Mitochondrial Ca2+-activated K+ channels more efficiently reduce mitochondrial Ca2+ overload in rat ventricular myocytes

We investigated the role of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel, the mitochondrial big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, and the mitochondrial permeability transition pore (MPTP) in the ouabain-induced increase of mitochondrial Ca(2+) in native rat ventricular myocytes by loading cells with rhod 2-AM. To overload mitochondrial Ca(2+), we pretreated cells with ouabain before applying mitochondrial K(ATP) or BK(Ca) channel and/or MPTP opener. Ouabain (1 mM) increased the rhod 2-sensitive fluorescence intensity (160 +/- 5.0% of control), which was dramatically decreased to the control level on application of diazoxide and NS-1619 in a dose-dependent manner (half-inhibition concentrations of 78.3 and 7.78 muM for diazoxide and NS-1619, respectively). This effect was reversed by selective inhibition of the mitochondrial K(ATP) channel by 5-hydroxydecanoate, the mitochondrial BK(Ca) channel by paxilline, and the MPTP by cyclosporin A. Although diazoxide did not efficiently reduce mitochondrial Ca(2+) during prolonged exposure to ouabain, NS-1619 reduced mitochondrial Ca(2+). These results suggest that although mitochondrial BK(Ca) and K(ATP) channels contribute to reduction of ouabain-induced mitochondrial Ca(2+) overload, activation of the mitochondrial BK(Ca) channel more efficiently reduces ouabain-induced mitochondrial Ca(2+) overload in our experimental model.     

Impairment of neurovascular coupling in type 1 diabetes mellitus in rats is linked to PKC modulation of BK(Ca) and Kir channels

We hypothesized that chronic hyperglycemia has a detrimental effect on neurovascular coupling in the brain and that this may be linked to protein kinase C (PKC)-mediated phosphorylation. Therefore, in a rat model of streptozotocin-induced chronic type 1 diabetes mellitus (T1DM), and in nondiabetic (ND) controls, we monitored pial arteriole diameter changes during sciatic nerve stimulation and topical applications of the large-conductance Ca(2+)-operated K(+) channel (BK(Ca)) opener, NS-1619, or the K(+) inward rectifier (Kir) channel agonist, K(+). In the T1DM vs. ND rats, the dilatory response associated with sciatic nerve stimulation was decreased by ～30%, whereas pial arteriolar dilations to NS-1619 and K(+) were largely suppressed. These responses were completely restored by the acute topical application of a PKC antagonist, calphostin C. Moreover, the suffusion of a PKC activator, phorbol 12,13-dibutyrate, in ND rats was able to reproduce the vascular reactivity impairments found in T1DM rats. Assay of PKC activity in brain samples from T1DM vs. ND rats revealed a significant gain in activity only in specimens harvested from the pial and superficial glia limitans tissue, but not in bulk cortical gray matter. Altogether, these findings suggest that the T1DM-associated impairment of neurovascular coupling may be mechanistically linked to a readily reversible PKC-mediated depression of BK(Ca) and Kir channel activity.     

Reverse electron flow-induced ROS production is attenuated by activation of mitochondrial Ca2+-sensitive K+ channels

Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O(2) concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH(2)-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain and that this process generates superoxide (O(2)(*-)); these effects are blocked by the complex I blocker rotenone. We demonstrated recently that succinate + rotenone-dependent H(2)O(2) production in isolated mitochondria increased mildly on activation of the putative big mitochondrial Ca(2+)-sensitive K(+) channel (mtBK(Ca)) by low concentrations of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619). In the present study we examined effects of NS-1619 on mitochondrial O(2) consumption, membrane potential (DeltaPsi(m)), H(2)O(2) release rates, and redox state in isolated guinea pig heart mitochondria respiring on succinate but without rotenone. NS-1619 (30 microM) increased state 2 and state 4 respiration by 26 +/- 4% and 14 +/- 4%, respectively; this increase was abolished by the BK(Ca) channel blocker paxilline (5 microM). Paxilline alone had no effect on respiration. NS-1619 did not alter DeltaPsi(m) or redox state but decreased H(2)O(2) production by 73% vs. control; this effect was incompletely inhibited by paxilline. We conclude that under substrate conditions that allow reverse electron flow, matrix K(+) influx through mtBK(Ca) channels reduces mitochondrial H(2)O(2) production by accelerating forward electron flow. Our prior study showed that NS-1619 induced an increase in H(2)O(2) production with blocked reverse electron flow. The present results suggest that NS-1619-induced matrix K(+) influx increases forward electron flow despite the high reverse electron flow, and emphasize the importance of substrate conditions on interpretation of effects on mitochondrial bioenergetics.     

