The beta subunit increases the Ca2+ sensitivity of large conductance Ca2+-activated potassium channels by retaining the gating in the bursting states

Coexpression of the beta subunit (KV,Cabeta) with the alpha subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the beta subunit increased open probability (Po) by increasing burst duration 20-100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the beta subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the beta subunit does not act by increasing all the Ca2+ binding rates proportionally. The beta subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the beta subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the beta subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.     

Membrane trafficking of large conductance Ca2+- and voltage-activated K+ (BK) channels is regulated by Rab4 GTPase

The large conductance voltage- and Ca²⁺-activated K⁺ (BK) channel is a major ionic determinant of vascular tone, vasodilation, and blood pressure. The activity of BK channels is regulated in part by membrane presentation. Rab GTPase (Rab) regulates important cellular processes, including ion channel membrane trafficking. We hypothesize that Rab4a participates in the regulation of BK channel α-subunit (BK-α) membrane trafficking. We found that vascular BK-α interacts physically with Rab4a. Co-expression of dominant-negative Rab4a reduced BK-α surface expression, whereas that of constitutively-active Rab4a augmented BK-α surface presentation. These novel findings suggest that vascular BK channel membrane expression is regulated by Rab4a through channel membrane trafficking.     

Down-regulation of N-type voltage-activated Ca2+ channels by gabapentin

1. Although the cellular and molecular mechanisms of the anticonvulsant action of gabapentin (GBP) remain incompletely described, in vitro studies have shown that GBP binds to the ₂ subunit of the high voltage-activated (HVA) Ca²⁺ channels.2. In this report, we analyzed the effects of GBP on the functional expression of HVA Ca²⁺ channels in the PC12 cell line model system. Negligible inhibition of Ca²⁺ channel activity was observed after acute treatment, but a significant decrease in Ca²⁺ current amplitude was promoted by chronic exposure to GBP.3. Consistent with this, radioligand binding experiments showed a comparable reduction in the total number of membrane HVA N-type channels after GBP treatment.     

Coupling between voltage sensor activation,Ca2+ binding and channel opening in large conductance (BK) potassium channels

To determine how intracellular Ca(2+) and membrane voltage regulate the gating of large conductance Ca(2+)-activated K(+) (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca(2+) over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305-336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). In 0 Ca(2+), the steady-state gating charge-voltage (Q(SS)-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (G(K)-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 microM Ca(2+). This change reflects a differential effect of Ca(2+) on voltage sensor activation and channel opening. Ca(2+) has only a small effect on the fast component of ON gating current, indicating that Ca(2+) binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than -80 mV) increases more than 1,000-fold in 70 microM Ca(2+), demonstrating that Ca(2+) increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca(2+) binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca(2+) sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca(2+) sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic I(K) kinetics indicate that Ca(2+) and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

Modulation of Ca2+- and voltage-activated K+ channels by internal Mg2+ in salivary acinar cells

High-conductance K+ channels are known to be activated by internal Ca2+ and membrane depolarization. The effects of changes in internal Mg2+ concentration have now been investigated in patch-clamp single-channel current experiments on excised membrane fragments from mouse acinar cells. It is shown that Mg2+ in the concentration range 10(-6)-10(-3) M evokes a dose-dependent K+ channel activation at a constant Ca2+ concentration of 10(-8) M. The demonstration that changes in [Mg2+]i between 2.5 X 10(-4) and 1.13 X 10(-3) M has effects on the channel open-state probability indicates that fluctuations in [Mg2+]i in intact cells may influence the control of channel opening.     

Bupivacaine inhibits large conductance,voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K⁺ channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca²⁺-activated K⁺ channels (BK_Ca). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K⁺ currents carried by BK_Ca channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC₅₀ 324 µM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 µM) can also block whole-cell K⁺ currents (~45% blockage) in which, under our working conditions, BK_Ca is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK_Ca channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Bupivacaine inhibits large conductance, voltage- and Ca2+- activated K+ channels in human umbilical artery smooth muscle cells

