A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans

The activities of many neuronal proteins are modulated by ethanol, but the fundamental mechanisms underlying behavioral effects of ethanol remain unclear. To identify mechanisms responsible for intoxication, we screened for Caenorhabditis elegans mutants with altered behavioral responses to ethanol. We found that slo-1 mutants, which were previously recognized as having slightly uncoordinated movement, are highly resistant to ethanol in two behavioral assays. Numerous loss-of-function slo-1 alleles emerged from our screens, indicating that slo-1 has a central role in ethanol responses. slo-1 encodes the BK potassium channel. Electrophysiological analysis shows that ethanol activates the channel in vivo, which would inhibit neuronal activity. Moreover, behaviors of slo-1 gain-of-function mutants resemble those of ethanol-intoxicated animals. These results demonstrate that selective activation of BK channels is responsible for acute intoxicating effects of ethanol in C. elegans. BK channel activation may explain a variety of behavioral responses to ethanol in invertebrate and vertebrate systems.     

Distinct mechanisms underlying cholesterol protection against alcohol-induced BK channel inhibition and resulting vasoconstriction

Alcohol (ethanol) at concentrations reached in blood following moderate to heavy drinking (30–80 mM) reduces cerebral artery diameter via inhibition of voltage- and calcium-gated potassium channels of large conductance (BK) in cerebral artery smooth muscle. These channels consist of channel-forming α and regulatory β1 subunits. A high-cholesterol diet protects against ethanol-induced constriction via accumulation of cholesterol within the vasculature. The molecular mechanisms of this protection remain unknown. In the present work, we demonstrate that in vitro cholesterol enrichment of rat middle cerebral arteries significantly increased cholesterol within arterial tissues and blunted constriction by 50 mM of ethanol. Ethanol-induced BK channel inhibition in inside-out patches excised from freshly isolated cerebral artery myocytes was also abolished by cholesterol enrichment. Enrichment of arteries with enantiomeric cholesterol (ent-cholesterol) also blunted BK channel inhibition and cerebral artery constriction in response to ethanol. The similar protection of cholesterol and ent-cholesterol against ethanol action indicates that this protection does not require protein site(s) that specifically sense natural cholesterol. Cholesterol-driven protection against ethanol-induced BK channel inhibition and vasoconstriction was replicated in myocytes and middle cerebral arteries of C57BL/6 mice. BK β1 subunits are known to regulate vascular diameter and its modification by ethanol. However, blunting of an ethanol effect by in vitro cholesterol enrichment was observed in arteries and myocyte membrane patches from BK β1 (KCNMB1) knockout mice. Thus, BK β1 subunits are not needed for cholesterol protection against ethanol effect on BK channel function and cerebral artery diameter.     

Alternative splicing switches potassium channel sensitivity to protein phosphorylation

Alternative exon splicing and reversible protein phosphorylation of large conductance calcium-activated potassium (BK) channels represent fundamental control mechanisms for the regulation of cellular excitability. BK channels are encoded by a single gene that undergoes extensive, hormonally regulated exon splicing. In native tissues BK channels display considerable diversity and plasticity in their regulation by cAMP-dependent protein kinase (PKA). Differential regulation of alternatively spliced BK channels by PKA may provide a molecular basis for the diversity and plasticity of BK channel sensitivities to PKA. Here we demonstrate that PKA activates BK channels lacking splice inserts (ZERO) but inhibits channels expressing a 59-amino acid exon at splice site 2 (STREX-1). Channel activation is dependent upon a conserved C-terminal PKA consensus motif (S869), whereas inhibition is mediated via a STREX-1 exon-specific PKA consensus site. Thus, alternative splicing acts as a molecular switch to determine the sensitivity of potassium channels to protein phosphorylation.     

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca²⁺- activated K⁺ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M₃ receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca²⁺ (Ca_V) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP₃Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions.     

A BK (Slo1) channel journey from molecule to physiology

Calcium and voltage-activated potassium (BK) channels are key actors in cell physiology, both in neuronal and non-neuronal cells and tissues. Through negative feedback between intracellular Ca²⁺ and membrane voltage, BK channels provide a damping mechanism for excitatory signals. Molecular modulation of these channels by alternative splicing, auxiliary subunits and post-translational modifications showed that these channels are subjected to many mechanisms that add diversity to the BK channel α subunit gene. This complexity of interactions modulates BK channel gating, modifying the energetic barrier of voltage sensor domain activation and channel opening. Regions for voltage as well as Ca²⁺ sensitivity have been identified, and the crystal structure generated by the 2 RCK domains contained in the C-terminal of the channel has been described. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, has been found to be relevant in many physiological processes. This review includes the hallmarks of BK channel biophysics and its physiological impact on specific cells and tissues, highlighting its relationship with auxiliary subunit expression.     

Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels

Large conductance Ca²⁺-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.     

Regulation of Guinea Pig Detrusor Smooth Muscle Excitability by 17β-Estradiol: The Role of the Large Conductance Voltage- and Ca2+-Activated K+ Channels

Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca²⁺-activated K⁺ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca²⁺ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability.     

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.     

Quantification and distribution of big conductance Ca2+-activated K+ channels in kidney epithelia

Big conductance Ca2+ activated K+ channels (BK channels) is an abundant channel present in almost all kind of tissue. The accurate quantity and especially the precise distribution of this channel in kidney epithelia are, however, still debated. The aim of the present study has therefore been to examine the presence of BK channels in kidney epithelia and determine the actual number and distribution of these channels. For this purpose, a selective peptidyl ligand for BK channels called iberiotoxin or the radiolabeled double mutant analog 125I-IbTX-D19Y/Y36F has been employed. The presence of BK channels were determined by a isotope flux assay where up to 44% of the total K+ channel activity could be inhibited by iberiotoxin indicating that BK channels are widely present in kidney epithelia. Consistent with these functional studies, 125I-IbTX-D19Y/Y36F binds to membrane vesicles from outer cortex, outer medulla and inner medulla with Bmax values (in fmol/mg protein) of 6.8, 2.6 and 21.4, respectively. These studies were performed applying rabbit kidney epithelia tissue. The distinct distribution of BK channels in both rabbit and rat kidney epithelia was confirmed by autoradiography and immunohistochemical studies. In cortical collecting ducts, BK channels were exclusively located in principal cells while no channels could be found in intercalated cells. The abundant and distinct distribution in kidney epithelia talks in favor for BK channels being important contributors in maintaining salt and water homeostasis.     

The BK channel: a vital link between cellular calcium and electrical signaling

Large-conductance Ca²⁺-activated K⁺ channels (BK channels) constitute an key physiological link between cellular Ca²⁺ signaling and electrical signaling at the plasma membrane. Thus these channels are critical to the control of action potential firing and neurotransmitter release in several types of neurons, as well as the dynamic control of smooth muscle tone in resistance arteries, airway, and bladder. Recent advances in our understanding of K⁺ channel structure and function have led to new insight toward the molecular mechanisms of opening and closing (gating) of these channels. Here we will focus on mechanisms of BK channel gating by Ca²⁺, transmembrane voltage, and auxiliary subunit proteins.     

A molecular switch for specific stimulation of the BKCa channel by cGMP and cAMP kinase

The cGMP and the cAMP pathways control smooth muscle tone by regulation of BK(Ca) (BK) channel activity. BK channels show considerable diversity and plasticity in their regulation by cyclic nucleotide-dependent protein kinases. The underlying molecular mechanisms are unclear but may involve expression of splice variants of the BK channel alpha subunit. Three isoforms, BK(A), BK(B), and BK(C), which were cloned from tracheal smooth muscle, differed only in their C terminus. When expressed in HEK293 cells, cGMP kinase (cGK) but not cAMP kinase (cAK) stimulated the activity of BK(A) and BK(B) by shifting the voltage dependence of the channel to more negative potentials. In contrast, BK(C) was exclusively stimulated by cAK. BK(C) lacks a C-terminal tandem phosphorylation motif for protein kinase C (PKC) with Ser(1151) and Ser(1154). Mutation of this motif in BK(A) switched channel regulation from cGK to cAK. Furthermore, inhibition of PKC in excised patches from cells expressing BK(A) abolished the stimulatory effect of cGK but allowed channel stimulation by cAK. cAK and cGK phosphorylated the channel at different sites. Thus, phosphorylation/dephosphorylation by PKC determines whether the BK channel is stimulated by cGK or cAK. The molecular mechanisms may be relevant for smooth muscle relaxation by cAMP and cGMP.     

