BK Channel Inhibitor Research Articles

Urine Stimulation Activates BK Channels in Mouse Vomeronasal Neurons

Most odor responses in mouse vomeronasal neurons are mediated by the phospholipase C (PLC) pathway, activation of which elevates diacylglycerol (DAG). Lucas et al. showed that DAG activates transient receptor potential channels, subfamily C, member 2 (TRPC2), resulting in a depolarizing Ca2+ influx. DAG can be subsequently converted to arachidonic acid (AA) by a DAG lipase, the role of which remains largely unknown. In this study, we found that urine stimulation of vomeronasal neurons activated large-conductance Ca2+-activated K+ (BK) channels via AA production. Using isolated neurons, we demonstrated that repetitive applications of AA potentiated a K+ current that required a Ca2+ influx and was sensitive to specific BK blockers. Using immunocytochemistry, we found that BK channels are present in vomeronasal neurons with labeling on the soma and heavy labeling on the dendrite with a BK channel antibody. We examined the role of these BK channels in regulating neuronal firing when the neuron was activated by membrane depolarization or urine. Contrary to a recent report, our data suggest that BK channels contribute to adaptation of urine/odor responses because the inhibition of BK channels during urine stimulation promoted repetitive firing. These data strongly support the hypothesis that AA mediates an inhibitory pathway through BK channels, a possible mechanism for odor adaptation in vomeronasal neurons.

β-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca2+-activated K+ channel

In urinary bladder smooth muscle (UBSM), stimulation of beta-adrenergic receptors (beta-ARs) leads to activation of the large-conductance Ca2+-activated K+ (BK) channel currents (Petkov GV and Nelson MT. Am J Physiol Cell Physiol 288: C1255-C1263, 2005). In this study we tested the hypothesis that the BK channel mediates UBSM relaxation in response to beta-AR stimulation using the highly specific BK channel inhibitor iberiotoxin (IBTX) and a BK channel knockout (BK-KO) mouse model in which the gene for the pore-forming subunit was deleted. UBSM strips isolated from wild-type (WT) and BK-KO mice were stimulated with 20 mM K+ or 1 microM carbachol to induce phasic and tonic contractions. BK-KO and WT UBSM strips pretreated with IBTX had increased overall contractility, and UBSM BK-KO cells were depolarized with approximately 12 mV. Isoproterenol, a nonspecific beta-AR agonist, and forskolin, an adenylate cyclase activator, decreased phasic and tonic contractions of WT UBSM strips in a concentration-dependent manner. In the presence of IBTX, the concentration-response curves to isoproterenol and forskolin were shifted to the right in WT UBSM strips. Isoproterenol- and forskolin-mediated relaxations were enhanced in BK-KO UBSM strips, and a leftward shift in the concentration-response curves was observed. The leftward shift was eliminated upon PKA inhibition with H-89, suggesting upregulation of the beta-AR-cAMP pathway in BK-KO mice. These results indicate that the BK channel is a key modulator in beta-AR-mediated relaxation of UBSM and further suggest that alterations in BK channel expression or function could contribute to some pathophysiological conditions such as overactive bladder and urinary incontinence.

M2 Muscarinic Receptors Induce Airway Smooth Muscle Activation via a Dual, Gβγ-mediated Inhibition of Large Conductance Ca2+-activated K+ Channel Activity

Airway smooth muscle is richly endowed with muscarinic receptors of the M(2) and M(3) subtype. Stimulation of these receptors inhibits large conductance calcium-activated K(+) (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M(2) or M(3) receptor (M(2)R or M(3)R). In M(2)R- but not M(3)R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M(2)R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gbetagamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nm purified Gbetagamma decreased channel open probability by 55%. The physical interaction of Gbetagamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (approximately 20%) extent. Mouse tracheal cells responded similarly to CCh, purified Gbetagamma and phospholipase C/protein kinase C inhibition as M(2)R-expressing HEK293 cells. Our results demonstrate that airway M(2)Rs inhibit BK channels by a dual, Gbetagamma-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.