Bupivacaine is a local anesthetic compound belonging to the amino amide group. Its anesthetic effect is commonly related to its inhibitory effect on voltage-gated sodium channels. However, several studies have shown that this drug can also inhibit voltage-operated K(+) channels by a different blocking mechanism. This could explain the observed contractile effects of bupivacaine on blood vessels. Up to now, there were no previous reports in the literature about bupivacaine effects on large conductance voltage- and Ca(2+) -activated K(+) channels (BK(Ca)). Using the patch-clamp technique, it is shown that bupivacaine inhibits single-channel and whole-cell K(+) currents carried by BK(Ca) channels in smooth muscle cells isolated from human umbilical artery (HUA). At the single-channel level bupivacaine produced, in a concentration- and voltage-dependent manner (IC(50) 324 μM at +80 mV), a reduction of single-channel current amplitude and induced a flickery mode of the open channel state. Bupivacaine (300 μM) can also block whole-cell K(+) currents (~45% blockage) in which, under our working conditions, BK(Ca) is the main component. This study presents a new inhibitory effect of bupivacaine on an ion channel involved in different cell functions. Hence, the inhibitory effect of bupivacaine on BK(Ca) channel activity could affect different physiological functions where these channels are involved. Since bupivacaine is commonly used during labor and delivery, its effects on umbilical arteries, where this channel is highly expressed, should be taken into account.     

Nitric oxide directly activates large conductance Ca2+-activated K+ channels (rSlo)

We investigated whether nitric oxide (NO) directly activates the cloned alpha-subunit of large conductance Ca2+-activated K+ (Maxi-K) channels from rat brain (rSlo), expressed either in HEK293 cells or Xenopus oocytes. In inside-out patches, the application of S-nitroso-N-acetylpenicillamine (SNAP), a NO-releasing compound, reversibly activated the channel shifting the voltage dependent activation curve of the macroscopic Maxi-K current to the left by about 15 mV. Pretreatment of the patches with N-ethylmaleimide to alkylate free sulfhydryl groups did not prevent the effect of SNAP, suggesting that NO may directly interact with the channels. These results suggest that Maxi-K channels might be one of the physiological targets of NO in the brain.     

Voltage- and Ca2+-activated potassium channels in Ca2+ store control Ca2+ release

Ca2+ release from Ca2+ stores is a 'quantal' process; it terminates after a rapid release of stored Ca2+. To explain the quantal nature, it has been supposed that a decrease in luminal Ca2+ acts as a 'brake' on store release. However, the mechanism for the attenuation of Ca2+ efflux remains unknown. We show that Ca2+ release is controlled by voltage- and Ca2+-activated potassium channels in the Ca2+ store. The potassium channel was identified as the big or maxi-K (BK)-type, and was activated by positive shifts in luminal potential and luminal Ca2+ increases, as revealed by patch-clamp recordings from an exposed nuclear envelope. The blockage or closure of the store BK channel due to Ca2+ efflux developed lumen-negative potentials, as revealed with an organelle-specific voltage-sensitive dye [DiOC5(3); 3,3'-dipentyloxacarbocyanine iodide], and suppressed Ca2+ release. The store BK channels are reactivated by Ca2+ uptake by Ca2+ pumps regeneratively with K+ entry to allow repetitive Ca2+ release. Indeed, the luminal potential oscillated bistably by approximately 45 mV in amplitude. Our study suggests that Ca2+ efflux-induced store BK channel closures attenuate Ca2+ release with decreases in counter-influx of K+.     

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.     

Differences in apparent pore sizes of low and high voltage-activated Ca2+ channels

Pore size is of considerable interest in voltage-gated Ca(2+) channels because they exemplify a fundamental ability of certain ion channels: to display large pore diameter, but also great selectivity for their ion of choice. We determined the pore size of several voltage-dependent Ca(2+) channels of known molecular composition with large organic cations as probes. T-type channels supported by the Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3 subunits; L-type channels encoded by the Ca(V)1.2, beta(1), and alpha(2)delta(1) subunits; and R-type channels encoded by the Ca(V)2.3 and beta(3) subunits were each studied using a Xenopus oocyte expression system. The weak permeabilities to organic cations were resolved by looking at inward tails generated upon repolarization after a large depolarizing pulse. Large inward NH(4)(+) currents and sizable methylammonium and dimethylammonium currents were observed in all of the channels tested, whereas trimethylammonium permeated only through L- and R-type channels, and tetramethylammonium currents were observed only in L-type channels. Thus, our experiments revealed an unexpected heterogeneity in pore size among different Ca(2+) channels, with L-type channels having the largest pore (effective diameter = 6.2 A), T-type channels having the tiniest pore (effective diameter = 5.1 A), and R-type channels having a pore size intermediate between these extremes. These findings ran counter to first-order expectations for these channels based simply on their degree of selectivity among inorganic cations or on the bulkiness of their acidic side chains at the locus of selectivity.     

