The effect of single cerebroside compounds on activation of BKCa channels

We have previously shown that a mixture of cerebrosides obtained from dried tubers of herb Typhonium giganteum Engl. plays a neuroprotective role in the ischemic brain through its effect on activation of BK(Ca) channels. It is very curious to know whether a single pure cerebroside compound could activate the BK(Ca) channel as well. This study explored the possible effects of pure cerebroside compounds, termitomycesphins A and B, on the BK(Ca) channel activation. Both termitomycesphins A and B activated the BK(Ca) channels at micromole concentration without significant difference. Termitomycesphin A increased the single channel open probability of the BK(Ca) channels in a dose-dependent manner without modifying the single channel conductance. Termitomycesphin A activated BK(Ca) channel more efficiently when it was applied to the cytoplasmic face of the membrane, suggesting that binding site for termitomycesphin A is located at the cytoplasmic side. Termitomycesphin A shifted the voltage-dependent activation curve to less positive membrane potentials and the Ca(2+)-dependent activation curve of the channel upwards, suggesting that termitomycesphin A could activate the channels even without intracellular free Ca(2+). Furthermore, STREX-deleted BK(Ca) channels were completely insensitive to termitomycesphin A, indicating that STREX domain is required for the activation of the BK(Ca) channel. These data provide evidence that termitomycesphins are potent in stimulating the activity of the BK(Ca) channels. As BK(Ca) channels are associated with pathology of many diseases, termitomycesphins might be used as therapeutic agents for treating these diseases through its regulatory effect on the BK(Ca) 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.     

Voltage dependence of the coupling of Ca(2+) sparks to BK(Ca) channels in urinary bladder smooth muscle

Large-conductance Ca(2+)-dependent K(+) (BK(Ca)) channels play a critical role in regulating urinary bladder smooth muscle (UBSM) excitability and contractility. Measurements of BK(Ca) currents and intracellular Ca(2+) revealed that BK(Ca) currents are activated by Ca(2+) release events (Ca(2+) sparks) from ryanodine receptors (RyRs) in the sarcoplasmic reticulum. The goals of this project were to characterize Ca(2+) sparks and BK(Ca) currents and to determine the voltage dependence of the coupling of RyRs (Ca(2+) sparks) to BK(Ca) channels in UBSM. Ca(2+) sparks in UBSM had properties similar to those described in arterial smooth muscle. Most Ca(2+) sparks caused BK(Ca) currents at all voltages tested, consistent with the BK(Ca) channels sensing approximately 10 microM Ca(2+). Membrane potential depolarization from -50 to -20 mV increased Ca(2+) spark and BK(Ca) current frequency threefold. However, membrane depolarization over this range had a differential effect on spark and current amplitude, with Ca(2+) spark amplitude increasing by only 30% and BK(Ca) current amplitude increasing 16-fold. A major component of the amplitude modulation of spark-activated BK(Ca) current was quantitatively explained by the known voltage dependence of the Ca(2+) sensitivity of BK(Ca) channels. We, therefore, propose that membrane potential, or any other agent that modulates the Ca(2+) sensitivity of BK(Ca) channels, profoundly alters the coupling strength of Ca(2+) sparks to BK(Ca) channels.     

Enhanced activity of a large conductance, calcium-sensitive K+ channel in the presence of Src tyrosine kinase