Cholesterol tuning of BK ethanol response is enantioselective, and is a function of accompanying lipids

In the search to uncover ethanol's molecular mechanisms, the calcium and voltage activated, large conductance potassium channel (BK) has emerged as an important molecule. We examine how cholesterol content in bilayers of 1,2-dioleoyl-3-phosphatidylethanolamine (DOPE)/sphingomyelin (SPM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) affect the function and ethanol sensitivity of BK. In addition, we examine how manipulation of cholesterol in biological membranes modulates ethanol's actions on BK. We report that cholesterol levels regulate the change in BK channel open probability elicited by 50 mM ethanol. Low levels of cholesterol (<20%, molar ratio) supports ethanol activation, while high levels of cholesterol leads to ethanol inhibition of BK. To determine if cholesterol affects BK and its sensitivity to ethanol through a direct cholesterol-protein interaction or via an indirect action on the lipid bilayer, we used the synthetic enantiomer of cholesterol (ent-CHS). We found that 20% and 40% ent-CHS had little effect on the ethanol sensitivity of BK, when compared with the same concentration of nat-CHS. We accessed the effects of ent-CHS and nat-CHS on the molecular organization of DOPE/SPM monolayers at the air/water interface. The isotherm data showed that ent-CHS condensed DOPE/SPM monolayer equivalently to nat-CHS at a 20% concentration, but slightly less at a 40% concentration. Atomic force microscopy (AFM) images of DOPE/SPM membranes in the presence of ent-CHS or nat-CHS prepared with LB technique or vesicle deposition showed no significant difference in topographies, supporting the interpretation that the differences in actions of nat-CHS and ent-CHS on BK channel are not likely from a generalized action on bilayers. We conclude that membrane cholesterol influences ethanol's modulation of BK in a complex manner, including an interaction with the channel protein. Finally, our results suggest that an understanding of membrane protein function and modulation is impossible unless protein and surrounding lipid are considered as a functional unit.     

Characterization of voltage-and Ca2+-activated K+ channels in rat dorsal root ganglion neurons

Auxiliary beta-subunits associated with pore-forming Slo1 alpha-subunits play an essential role in regulating functional properties of large-conductance, voltage- and Ca(2+)-activated K(+) channels commonly termed BK channels. Even though both noninactivating and inactivating BK channels are thought to be regulated by beta-subunits (beta1, beta2, beta3, or beta4), the molecular determinants underlying inactivating BK channels in native cells have not been extensively demonstrated. In this study, rbeta2 (but not rbeta3-subunit) was identified as a molecular component in rat lumbar L4-6 dorsal root ganglia (DRG) by RT-PCR responsible for inactivating large-conductance Ca(2+)-dependent K(+) currents (BK(i) currents) in small sensory neurons. The properties of native BK(i) currents obtained from both whole-cell and inside-out patches are very similar to inactivating BK channels produced by co-expressing mSlo1 alpha- and hbeta2-subunits in Xenopus oocytes. Intracellular application of 0.5 mg/ml trypsin removes inactivation of BK(i) channels, and the specific blockers of BK channels, charybdotoxin (ChTX) and iberiotoxin (IbTX), inhibit these BK(i) currents. Single BK(i) channel currents derived from inside-out patches revealed that one BK(i) channel contained three rbeta2-subunits (on average), with a single-channel conductance about 217 pS under 160 K(+) symmetrical recording conditions. Blockade of BK(i) channels by 100 nM IbTX augmented firing frequency, broadened action potential waveform and reduced after-hyperpolarization. We propose that the BK(i) channels in small diameter DRG sensory neurons might play an important role in regulating nociceptive input to the central nervous system (CNS).     

Neuronal Ca2+-activated K+ channels limit brain infarction and promote survival

Neuronal calcium-activated potassium channels of the BK type are activated by membrane depolarization and intracellular Ca(2+) ions. It has been suggested that these channels may play a key neuroprotective role during and after brain ischemia, but this hypothesis has so far not been tested by selective BK-channel manipulations in vivo. To elucidate the in vivo contribution of neuronal BK channels in acute focal cerebral ischemia, we performed middle cerebral artery occlusion (MCAO) in mice lacking BK channels (homozygous mice lacking the BK channel alpha subunit, BK(-/-)). MCAO was performed in BK(-/-) and WT mice for 90 minutes followed by a 7-hour-reperfusion period. Coronal 1 mm thick sections were stained with 2,3,5-triphenyltetrazolium chloride to reveal the infarction area. We found that transient focal cerebral ischemia by MCAO produced larger infarct volume, more severe neurological deficits, and higher post-ischemic mortality in BK(-/-) mice compared to WT littermates. However, the regional cerebral blood flow was not significantly different between genotypes as measured by Laser Doppler (LD) flowmetry pre-ischemically, intra-ischemically, and post-ischemically, suggesting that the different impact of MCAO in BK(-/-) vs. WT was not due to vascular BK channels. Furthermore, when NMDA was injected intracerebrally in non-ischemic mice, NMDA-induced neurotoxicity was found to be larger in BK(-/-) mice compared to WT. Whole-cell patch clamp recordings from CA1 pyramidal cells in organotypic hippocampal slice cultures revealed that BK channels contribute to rapid action potential repolarization, as previously found in acute slices. When these cultures were exposed to ischemia-like conditions this induced significantly more neuronal death in BK(-/-) than in WT cultures. These results indicate that neuronal BK channels are important for protection against ischemic brain damage.     