Suppression of human detrusor smooth muscle excitability and contractility via pharmacological activation of large conductance Ca2+-activated K+ channels

Overactive bladder syndrome is frequently associated with increased detrusor smooth muscle (DSM) contractility. We tested the hypothesis that pharmacological activation of the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel with NS-1619, a selective BK channel opener, reduces the excitability and contractility of human DSM. We used the amphotericin-perforated whole cell patch-clamp technique on freshly isolated human DSM cells, live-cell Ca(2+) imaging, and isometric DSM tension recordings of human DSM strips obtained from open bladder surgeries. NS-1619 (30 μM) significantly increased the amplitude of the voltage step-induced whole cell BK currents, and this effect was abolished by pretreatment with 200 nM iberiotoxin (IBTX), a selective BK channel inhibitor. In current-clamp mode, NS-1619 (30 μM) significantly hyperpolarized the resting membrane potential, and the hyperpolarization was reversed by IBTX (200 nM). NS-1619 (30 μM) significantly decreased the intracellular Ca(2+) level in isolated human DSM cells. BK channel activation with NS-1619 (30 μM) significantly inhibited the amplitude, muscle force, frequency, duration, and tone of the spontaneous phasic and pharmacologically induced DSM contractions from human DSM isolated strips. IBTX (200 nM) suppressed the inhibitory effects of NS-1619 on spontaneous contractions. The amplitude of electrical field stimulation (0.5-50 Hz)-induced contractions was significantly reduced by NS-1619 (30 μM). Our data suggest that pharmacological activation of BK channels could represent a novel treatment option to control bladder dysfunction in humans.     

Carbon monoxide and Ca2+-activated K+ channels in cerebral arteriolar responses to glutamate and hypoxia in newborn pigs

Large-conductance calcium-activated potassium (K(Ca)) channels regulate the physiological functions of many tissues, including cerebrovascular smooth muscle. l-Glutamic acid (glutamate) is the principal excitatory neurotransmitter in the central nervous system, and oxygen tension is a dominant local regulator of vascular tone. In vivo, glutamate and hypoxia dilate newborn pig cerebral arterioles, and both dilations are blocked by inhibition of carbon monoxide (CO) production. CO dilates cerebral arterioles by activating K(Ca) channels. Therefore, the present study was designed to investigate the effects of glutamate and hypoxia on cerebral CO production and the role of K(Ca) channels in the cerebral arteriolar dilations to glutamate and hypoxia. In the presence of iberiotoxin or paxilline that block dilation to the K(Ca) channel opener, NS-1619, neither CO nor glutamate dilated pial arterioles. Conversely, neither paxilline nor iberiotoxin inhibited dilation to acute severe or moderate prolonged hypoxia. Both glutamate and hypoxia increased cerebrospinal fluid (CSF) CO concentration. Iberiotoxin that blocked dilation to glutamate did not attenuate the increase in CSF CO. The guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), which blocked dilation to sodium nitroprusside, did not inhibit dilation to hypoxia. These data suggest that dilation of newborn pig pial arterioles to glutamate is mediated by activation of K(Ca) channels, consistent with the intermediary signal being CO. Surprisingly, although 1) heme oxygenase (HO) inhibition attenuates dilation to hypoxia, 2) hypoxia increases CSF CO concentration, and 3) K(Ca) channel antagonists block dilation to CO, neither K(Ca) channel blockers nor ODQ altered dilation to hypoxia, suggesting the contribution of the HO/CO system to hypoxia-induced dilation is not by stimulating vascular smooth muscle K(Ca) channels or guanylyl cyclase.     