Large conductance, calcium-sensitive K(+) channels (BK(Ca) channels) contribute to the control of membrane potential in a variety of tissues, including smooth muscle, where they act as the target effector for intracellular "calcium sparks" and the endothelium-derived vasodilator nitric oxide. Various signal transduction pathways, including protein phosphorylation can regulate the activity of BK(Ca) channels, along with many other membrane ion channels. In our study, we have examined the regulation of BK(Ca) channels by the cellular Src gene product (cSrc), a soluble tyrosine kinase that has been implicated in the regulation of both voltage- and ligand-gated ion channels. Using a heterologous expression system, we observed that co-expression of murine BK(Ca) channel and the human cSrc tyrosine kinase in HEK 293 cells led to a calcium-sensitive enhancement of BK(Ca) channel activity in excised membrane patches. In contrast, co-expression with a catalytically inactive cSrc mutant produced no change in BK(Ca) channel activity, demonstrating the requirement for a functional cSrc molecule. Furthermore, we observed that BK(Ca) channels underwent direct tyrosine phosphorylation in cells co-transfected with BK(Ca) channels and active cSrc but not in cells co-transfected with the kinase inactive form of the enzyme. A single Tyr to Phe substitution in the C-terminal half of the channel largely prevented this observed phosphorylation. Given that cSrc may become activated by receptor tyrosine kinases or G-protein-coupled receptors, these findings suggest that cSrc-dependent tyrosine phosphorylation of BK(Ca) channels in situ may represent a novel regulatory mechanism for altering membrane potential and calcium entry.     

A catalytically inactive mutant of type I cGMP-dependent protein kinase prevents enhancement of large conductance, calcium-sensitive K+ channels by sodium nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.     

Differential effects of estrogen and progesterone on potassium channels expressed in Xenopus oocytes

HYPOTHESIS: Potassium (K(+)) channel activation contributes in part to estrogen-mediated vasorelaxation. However, the underlying mechanism is still unclear. We hypothesize that estrogen increases K(+) currents via membrane-associated, non-genomic interaction and that steroid hormones have differential effects on different types of K(+) channels. EXPERIMENTAL: Human large-conductance Ca(2+)-activated K(+) channels (BK(Ca)) and human voltage-gated K(+) channels (K(V1.5)) were expressed in Xenopus oocytes, and K(+) currents elicited by voltage clamp were measured. RESULTS: Both 17beta-estradiol and BSA-conjugated 17beta-estradiol increased the BK(Ca) current in a concentration-dependent manner and this effect was abolished by tetraethylammonium ions and iberiotoxin (putative BK(Ca) channel blockers). 17beta-estradiol-stimulated increase in the BK(Ca) current was unaffected by treatment with ICI 182,780 (classic estrogen receptor antagonist), tamoxifen (estrogen receptor agonist/antagonist), actinomycin D (RNA synthesis inhibitor), or cycloheximide (protein synthesis inhibitor). In contrast, progesterone reduced the BK(Ca) current in the absence or presence of NS 1619 (BK(Ca) channel activator). Progesterone also inhibited 17beta-estradiol-stimulated increase in the BK(Ca) current. Finally, progesterone but not 17beta-estradiol reduced the K(V1.5) current. CONCLUSIONS: The present results show that 17beta-estradiol stimulates BK(Ca) channels without affecting K(V1.5) channels. This effect is ICI 182,780-insensitive and is likely mediated via a membrane-bound binding site. Progesterone inhibits both BK(Ca)- and K(V1.5)-encoded currents. The present results suggest that inhibition of K(+) channels may contribute in part to its reported antagonism against 17beta-estradiol-mediated vascular relaxation via BK(Ca) channels.     

Changes in membrane cholesterol of pituitary tumor (GH3) cells regulate the activity of large-conductance Ca2+-activated K+ channels

The effects of changes in membrane cholesterol on ion currents were investigated in pituitary GH3 cells. Depletion of membrane cholesterol by exposing cells to methyl-beta-cyclodextrin (MbetaCD), an oligosaccharide, resulted in an increase in the density of Ca2+-activated K+ current (IK(Ca)). However, no significant change in IK(Ca) density was demonstrated in GH3 cells treated with a mixture of MbetaCD and cholesterol. Cholesterol depletion with MbetaCD (1.5 mg/ml) slightly suppressed the density of voltage-dependent L-type Ca2+ current. In inside-out patches recorded from MbetaCD-treated cells, the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels was enhanced with no change in single-channel conductance. In MbetaCD-treated cells, voltage-sensitivity of BK(Ca) channels was increased; however, no change in Ca2+-sensitivity could be demonstrated. A negative correlation between adjacent closed and open times in BK(Ca) channels was observed in MbetaCD-treated cells. In inside-out patches from MbetaCD-treated cells, dexamethasone (30 microM) applied to the intracellular surface did not increase BK(Ca)-channel activity, although caffeic acid phenethyl ester and cilostazol still opened its probability effectively. However, no modification in the activity of ATP-sensitive K+ channels could be seen in MbetaCD-treated cells. Current-clamp recordings demonstrated that the cholesterol depletion maneuver with MbetaCD reduced the firing of action potentials. Therefore, the increase in BK(Ca)-channel activity induced by membrane depletion may influence the functional activities of neurons or neuroendocrine cells if similar results occur in vivo.     