1-[1-Hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone, a Potent and Highly Selective Small Molecule Blocker of the Large-Conductance Voltage-Gated and Calcium-Dependent K+Channel

The large-conductance voltage-gated and calcium-dependent K(+) (BK) channels are widely distributed and play important physiological roles. Commonly used BK channel inhibitors are peptide toxins that are isolated from scorpion venoms. A high-affinity, nonpeptide, synthesized BK channel blocker with selectivity against other ion channels has not been reported. We prepared several compounds from a published patent application (Doherty et al., 2004) and identified 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP) as a potent and selective BK channel blocker. The patch-clamp technique was used for characterizing the activity of HMIMP on recombinant human BK channels (alpha subunit, alpha+beta1 and alpha+beta4 subunits). HMIMP blocked all of these channels with an IC(50) of approximately 2 nM. The inhibitory effect of HMIMP was not voltage-dependent, nor did it require opening of BK channels. HMIMP also potently blocked BK channels in freshly isolated detrusor smooth muscle cells and vagal neurons. HMIMP (10 nM) reduced the open probability significantly without affecting single BK-channel current in inside-out patches. HMIMP did not change the time constant of open states but increased the time constants of the closed states. More importantly, HMIMP was highly selective for the BK channel. HMIMP had no effect on human Na(V)1.5 (1 microM), Ca(V)3.2, L-type Ca(2+), human ether-a-go-go-related gene potassium channel, KCNQ1+minK, transient outward K(+) or voltage-dependent K(+) channels (100 nM). HMIMP did not change the action potentials of ventricular myocytes, confirming its lack of effect on cardiac ion channels. In summary, HMIMP is a highly potent and selective BK channel blocker, which can serve as an important tool in the pharmacological study of the BK channel.

BK Channel Modulators: A Comprehensive Overview

The large Ca(2+)-activated K(+) channel (BK channel) reflects per excellence the dilemma of the molecular target driven drug discovery process. Significant experimental evidence suggests that the BK channels play a pivotal and specific role in many pathophysiological conditions supporting the notion that the channel represents an innovative and promising drug target. However, after more than ten years of intense research effort both in academia and industry, scientists have yet to witness the approval of a single BK channel modulator for clinical use. On the contrary, three BK openers that were progressed to clinical development have recently been discontinued (NS8, BMS204352 and TA1702) and, at the present time, only one drug candidate targeting BK channels (andolast) remains in the early phases of clinical development. Since biological studies keep strengthening the concept of BK channels as a potentially attractive target, the design and synthesis of potent and selective BK modulators continue based on novel chemical ideas. A comprehensive overview of BK channel modulators is therefore timely and important to the current medicinal chemist for review, summary, and classification of the multitude of chemical entities claimed to be BK-modulating agents. Such chemical entities are, herein, classified by both origin and chemical structure in 1) Endogenous BK channel modulators and structural analogues 2) Naturally-occurring BK channel inhibitors and blockers 3) Synthetic BK channel inhibitors and blockers 4) Marketed and/or investigational drugs with BK-modulating side properties and structural analogues 5) Naturally-occurring BK channel openers and structural analogues 6) Synthetic BK channel openers. This review is intended to provide readers with current opinion on the BK channel as a drug target, the chemical structures of BK channel modulators, the structural and chemical features involved in the BK channel modulating activity and, where and when possible, with highlights of structure-activity relationships.

ASIC Channels Inhibit BK Potassium Channels by a Toxin‐Like Extracellular Motif

Acid‐sensing ion channels (ASICs) contain a large extracellular domain whose function has remained largely unknown. Within this domain sits a highly conserved sequence which resembles the scorpion toxin sequence that binds and inhibits large conductance calcium‐activated potassium channel (BK). We hypothesized that ASICs might interact with and inhibit BK. In transfected HEK 293 cells, we found that ASIC1a inhibited BK currents. Lowering extracellular pH relieved the inhibition.ASIC2a, −2b and −3 also inhibited BK currents, and the pH‐dependent relief of inhibition varied between ASIC channels. In scorpion toxins, the cationic residues in the RGKC/RFGKC sequence are critical for inhibition. When we mutated the equivalent R and K residues in ASICs to A or E, the inhibition of BK channels was abolished. To test for an interaction of the endogenous proteins, we studied cultured cortical neurons and found an ASIC‐dependent inhibition of BK current and its pH‐dependent relief. BK currents were larger in ASIC1a/2/3 knockout mice, consistent with the lack of inhibition. In addition, the two proteins co‐precipitated from transfected cells and whole brain. These data indicate that a toxin‐like sequence in the extracellular domain of ASICs inhibits BK channels. This novel mechanism for regulating potassium channel activity may influence neuronal excitability. Support: HL014388 to FMA.