Heat shock protein modulation of KATP and KCa channel cerebrovasodilation after brain injury

Fluid percussion brain injury (FPI) impairs pial artery dilation to activators of the ATP-sensitive (K(ATP)) and calcium-activated (K(Ca)) K(+) channels. This study investigated the role of heat shock protein (HSP) in the modulation of K(+) channel-induced pial artery dilation after FPI in newborn pigs equipped with a closed cranial window. Under nonbrain injury conditions, topical coadministration of exogenous HSP-27 (1 mug/ml) blunted dilation to cromakalim, CGRP, and NS-1619 (10(-8) and 10(-6) M; cromakalim and CGRP are K(ATP) agonists and NS-1619 is a K(Ca) agonist). In contrast, coadministration of exogenous HSP-70 (1 mug/ml) potentiated dilation to cromakalim, CGRP, and NS-1619. FPI increased the cerebrospinal fluid (CSF) concentration of HSP-27 from 0.051 +/- 0.012 to 0.113 +/- 0.035 ng/ml but decreased the CSF concentration of HSP-70 from 50.42 +/- 8.96 to 30.9 +/- 9.9 ng/ml at 1 h postinsult. Pretreatment with topical exogenous HSP-70 (1 mug/ml) before FPI fully blocked injury-induced impairment of cromakalim and CGRP dilation and partially blocked injury-induced impairment of dilation to NS-1619. These data indicate that HSP-27 and HSP-70 contribute to modulation of K(+) channel-induced pial artery dilation. These data suggest that HSP-70 is an endogenous protectant of which its actions may be unmasked and/or potentiated with exogenous administration before brain injury.     

Role of large-conductance Ca²+-activated K+ channels in adenosine A₁ receptor-mediated pharmacological postconditioning in H9c2 cells

Ischaemic postconditioning is a phenomenon whereby short periods of ischaemia applied during the start of reperfusion protect the myocardium from the damaging consequences of reperfusion. As such, pharmacological-induced postconditioning represents an attractive therapeutic strategy for reducing reperfusion injury during cardiac surgery and following myocardial infarction. The primary aim of this study was to determine the role of large-conductance Ca2(+)-activated potassium channels (BK(Ca) channels) in adenosine A? receptor-induced pharmacological postconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. H9c2 cells were exposed to 6 h hypoxia (0.5% O?) followed by 18 h reoxygenation (H/R) after which cell viability was assessed by monitoring lactate dehydrogenase (LDH) release and caspase-3 activation. The adenosine A? receptor agonist N?-cyclopentyladenosine (CPA; 100 nmol/L) or the BK(Ca) channel opener NS1619 (10 μmol/L) were added for 30 min at the start of reoxygenation following 6 h hypoxic exposure. Where appropriate, cells were treated (15 min) before pharmacological postconditioning with the BK(Ca) channel blockers paxilline (1 μmol/L) or iberiotoxin (100 nmol/L). Pharmacological postconditioning with CPA or NS1619 significantly reduced H/R-induced LDH release. Treatment with paxilline or iberiotoxin attenuated adenosine A? receptor and NS1619-induced pharmacological postconditioning. These results have shown for the first time that BK(Ca) channels are involved in adenosine A? receptor-induced pharmacological postconditioning in a cell model system.     

Reduced Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels from arteries of Type 2 diabetic Zucker diabetic fatty rats

