTT did completely reverse the decrease in P(o) induced by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of K(V,Ca) channel activity promoted by DTT suggests that H(2)O(2) treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing hSlo channels showed that H(2)O(2) induced a decrease in current mediated by a decrease both in the number of active channels and P(o).     

Effects of intracellular K+ and Rb+ on gating of embryonic rat telencephalon Ca(2+)-activated K+ channels.

We have investigated the effects of intracellular K+ and Rb+ on single-channel currents recorded from the large-conductance Ca(2+)-activated K+ (BK) channel of the embryonic rat telencephalon using the inside-out patch-clamp technique. Our novel observation concerns the effects of these ions on rapid flickering of channel openings. Specifically, flicker gating was voltage dependent, i.e., it was reduced by depolarization in the -60 to -10 mV range with equimolar concentrations of K+ ions (150 Ko+/150 Ki+). Removal of Ki+ resulted in significant flickering at all potentials in this voltage range. In other words, the voltage dependence of flicker gating was effectively eliminated by the removal of Ki+. This suggests that a K+ ion entering the channel from the intracellular medium binds, in a voltage-dependent manner, at a site that locks the flicker gate in its open position. No effects of changes in Ki+ were observed on the primary, voltage-dependent gate of the channel. The change in flickering did not cause a change in the mean burst duration, which indicates that the primary gate is stochastically independent of the flicker gate. Intracellular Rb+ can substitute for--and is even more effective than--Ki+ with regard to suppression of flickering. Substitution of Rbi+ for Ki+ also increased the mean burst duration for V > or = -30 mV. Both effects of Rbi+ were removed by membrane hyperpolarization.     

Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage

Large conductance Ca2+-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 NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 microM-100 microM) 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 Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.     

Proton modulation of a Ca(2+)-activated K+ channel from rat skeletal muscle incorporated into planar bilayers

The effect of pH on the activation of a Ca-activated K+ [K(Ca)] channel from rat skeletal muscle incorporated into planar lipid bilayers was studied. Experiments were done at different intracellular Ca2+ and proton concentrations. Changes in pH modified channel kinetics only from the Ca-sensitive face of the channel. At constant Ca2+ concentration, intracellular acidification induced a decrease in the open probability (Po) and a shift of the channel activation curves toward the right along the voltage axis. The displacement was 23.5 mV per pH unit. This displacement was due to a change in the half saturation voltage (Vo) and not to a change in channel voltage dependence. The shifts in Vo induced by protons appeared to be independent of Ca2+ concentration. The slope of the Hill plot of the open-closed equilibrium vs. pH was close to one, suggesting that a minimum of one proton is involved in the proton-driven channel closing reaction. The change in Po with variations in pH was due to both a decrease in the mean open time (To) and an increase in the mean closed time (Tc). At constant voltage, the mean open time of the channel was a linear function of [Ca2+] and the mean closed time was a linear function of 1/[Ca2+]2. Changes in the internal pH modified the slope, but not the intercept of the linear relations To vs. [Ca2+] and Tc vs. 1/[Ca2+]2. On the basis of these results an economical kinetic model of the effect of pH on this channel is proposed. It is concluded that protons do not affect the open-closed reaction, but rather weaken Ca2+ binding to all the conformational states of the channel. Moreover, competitive models in which Ca2+ and H+ cannot bind to the same open or closed state are inconsistent with the data.     

Shaking stack model of ion conduction through the Ca(2+)-activated K+ channel.

Motivated by the results of Neyton and Miller (1988. J. Gen. Physiol. 92:549-586), suggesting that the Ca(2+)-activated K+ channel has four high affinity ion binding sites, we propose a physically attractive variant of the single-vacancy conduction mechanism for this channel. Simple analytical expressions for conductance, current, flux ratio exponent, and reversal potential under bi-ionic conditions are found. A set of conductance data are analyzed to determine a realistic range of parameter values. Using these, we find qualitative agreement with a variety of experimental results previously reported in the literature. The exquisite selectivity of the Ca(2+)-activated K+ channel may be explained as a consequence of the concerted motion of the "stack" in the proposed mechanism.     