SK4 mediated K+ secretion depends on P2Y receptor localization in human mammary epithelial cells

Immortalized human mammary epithelial cells express multiple P2Y receptors that are localized in both apical and basolateral membranes. Basolateral UTP (10 μM) stimulation of monolayers mounted in Ussing chambers produced an increase in ENaC‐dependent Na+ absorption resulting in part from SK4 channel activation in the basolateral membrane. In contrast apical stimulation with UTP (10 μM) produced a transient decrease in Isc consistent with transepithelial K+ secretion. Inhibition of Ca2+ mobilization evoked by UTP using BAPTA‐AM abolished the decrease in Isc. In addition, pretreatment with a known Ca2+‐activated K+ channel toxin, charybdotoxin (200 nM), blocked the Isc response by >70%. A similar degree of inhibition was produced by pretreatment with clotrimazole (10 μM) and TRAM‐34 (0.5 μM). Apical addition of the selective BK channel inhibitor, iberiotoxin also affected the response to apical UTP by stimulating oscillations in the Isc. These results suggest that apical UTP stimulation elicits K+ secretion that is mediated by BK and SK4 potassium channels. Moreover, apical UTP failed to stimulate ENaC‐dependent Na+ absorption observed following basolateral UTP treatment, suggesting that the Ca2+‐mobilizing effects of apical UTP are limited to the apical membrane.

Stimulation of β3–adrenoceptors activates spontaneous transient outward BK currents and causes relaxation in rat urinary bladder smooth muscle

The role of large conductance Ca2+‐activated K+ (BK) channels in the β3‐adrenoceptor (β3‐AR)‐induced relaxation in rat urinary bladder smooth muscle (UBSM) was studied. BRL 37344, a specific β3‐AR agonist, inhibits spontaneous contractions of isolated UBSM strips. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration‐response curves for contraction amplitude, muscle force, and tone to the right. Freshly isolated UBSM cells and the perforated patch‐clamp technique were used to determine further the effect of β3‐AR stimulation on the BK channel activity. BRL 37344 increased spontaneous transient outward BK current (STOC) frequency by 46%. In the presence of ryanodine and nifedipine, the single BK channel amplitude and open probability recorded in cell‐attached mode at Vh=0 mV as well as the current‐voltage relationship in response to depolarization steps were not affected by BRL37344. In current‐clamp mode, BRL 37344 caused membrane potential hyperpolarization of about 3 mV. The BRL 37344‐induced hyperpolarization was eliminated by application of iberiotoxin or 1 mM tetraethylammonium. This study provides evidence that in rat UBSM, β3‐ARs and BK channels are functionally coupled so that stimulation of β3‐AR leads to an increase in the BK channel STOC frequency, which causes membrane hyperpolarization and relaxation. Supported by DK070909 to G.V.P.

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-β

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.

Hypoxia Increases Activity of the BK-Channel in the Inner Mitochondrial Membrane and Reduces Activity of the Permeability Transition Pore

Hypoxia can cause severe damage to cells by initiating signaling cascades that lead to cell death. A cellular oxygen sensor, other than the respiratory chain, might exist in sensitive components of these signaling cascades. Recently, we found evidence that mitochondrial ion channels are sensitive to low levels of oxygen. We therefore studied the effects of hypoxia on the mitochondrial BK-channel (mtBK), on the mitochondrial permeability transition pore (PTP), and on their possible interaction. Using single-channel patch-clamp techniques we found that hypoxia inhibited the PTP but substantially increased the mtBK activity of mitoplasts from rat liver and astrocytes. Experiments measuring the mitochondrial membrane potential of intact rat brain mitochondria (using the fluorescence dye safranine O) during hypoxia exhibited an increased Ca²⁺-retention capacity implying an impaired opening of the PTP. We also found a reduced Ca²⁺-retention capacity with 100 nM iberiotoxin, a selective inhibitor of BK-channels. We therefore conclude that there is interaction between the mtBK and the PTP in a way that an open mtBK keeps the PTP closed. Thus, the response of mitochondrial ion channels to hypoxia could be interpreted as anti-apoptotic.