Although it is well established that diabetes impairs endothelium-dependent vasodilation, including those pathways involving vascular myocyte large-conductance Ca(2+)-activated K(+) channels (BK(Ca)), little is known about the effects of diabetes on BK(Ca) activation as an intrinsic response to contractile stimulation. We have investigated this mechanism in a model of Type 2 diabetes, the male Zucker diabetic fatty (ZDF) rat. BK(Ca) function in prediabetic (5-7 wk) and diabetic (17-20 wk) ZDF and lean control animals was assessed in whole arteries using myograph and electrophysiology techniques and in freshly dissociated myocytes by patch clamping. Log EC(25) values for phenylephrine concentration-tension curves were shifted significantly to the left by blockade of BK(Ca) with iberiotoxin (IBTX) in arteries from non- and prediabetic animals but not from diabetic animals. Smooth muscle hyperpolarizations of arteries evoked by the BK(Ca) opener NS-1619 were significantly reduced in the diabetic group. Voltage-clamp recordings indicated that IBTX-sensitive currents were not enhanced to the extent observed in nondiabetic controls by increasing the Ca(2+) concentration in the pipette solution or the application of NS-1619 in myocytes from diabetic animals. An alteration in the expression of BK(Ca) beta(1) subunits was not evident at either the mRNA or protein level in arteries from diabetic animals. Collectively, these results suggest that myocyte BK(Ca) of diabetic animals does not significantly oppose vasoconstriction, unlike that of prediabetic and control animals. This altered function was related to a reduced Ca(2+)-dependent activation of the channel not involving beta(1) subunits.     

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.     

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.     

Functional and molecular characterization of maxi K+ -channels (BK(Ca)) in buffalo myometrium

Large conductance potassium channels (BK(Ca) channels) play a central role in maintaining myometrial tone, thus activation of these channels proved to have therapeutic potential in preterm labor. Present study aims to unravel the presence of BK(Ca) (maxi-K) channels in buffalo myometrium. Tension experiments, mRNA and protein expression studies were done to characterize BK(Ca) channels in buffalo myometrium. Isolated myometrial preparations exhibited rhythmic spontaneity with regular pattern of amplitude and frequency. Selective blockers of BK(Ca) channels iberiotoxin (IbTx; 100nM) and tetraethylammonium (TEA; 1mM) produced excitatory effects as evidenced by increase in amplitude and frequency of myogenic activity. 1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimi-dazol-2-one (NS-1619; 10(-7)-10(-4)M), a BK(Ca) channel opener, produced concentration-dependent relaxation of myometrium with pD(2) of 5.02±0.19 and R(max) of 31.35±3.5% (n=5). TEA significantly antagonized NS-1619-induced relaxation (pD(2) of 4.72±0.12 and R(max) of 22.72±1.78%; n=5). IbTx also significantly shifted the dose response curve of NS-1619 towards right (pD(2) of 3.98±0.16; n=4) without significant change in the per cent maximal response. Further, RT-PCR study detected mRNA encoding BK(Ca) α-subunit and Western blot analysis detected its protein expression in myometrium. Based on the results of the present investigation, it is suggested that BK(Ca) channels are present in the buffalo myometrium and are open in the resting state. Thus, their activation by potassium channel opener/β(2)-adrenoceptor agonist (tocolytic drug) may lead to uterine relaxation in preterm labor.     

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.     

Role of angiotensin II in sympathetic nervous system induced left ventricular dysfunction

Experiments were undertaken to determine whether angiotensin (Ang) II concentration increases during massive sympathetic nervous system (SNS) activation and whether such an increase plays a role in the pathogenesis of SNS-induced left ventricular (LV) dysfunction. We also sought to determine whether excessive Ca2+ uptake through L-type channels due to intense adrenoceptor activation is responsible for the LV dysfunction. AngII concentration was measured in the plasma and myocardium before and after massively activating the SNS with an intracisternal injection of veratrine. In separate experiments, rabbits were given losartan, enalaprilat, enalaprilat plus HOE-140, nifedipine, -Bay K 4866, or saline before massively activating the SNS. LV function was evaluated 2.5 h later. The intense SNS activity caused plasma and myocardial AngII to increase by 400 and 437%, respectively. AngII receptor blockade did not prevent LV dysfunction. In contrast, enalaprilat reduced the degree of dysfunction, but its cardioprotection was abolished by HOE-140. Although nifedipine prevented SNS-induced LV dysfunction, administration of the Ca2+ channel opener, -Bay K 4866, did not increase its severity. Our results indicate that AngII is not involved in the pathogenesis of SNS-induced LV dysfunction and that the cardioprotection provided by angiotensin converting enzyme (ACE) inhibition is due to activation of a bradykinin pathway. Furthermore, the finding that the magnitude of the LV dysfunction was reduced by enalaprilat, and not increased by -Bay K 4866, suggests that intense adrenoceptor activation of L-type Ca2+ channels is not the primary pathogenetic mechanism.     