Renovascular BK(Ca) channels are not activated in vivo under resting conditions and during agonist stimulation

We investigated the role of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels for the basal renal vascular tone in vivo. Furthermore, the possible buffering by BK(Ca) of the vasoconstriction elicited by angiotensin II (ANG II) or norepinephrine (NE) was investigated. The possible activation of renal vascular BK(Ca) channels by cAMP was investigated by infusing forskolin. Renal blood flow (RBF) was measured in vivo using electromagnetic flowmetry or ultrasonic Doppler. Renal preinfusion of tetraethylammonium (TEA; 3.0 mumol/min) caused a small reduction of baseline RBF, but iberiotoxin (IBT; 0.3 nmol/min) did not have any effect. Renal injection of ANG II (1-4 ng) or NE (10-40 ng) produced a transient decrease in RBF. These responses were not affected by preinfusion of TEA or IBT. Renal infusion of the BK(Ca) opener NS-1619 (90.0 nmol/min) did not affect basal RBF or the response to NE, but it attenuated the response to ANG II. Coadministration of NS-1619 with TEA or IBT abolished this effect. Forskolin caused renal vasodilation that was not inhibited by IBT. The presence of BK(Ca) channels in the preglomerular vessels was confirmed by immunohistochemistry. Despite their presence, there is no indication for a major role for BK(Ca) channels in the control of basal renal tone in vivo. Furthermore, BK(Ca) channels do not have a buffering effect on the rat renal vascular responses to ANG II and NE. The fact that NS-1619 attenuates the ANG II response indicates that the renal vascular BK(Ca) channels can be activated under certain conditions.     

Activity of BK(Ca) channel is modulated by membrane cholesterol content and association with Na+/K+-ATPase in human melanoma IGR39 cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.     

Redistribution of the tight junction protein ZO-1 during physiological shedding of mouse intestinal epithelial cells

A novel vasodilatory influence of endothelial cell (EC) large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels is present following in vivo exposure to chronic hypoxia (CH) and may exist in other pathological states. However, the mechanism of channel activation that results in altered vasoreactivity is unknown. We tested the hypothesis that CH removes an inhibitory effect of the scaffolding domain of caveolin-1 (Cav-1) on EC BK(Ca) channels to permit activation, thereby affecting vasoreactivity. Experiments were performed on gracilis resistance arteries and ECs from control and CH-exposed (380 mmHg barometric pressure for 48 h) rats. EC membrane potential was hyperpolarized in arteries from CH-exposed rats and arteries treated with the cholesterol-depleting agent methyl-β-cyclodextrin (MBCD) compared with controls. Hyperpolarization was reversed by the BK(Ca) channel antagonist iberiotoxin (IBTX) or by a scaffolding domain peptide of Cav-1 (AP-CAV). Patch-clamp experiments documented an IBTX-sensitive current in ECs from CH-exposed rats and in MBCD-treated cells that was not present in controls. This current was enhanced by the BK(Ca) channel activator NS-1619 and blocked by AP-CAV or cholesterol supplementation. EC BK(Ca) channels displayed similar unitary conductance but greater Ca(2+) sensitivity than BK(Ca) channels from vascular smooth muscle. Immunofluorescence imaging demonstrated greater association of BK(Ca) α-subunits with Cav-1 in control arteries than in arteries from CH-exposed rats, although fluorescence intensity for each protein did not differ between groups. Finally, AP-CAV restored myogenic and phenylephrine-induced constriction in arteries from CH-exposed rats without affecting controls. AP-CAV similarly restored diminished reactivity to phenylephrine in control arteries pretreated with MBCD. We conclude that CH unmasks EC BK(Ca) channel activity by removing an inhibitory action of the Cav-1 scaffolding domain that may depend on cellular cholesterol levels.     