Abstract 971: Inhibition Of Vascular Bk Channel Activity By Caveolae-mediated Angiotensin II Type I Receptor Signaling In Diabetic Vessels

Angiotensin II (Ang II) type I receptor (ATR 1 ) trafficking into caveolae is essential for Ang II signaling, which is known to be abnormal in diabetic vessels. We have shown that the large conductance Ca 2+ actviated K + (BK) channels are also targeted to caveolae in vascular cells. The potential interaction between Ang II signaling and BK channel function in normal and diabetic vessels is unknown. Using whole-cell patch clamp recordings and molecular biology techniques, we examined the mechanisms through which caveolae targeting facilitates the regulation of BK channels by Ang II signaling. We found that in cultured human coronary arterial smooth muscle cells (CASMC) and in freshly isolated rat CASMC, BK channels, ATR 1 , and Src-family protein tyrosine kinases (Src-PTK) were colocalized and enriched in the low buoyant density, caveolae-rich fractions. 2 μM Ang II inhibited BK channel activity by ∼50% in rat and human CASMC and these effects were completely abolished by 2 μM Losartan (a selective ATR 1 inhibitor), 10 μM PP2 (a selective Src-PTK inhibitor), and by caveolin-1 (cav-1) knockdown using 60 nM siRNA. Similar results were obtained in HEK293 cells coexpressing hSlo, BK-β 1 subunit, ATR 1 , cav-1, and Src-PTK, indicating that inhibition of BK channels by Ang II was mediated through ATR 1 activation of Src-PTK and the integrity of caveolae is critical for Ang II signaling. Culturing human CASMC with high glucose (HG, 22 mM) enhanced Ang II-mediated BK channel inhibition (78.8±16.8% vs. 54.5±15.7% in 5 mM glucose, n=3, p<0.05). Analysis of ATR 1 , Src-PTK, and BK channel distribution by sucrose gradient fractionation and by co-immunoprecipitation with anti-cav-1 antibodies showed that expression of ATR 1 and Src-PTK were up-regulated in human CASMC cultured in HG and in CASMC from streptozotocin-induced diabetic rats. Total BK channel protein in these cells was diminished, but the amount of BK channels co-immunoprecipitated with anti-cav-1 antibody was increased, suggesting increased caveolae targeting of BK channels in diabetes, which leads to enhanced Ang II-mediated BK channel inhibition. These results indicate that Ang II-BK channel interaction is critically dependent upon caveolae targeting under normal conditions and in disease states such as diabetes.

Regulation of STREX exon large conductance, calcium-activated potassium channels by the β4 accessory subunit

Large conductance (BK-type) calcium-activated potassium channels utilize alternative splicing and association with accessory β subunits to tailor BK channel properties to diverse cell types. Two important modulators of BK channel gating are the neuronal-specific beta4 accessory subunit (β4) and alternative splicing at the stress axis hormone-regulated exon (STREX). Individually, these modulators affect the gating properties of the BK channel as well as its response to phosphorylation. In this study, the combined functional consequences of STREX and the mouse β4 subunit on mouse BK channel biophysical properties were investigated in transfected HEK 293 cells. Surprisingly, we found that the combined effects of STREX and β4 are non-additive and even opposite for some properties. At high calcium, β4 and the STREX individually share properties that promote BK channel opening via slowing of deactivation. However, the combined effects are a speeding of deactivation and a decreased open probability. β4 also inhibits BK channel opening by a slowing of activation. This effect occurs across calcium concentrations in the absence of STREX, but predominates only at low calcium for STREX containing channels. BK channel responses to phosphorylation status are also altered by the combination of the β4 subunit and STREX. β4/STREX channels show a slowing of activation kinetics following dephosphorylation whereas β4 channels lacking STREX do not. In contrast, β4 confers a speeding of activation in response to cyclic AMP–dependent phosphorylation in channels lacking STREX, but not in channels containing STREX. These results indicate that the combination of the β4 subunit and STREX confers non-additive and unique properties to BK channels. Analysis of expression in brain slices suggests that STREX and β4 mRNA overlap expression in the dentate gyrus of the hippocampus and the cerebellar Purkinje cells, suggesting that these unique properties of BK channels may underlie BK channel gating in these cells.