Hydrophobic interface between two regulators of K+ conductance domains critical for calcium-dependent activation of large conductance Ca2+-activated K+ channels

It has been suggested that the large conductance Ca(2)+-activated K(+) channel contains one or more domains known as regulators of K(+) conductance (RCK) in its cytosolic C terminus. Here, we show that the second RCK domain (RCK2) is functionally important and that it forms a heterodimer with RCK1 via a hydrophobic interface. Mutant channels lacking RCK2 are nonfunctional despite their tetramerization and surface expression. The hydrophobic residues that are expected to form an interface between RCK1 and RCK2, based on the crystal structure of the bacterial MthK channel, are well conserved, and the interactions of these residues were confirmed by mutant cycle analysis. The hydrophobic interaction appears to be critical for the Ca(2+)-dependent gating of the large conductance Ca(2+)-activated K(+) channel.     

Cysteine modification alters voltage- and Ca(2+)-dependent gating of large conductance (BK) potassium channels

The Ca(2+)-activated K+ (BK) channel alpha-subunit contains many cysteine residues within its large COOH-terminal tail domain. To probe the function of this domain, we examined effects of cysteine-modifying reagents on channel gating. Application of MTSET, MTSES, or NEM to mSlo1 or hSlo1 channels changed the voltage and Ca2+ dependence of steady-state activation. These reagents appear to modify the same cysteines but have different effects on function. MTSET increases I(K) and shifts the G(K)-V relation to more negative voltages, whereas MTSES and NEM shift the G(K)-V in the opposite direction. Steady-state activation was altered in the presence or absence of Ca2+ and at negative potentials where voltage sensors are not activated. Combinations of [Ca2+] and voltage were also identified where P(o) is not changed by cysteine modification. Interpretation of our results in terms of an allosteric model indicate that cysteine modification alters Ca2+ binding and the relative stability of closed and open conformations as well as the coupling of voltage sensor activation and Ca2+ binding and to channel opening. To identify modification-sensitive residues, we examined effects of MTS reagents on mutant channels lacking one or more cysteines. Surprisingly, the effects of MTSES on both voltage- and Ca(2+)-dependent gating were abolished by replacing a single cysteine (C430) with alanine. C430 lies in the RCK1 (regulator of K+ conductance) domain within a series of eight residues that is unique to BK channels. Deletion of these residues shifted the G(K)-V relation by > -80 mV. Thus we have identified a region that appears to strongly influence RCK domain function, but is absent from RCK domains of known structure. C430A did not eliminate effects of MTSET on apparent Ca2+ affinity. However an additional mutation, C615S, in the Haem binding site reduced the effects of MTSET, consistent with a role for this region in Ca2+ binding.     

Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes

K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could contribute to the voltage-dependent Ca2+-activated macroscopic K+ current (IC) that has been observed in several neuronal somata preparations, as well as in other cells. Some of the properties reported here may serve to distinguish which type contributes in each case. A third class of smaller (40-50 pS) channels was also studied. These channels were independent of calcium over the concentration range examined (10(-7)-10(-3) M), and were also independent of voltage over the range of pipette potentials of -60 to +60 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume

Large conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca(2+) are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K(+) channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K(+) flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl(-) efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration.     

Physiological roles of the intermediate conductance, Ca2+-activated potassium channel Kcnn4

Three broad classes of Ca(2+)-activated potassium channels are defined by their respective single channel conductances, i.e. the small, intermediate, and large conductance channels, often termed the SK, IK, and BK channels, respectively. SK channels are likely encoded by three genes, Kcnn1-3, whereas IK and most BK channels are most likely products of the Kcnn4 and Slo (Kcnma1) genes, respectively. IK channels are prominently expressed in cells of the hematopoietic system and in organs involved in salt and fluid transport, including the colon, lung, and salivary glands. IK channels likely underlie the K(+) permeability in red blood cells that is associated with water loss, which is a contributing factor in the pathophysiology of sickle cell disease. IK channels are also involved in the activation of T lymphocytes. The fluid-secreting acinar cells of the parotid gland express both IK and BK channels, raising questions about their particular respective roles. To test the physiological roles of channels encoded by the Kcnn4 gene, we constructed a mouse deficient in its expression. Kcnn4 null mice were of normal appearance and fertility, their parotid acinar cells expressed no IK channels, and their red blood cells lost K(+) permeability. The volume regulation of T lymphocytes and erythrocytes was severely impaired in Kcnn4 null mice but was normal in parotid acinar cells. Despite the loss of IK channels, activated fluid secretion from parotid glands was normal. These results confirm that IK channels in red blood cells, T lymphocytes, and parotid acinar cells are indeed encoded by the Kcnn4 gene. The role of these channels in water movement and the subsequent volume changes in red blood cells and T lymphocytes is also confirmed. Surprisingly, Kcnn4 channels appear to play no required role in fluid secretion and regulatory volume decrease in the parotid gland.