Differential role of PTK and ERK MAPK in superoxide impairment of K(ATP) and K(Ca) channel cerebrovasodilation

Previously, superoxide (O2 -) has been observed to impair pial artery dilation (PAD) to activators of the ATP-sensitive (KATP) and calcium-sensitive (KCa) K+ channels. This study tested the hypothesis that activation of protein tyrosine kinase (PTK) and the ERK isoform of MAPK by O2 - contribute to impairment of KATP and KCa channel PAD. Exposure of the cerebral cortex to a xanthine oxidase O2 --generating system (OX) blunted PAD to cromakalim, a KATP agonist, but preadministration of genistein, a PTK antagonist, or U-0126, an ERK MAPK inhibitor, almost completely prevented such impairment (11 +/- 1 and 22 +/- 1 vs. 3 +/- 1 and 7 +/- 1 vs. 10 +/- 1 and 16 +/- 2% for cromakalim with 10-8 and 10-6 M PAD during control, OX, and OX + genistein conditions). In contrast, neither genistein nor U-0126 robustly protected PAD to NS-1619, a KCa agonist, after OX exposure (11 +/- 1 and 18 +/- 2 vs. 1 +/- 1 and 2 +/- 1 vs. 4 +/- 1 and 6 +/- 1% for 10-8 and 10-6 M NS-1619 during control, OX, and OX + genistein conditions). These data show that PTK and ERK MAPK activation contribute to O2 --induced KATP and KCa channel PAD impairment and suggest a differential greater role for PTK and ERK MAPK in KATP vs. KCa channel PAD impairment.     

Effects of potassium channel modulators on myotropic responses of aortic rings of pregnant rats

The contribution of potassium channels [ATP-sensitive potassium (K(ATP)) and high-conductance calcium-activated potassium (BK(Ca)) channels] in the resistance of aortic rings of term pregnant rats to phenylephrine (Phe), arginine vasopressin (AVP), and KCl was investigated. Concentration-response curves to tetraethylammonium (TEA), a nonselective K(+) channel inhibitor, were obtained in the absence or presence of KCl. TEA induced by itself concentration-dependent responses only in aortic rings of nonpregnant rats. These responses to TEA could be modulated in both groups of rings by preincubation with different concentrations of KCl. Concentration-response curves to Phe, AVP, and KCl were obtained in the absence or presence of cromakalim or NS-1619 (K(ATP) and BK(Ca) openers, respectively) and glibenclamide or iberiotoxin (K(ATP) and BK(Ca) inhibitors, respectively). Cromakalim significantly inhibited the responses to the three agonists in a concentration-dependent manner in both groups of rats. Alternatively, in the pregnant group of rats, glibenclamide increased the sensitivity to all three agonists. NS-1619 also inhibited the response to all agonists. With AVP and KCl, its effect was greater in aortic rings of pregnant than nonpregnant rats. Finally, iberiotoxin increased the sensitivity to all three agents. This effect was more important in aortic rings of nonpregnant rats and was accompanied by an increase of the maximal response to Phe and AVP. These results suggest that potassium channels are implicated in the control of basal membrane potential and in the blunted responses to these agents during pregnancy.     

BK channels are linked to inositol 1,4,5-triphosphate receptors via lipid rafts: a novel mechanism for coupling [Ca(2+)](i) to ion channel activation

Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K(+) conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca(2+) increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca(2+)-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP(3)R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-beta-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K(+) conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca(2+) in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP(3)Rs negated the effect of methyl-beta-cyclodextrin, providing further support that IP(3)Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca(2+)-sensitive channels to their second messenger source.     

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.     