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.     

The calcium-activated potassium channels of turtle hair cells

A major factor determining the electrical resonant frequency of turtle cochlear hair cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987. Journal of Physiology. 385:207- 242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair bundle. All cells possess BK channels with a similar unit conductance of approximately 320 pS but with different mean open times of 0.25-12 ms. The time constant of relaxation of the average single- channel current at -50 mV in 4 microM Ca varied between cells from 0.4 to 13 ms and was correlated with the hair bundle height. The magnitude and voltage dependence of the time constant agree with the expected behavior of the macroscopic K(Ca) current, whose speed may thus be limited by the channel kinetics. All BK channels had similar sensitivities to Ca which produced half-maximal activation for a concentration of approximately 2 microM at +50 mV and 12 microM at -50 mV. We estimate from the voltage dependence of the whole-cell K(Ca) current that the BK channels may be fully activated at -35 mV by a rise in intracellular Ca to 50 microM. BK channels were occasionally observed to switch between slow and fast gating modes which raises the possibility that the range of kinetics of BK channels observed in different hair cells reflects a common channel protein whose kinetics are regulated by an unidentified intracellular factor. Membrane patches also contained 30 pS SK channels which were approximately 5 times more Ca-sensitive than BK channels at -50 mV. The SK channels may underlie the inhibitory synaptic potential produced in hair cells by efferent stimulation.     

Conditional and unconditional inhibition of calcium-activated potassium channels by reversible protein phosphorylation

Large conductance, calcium-activated potassium channels (BK(Ca) or maxi-K) are important determinants of membrane excitability in many cell types. We used patch clamp techniques to study the biochemical regulation of native BK(Ca) channel proteins by endogenous Ser/Thr-directed protein kinases and phosphatases in cell-free membrane patches from rat pituitary tumor cells (GH(4)C(1)). When protein kinase activity was blocked by removing ATP, endogenous protein phosphatases slowly increased BK(Ca) channel activity approximately 3-fold. Dephosphorylated channels could be activated fully by physiological increases in cytoplasmic calcium or membrane depolarization. In contrast, endogenous protein kinases inhibited BK(Ca) channel activity at two functionally distinct sites. A closely associated, cAMP-dependent protein kinase rapidly reduced channel activity in a conditional manner that could be overcome completely by increasing cytoplasmic free calcium 3-fold or 20 mV further depolarization. Phosphorylation at a pharmacologically distinct site inhibited channel activity unconditionally by reducing availability to approximately half that of maximum at all physiological calcium and voltages. Conditional versus unconditional inhibition of BK(Ca) channel activity through different protein kinases provides cells with a powerful computational mechanism for regulating membrane excitability.     

三磷酸肌醇对猪冠状动脉平滑肌细胞大电导钙激活钾通道的作用

应用膜片钳单通道电流记录技术,研究三磷酸肌醇(trisphosphateinositol,IP3)对猪冠状动脉平滑肌细胞大电导钙激活钾通道(large-conductanceCa2+-activatedpotassiumchannels,BKchannels)的作用。结果显示:在内面向外式(inside-out)膜片下,IP3(10~50μmol/L)可以浓度依赖性地增加通道的开放概率,而对电流幅值无明显影响,开放概率的增加是通过明显缩短平均关闭时间实现的(n=11,P<0.01);洗去药物后通道活性可以恢复到对照水平;IP3对通道的激活作用不随时间而衰减;IP3的降解产物对通道没有明显的激活作用。结果表明:在inside-out膜片下,IP3能够激活猪冠状动脉平滑肌细胞BK通道。     

Effects of insulin and high glucose on mobilization of slo1 BKCa channels in podocytes