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism

Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, giving an approximately 10-fold increase in open probability (P(o)) in 0 Ca(2+)(i). The effect of the beta(1) subunit on increasing burst duration was little changed over a wide range of P(o) achieved by varying either Ca(2+)(i) or depolarization. The effect of the beta(1) subunit on increasing the durations of the gaps between bursts in 0 Ca(2+)(i) was preserved over a range of voltage, but was switched off as Ca(2+)(i) was increased into the activation range. The Ca(2+)-independent, beta(1) subunit-induced increase in burst duration accounted for 80% of the leftward shift in the P(o) vs. Ca(2+)(i) curve that reflects the increased Ca(2+) sensitivity induced by the beta(1) subunit. The Ca(2+)-dependent effect of the beta(1) subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the beta(1) subunit are independent of Ca(2+)(i) suggests that the beta(1) subunit mainly alters the energy barriers of Ca(2+)-independent transitions. The changes in gating induced by the beta(1) subunit differ from those induced by depolarization, as increasing P(o) by depolarization or by the beta(1) subunit gave different gating kinetics. The complex gating kinetics for both alpha and alpha+beta(1) channels in 0 Ca(2+)(i) arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca(2+)(i).     

Ca(2+)-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca(2+)-activated Ca2+ release.

We examined the possibility that Ca2+ released from intracellular stores could activate K+ currents underlying the afterhyperpolarization (AHP) in neurons. In neurons of the dorsal motor nucleus of the vagus, the current underlying the AHP had two components: a rapidly decaying component that was maximal following the action potential (GkCa,1) and a slower component that had a distinct rising phase (GkCa,2). Both components required influx of extracellular Ca2+ for their activation, and neither was blocked by extracellular TEA (10 mM). GkCa,1 was selectively blocked by apamin, whereas GkCa,2 was selectively reduced by noradrenaline. The time course of GkCa,2 was markedly temperature sensitive. GkCa,2 was selectively blocked by application of ryanodine or sodium dantrolene, or by loading cells with ruthenium red. These results suggest that influx of Ca2+ directly gates one class of K+ channels and leads to release of Ca2+ from intracellular stores, which activates a different class of K+ channel.     

Aminoglycoside blockade of Ca2+-activated K+ channel from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Ca²⁺-activated K⁺ channels from rat brain synaptosomal membranes were incorporated into planar lipid bilayers, and the effects of aminoglycoside antibiotics on the single channel conductance (258±13 pS at 100mm K⁺) were investigated. Aminoglycosides reduced the single channel conductance from the cis (cytoplasmic) side in a dose- and voltage-dependent manner. Voltage dependence of the blockade indicated an interaction between positively charged amino residues of aminoglycoside antibiotics and a binding site located within the electric field of the ion-conducting pathway. The order of blocking potency was consistent with that of the number of amino residues of aminoglycosides (neomycin (6)>dibekacin (5)>ribostamycin (4)=kanamycin (4)), while the electrical distance (z=0.46–0.49) of the binding site kept almost constant for each drug. Thesezs were almost the same with those (0.46–0.51) of alkyldiamine blockers with two amino residues (total net charge of +2) and approximately twice of those (0.25–0.26) of alkylmonoamine blockers (total net charge of +1). Assuming that amino residues of aminoglycosides and alkylamines shared the same binding site located at 25% voltage drop from the cytoplasmic surface of the channel, the site would have to be at least large enough to accommodate one diamino sugar residue of the aminoglycoside in order to simultaneously interact with two positively charged amino groups. Dose- and voltage-dependent blockade of the channel by gallamine, an extremely bulky trivalent organic cation, supported the picture that the channel has a wide mouth on the cytoplasmic side and its pore region, where voltage drop occurs, may also be quite wide and nonselective, suddenly tapering to a constriction where most charged cations block the channel by occluding the K⁺-conducting pathway.