Opening of Ca2+-activated K+ channels triggers early and delayed preconditioning against I/R injury independent of NOS in mice

Opening of Ca2+-activated K+ (KCa) channels has been shown to confer early cardioprotection. It is unknown whether the opening of these channels also induces delayed cardioprotection. In addition, we determined the involvement of nitric oxide synthases (NOSs), which have been implicated in cardioprotection induced by opening of mitochondrial ATP-sensitive K+ (KATP) channels. Adult male ICR mice were pretreated with the KCa-channel opener NS-1619 either 10 min or 24 h before 30 min of global ischemia and 60 min of reperfusion (I/R) in Langendorff mode. Infusion of NS-1619 (10 microM) for 10 min before I/R led to smaller infarct sizes as compared with the vehicle (DMSO)-treated group (P <0.05). This infarct-limiting effect of NS-1619 was associated with improvement in ventricular functional recovery after I/R. The NS-1619-induced protection was abolished by coadministration with the KCa-channel blocker paxilline (1 microM). Similarly, pretreatment with NS-1619 (1 mg/kg ip) induced delayed protection 24 h later (P <0.05). Interestingly, the NS-1619-induced late protection was not blocked by the NOS inhibitor Nomega-nitro-L-arginine methyl ester (15 mg/kg ip). Unlike diazoxide (the opener of mitochondrial KATP channels), NS-1619 did not increase the expression of inducible or endothelial NOS. Western blot analysis demonstrated the existence of alpha- and beta-subunits of KCa channels in mouse heart tissue. We conclude that opening of KCa channels leads to both early and delayed preconditioning effects through a mechanism that is independent of nitric oxide.     

Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation

ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BK(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BK(Ca) channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2(-*)), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BK(Ca) channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2(-*), or an antagonist of the BK(Ca) channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2(-*) generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBK(Ca) channels; 2) NS effects are blocked by BK(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.     

Maternal diabetes increases large conductance Ca2+-activated K+ outward currents that alter action potential properties but do not contribute to attenuated excitability of parasympathetic cardiac motoneurons in the nucleus ambiguus of neonatal mice

Previously, we demonstrated that maternal diabetes reduced the excitability and increased small-conductance Ca(2+)-activated K(+) (SK) currents of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA). In addition, blockade of SK channels with apamin completely abolished this reduction. In the present study, we examined whether maternal diabetes affects large-conductance Ca(2+)-activated K(+) (BK) channels and whether BK channels contribute to the attenuation of PCMN excitability observed in neonates of diabetic mothers. Neonatal mice from OVE26 diabetic mothers (NMDM) and normal FVB mothers (control) were used. The pericardial sac of neonatal mice at postnatal days 7-9 was injected with the tracer X-rhodamine-5 (and 6)-isothiocyanate 2 days prior to the experiment to retrogradely label PCMNs in the NA. Whole cell current- and voltage-clamps were used to measure spike frequency, action potential (AP) repolarization (half-width), afterhyperpolarization potential (AHP), transient outward currents, and afterhyperpolarization currents (I(AHP)). In whole cell voltage clamp mode, we confirmed that maternal diabetes increased transient outward currents and I(AHP) compared with normal cells. Using BK channel blockers charybdotoxin (CTx) and paxilline, we found that maternal diabetes increased CTx- and paxilline-sensitive transient outward currents but did not change CTx- and paxilline-sensitive I(AHP). In whole cell current-clamp mode, we confirmed that maternal diabetes increased AP half-width and AHP, and reduced excitability of PCMNs. Furthermore, we found that after blockade of BK channels with CTx or paxilline, maternal diabetes induced a greater increase of AP half-width but similarly decreased fast AHP without affecting medium AHP. Finally, blockade of BK channels decreased spike frequency in response to current injection in both control and NMDM without reducing the difference of spike frequency between the two groups. Therefore, we conclude that although BK transient outward currents, which may alter AP repolarization, are increased in NMDM, BK channels do not directly contribute to maternal diabetes-induced attenuation of PCMN excitability. In contrast, based on evidence from our previous and present studies, reduction of PCMN excitability in neonates of diabetic mothers is largely dependent on altered SK current associated with maternal diabetes.