Podocytes are dynamic polarized cells that lie on the surface of glomerular capillaries and comprise an essential component of the glomerular filtration barrier. Podocytes are affected in the earliest stages of diabetic nephropathy and insulin signaling to podocytes is essential for normal glomerular function. Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) encoded by the Slo1 gene are expressed in podocytes in a complex with multiple glomerular slit diaphragm proteins including nephrin, TRPC6 channels, and several different actin-binding proteins. Here we show that insulin increases cell surface expression of podocyte BK(Ca) channels, which is accompanied by a corresponding increase in the density of current flowing through these channels. Insulin stimulation of BK(Ca) channels was detectable in 15 min and required activation of both Erk and Akt signaling cascades. Exposure to high glucose (36.1 mM) for 24 h caused a marked reduction in the steady-state surface expression of BK(Ca) channels as well as of the slit diaphragm signaling molecule nephrin. High glucose treatment also abolished the stimulatory effects of insulin on BK(Ca) current density, although insulin continued to increase phosphorylation of Erk and Akt under those conditions. Therefore, in contrast to most other cell types, high glucose abrogates the effects of insulin in podocytes at relatively distal steps in its signaling pathway. Insulin stimulation of BK(Ca) channels in podocytes may prepare podocytes to adapt to changes in pressure gradients that occur during postprandial hyperfiltration.     

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).     

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.     

Alterations in blood-brain barrier ICAM-1 expression and brain microglial activation after lambda-carrageenan-induced inflammatory pain

Large-conductance Ca2+-sensitive K+ (BK) channel activity is greater in basilar artery smooth muscle cells (SMCs) of the fetus than the adult, and this increased activity is associated with a lower BK channel Ca2+ set point (Ca0). Associated PKG activity is three times greater in BK channels from fetal than adult myocytes, whereas associated PKA activity is three times greater in channels from adult than fetal myocytes. We hypothesized that the change in Ca0 during development results from different levels of channel phosphorylation. In inside-out membrane patch preparations of basilar artery SMCs from adult and fetal sheep, we measured BK channel activity in four states of phosphorylation: native, dephosphorylated, PKA phosphorylated, and PKG phosphorylated. BK channels from adult and fetus exhibited similar voltage-activation curves, Ca0 values, and Ca2+ dissociation constants (Kd) for the dephosphorylated, PKA phosphorylated, and PKG phosphorylated states. However, voltage-activation curves of native fetal BK channels shifted significantly to the left of those of the adult, with Ca0 and Kd values half those of the adult. For the two age groups at each of the phosphorylation states, Ca0 and Kd produced linear relations when plotted against voltage at half-maximal channel activation. We conclude that the Ca0 and Kd values of the BK channel can be modulated by differential channel phosphorylation. Lower Ca0 and Kd values in BK channels of fetal myocytes can be explained by a greater extent of channel phosphorylation of fetal than adult myocytes.     

H(2)O(2) opens BK(Ca) channels via the PLA(2)-arachidonic acid signaling cascade in coronary artery smooth muscle

H(2)O(2) is a reactive oxygen species that contracts or relaxes vascular smooth muscle, but the molecular basis of these effects remains obscure. We previously demonstrated that H(2)O(2) opens the large-conductance, calcium- and voltage-activated (BK(Ca)) potassium channel of coronary myocytes (2) and now report physiological and biochemical evidence that the effect of H(2)O(2) on coronary smooth muscle involves the phospholipase A(2) (PLA(2))/arachidonic acid (AA) signaling cascades. H(2)O(2) stimulation of BK(Ca) channel activity was inhibited by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA(2). Furthermore, H(2)O(2) stimulated release of [(3)H]AA from coronary myocytes, and exogenous AA mimicked the effect of H(2)O(2) on BK(Ca) channels. Inhibitors of protein kinase C activity attenuated the effect of H(2)O(2) on BK(Ca) channels, [(3)H]AA release, or intact coronary arteries. In addition, the effect of H(2)O(2) or AA on BK(Ca) channels was inhibited by blockers of lipoxygenase metabolism. In contrast, inhibitors of cyclooxygenase or cytochrome P-450 had no effect. We propose that H(2)O(2) relaxes coronary arteries by stimulating BK(Ca) channels via the PLA(2)/AA signaling cascade and that lipoxygenase metabolites mediate this response.