BK channels mediate cholinergic inhibition of high frequency cochlear hair cells

Background

Outer hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K⁺ channels that control the membrane potential and also ligand-gated K⁺ channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea.

Methodology/Principal

Findings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K⁺ currents.

Conclusions/Significance

Our findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.     

Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites

Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming alpha subunit. Two mechanisms account for physiological regulation of BK channels by microM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10-300 microM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.     

BKCa channels activating at resting potential without calcium in LNCaP prostate cancer cells

Large-conductance Ca2+-dependent K+ (BK(Ca)) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK(Ca)-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK(L). K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK(Ca) channels. However, unlike conventional BK(Ca) channels, BK(L) channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about -100 mV compared with BK(Ca) channels. Half-maximal Ca2+-dependent activation was observed at 0.4 microM: for BK(L) (at -20 mV) and at 4.1 microM: for BK(Ca) channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK(L) conductance. Expression of hSlo-beta1 in LNCaP cells shifted voltage-dependent activation to values between that of BK(L) and BK(Ca) channels and reduced the slope of the P (open) (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK(Ca) subunit, which is responsible for the BK(L) phenotype in a dominant manner. BK(L)-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK(L) in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Quantification and distribution of big conductance Ca-activated K channels in kidney epithelia

Big conductance Ca²⁺ activated K⁺ channels (BK channels) is an abundant channel present in almost all kind of tissue. The accurate quantity and especially the precise distribution of this channel in kidney epithelia are, however, still debated. The aim of the present study has therefore been to examine the presence of BK channels in kidney epithelia and determine the actual number and distribution of these channels. For this purpose, a selective peptidyl ligand for BK channels called iberiotoxin or the radiolabeled double mutant analog ¹²⁵I-IbTX-D19Y/Y36F has been employed. The presence of BK channels were determined by a isotope flux assay where up to 44% of the total K⁺ channel activity could be inhibited by iberiotoxin indicating that BK channels are widely present in kidney epithelia. Consistent with these functional studies, ¹²⁵I-IbTX-D19Y/Y36F binds to membrane vesicles from outer cortex, outer medulla and inner medulla with B_max values (in fmol/mg protein) of 6.8, 2.6 and 21.4, respectively. These studies were performed applying rabbit kidney epithelia tissue. The distinct distribution of BK channels in both rabbit and rat kidney epithelia was confirmed by autoradiography and immunohistochemical studies. In cortical collecting ducts, BK channels were exclusively located in principal cells while no channels could be found in intercalated cells. The abundant and distinct distribution in kidney epithelia talks in favor for BK channels being important contributors in maintaining salt and water homeostasis.     

Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca(2+) sparks in airway myocytes

Short-lived, localized Ca(2+) events mediate Ca(2+) signaling with high efficiency and great fidelity largely as a result of the close proximity between Ca(2+)-permeable ion channels and their molecular targets. However, in most cases, direct evidence of the spatial relationship between these two types of molecules is lacking, and, thus, mechanistic understanding of local Ca(2+) signaling is incomplete. In this study, we use an integrated approach to tackling this issue on a prototypical local Ca(2+) signaling system composed of Ca(2+) sparks resulting from the opening of ryanodine receptors (RYRs) and spontaneous transient outward currents (STOCs) caused by the opening of Ca(2+)-activated K(+) (BK) channels in airway smooth muscle. Biophysical analyses of STOCs and Ca(2+) sparks acquired at 333 Hz demonstrate that these two events are associated closely in time, and approximately eight RYRs open to give rise to a Ca(2+) spark, which activates ～15 BK channels to generate a STOC at 0 mV. Dual immunocytochemistry and 3-D deconvolution at high spatial resolution reveal that both RYRs and BK channels form clusters and RYR1 and RYR2 (but not RYR3) localize near the membrane. Using the spatial relationship between RYRs and BK channels, the spatial-temporal profile of [Ca(2+)] resulting from Ca(2+) sparks, and the kinetic model of BK channels, we estimate that an average Ca(2+) spark caused by the opening of a cluster of RYR1 or RYR2 acts on BK channels from two to three clusters that are randomly distributed within an ～600-nm radius of RYRs. With this spatial organization of RYRs and BK channels, we are able to model BK channel currents with the same salient features as those observed in STOCs across a range of physiological membrane potentials. Thus, this study provides a mechanistic understanding of the activation of STOCs by Ca(2+) sparks using explicit knowledge of the spatial relationship between RYRs (the Ca(2+) source) and BK channels (the Ca(2+) target).