Electrophysiology of the phagocyte respiratory burst. Focus on “Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity”

there is a trend, which seems quite reasonable to me, toward giving papers titles that are declarative summaries of the main conclusion of the paper, in contrast to traditional titles, which simply describe the topic studied. A busy (or lazy) reader can extract the gist of the story without even

Synaptic plasticity in myenteric neurons of the guinea‐pig distal colon: presynaptic mechanisms of inflammation‐induced synaptic facilitation

The purpose of this study was to investigate the pre- and postsynaptic mechanisms that contribute to synaptic facilitation in the myenteric plexus of the trinitrobenzene sulphonic acid-inflamed guinea-pig distal colon. Intracellular recordings of evoked fast excitatory postsynaptic potentials (fEPSPs) in myenteric S neurons were evaluated, and the density of synaptic terminals was morphometrically analysed by transmission electron microscopy. In inflamed tissue, fEPSPs were reduced to control levels by the protein kinase A (PKA) inhibitor, H89, but H89 did not affect the fEPSPs in control tissue. This PKA activation in inflamed tissue did not appear to involve 5-HT(4) receptors because the antagonist/inverse agonist, GR 125487, caused comparable decreases of fEPSPs in both tissues. Inhibition of BK channels with iberiotoxin did not alter the fEPSPs in inflamed tissue, but increased the fEPSPs in control tissue to the amplitude detected in inflamed tissue. During trains of stimuli, run-down of EPSPs was less extensive in inflamed tissue and there was a significant increase in the paired pulse ratio. Depolarizations in response to exogenous neurotransmitters were not altered in inflamed tissue. These inflammation-induced changes were not accompanied by alterations in the pharmacological profile of EPSPs, and no changes in synaptic density were detected by electron microscopy. Collectively, these data indicate that synaptic facilitation in the inflamed myenteric plexus involves a presynaptic increase in PKA activity, possibly involving an inhibition of BK channels, and an increase in the readily releasable pool of synaptic vesicles.

Depletion of membrane cholesterol eliminates the Ca2+‐activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes

Changes in membrane cholesterol content have potent effects on cell signalling and contractility in rat myometrium and other smooth muscles. We have previously shown that depletion of cholesterol with methyl-beta-cyclodextrin (MCD) disrupts caveolar microdomains. The aim of this work was to determine the mechanism underlying the increase in Ca(2+) signalling and contractility occurring in the myometrium with MCD. Patch clamp data obtained on freshly isolated myocytes from the uterus of day 19-21 rats showed that outward K(+) current was significantly reduced by MCD. Membrane capacitance was also reduced. Cholesterol-saturated MCD had no effect on the amplitude of outward current suggesting that the reduction in the outward current was due to cholesterol depletion induced by MCD rather than a direct inhibitory action of MCD on the K(+) channels. Confocal visualization of the membrane bound indicator Calcium Green C18, revealed internalization of the surface membrane with MCD treatment. Large conductance, Ca(2+)-sensitive K(+) channel proteins have been shown to localize to caveolae. When these channels were blocked by iberiotoxin outward current was significantly reduced in the uterine myocytes; MCD treatment reduced the density of outward current. Following reduction of outward current by MCD pretreatment, iberiotoxin was unable to produce any additional decrease in the current, suggesting a common target. MCD treatment also increased the amplitude and frequency of spontaneous rises in cytosolic Ca(2+) level ([Ca(2+)](i) transients) in isolated myocytes. In intact rat myometrium, MCD treatment increased Ca(2+) signalling and contractility, consistent with previous findings, and this effect was also found to be reduced by BK channel inhibition. These data suggest that (1) disruption of cholesterol-rich microdomains and caveolae by MCD leads to a decrease in the BK channel current thus increasing cell excitability, and (2) the changes in membrane excitability produced by MCD underlie the changes found in Ca(2+) signalling and uterine contractility.

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca(2+)-activated K(+) (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK(-/-)) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O(2)(-) and H(2)O(2) production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn(2+), which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca(2+) and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca(2+) by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK(-/-) and BK(+/+) mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity.

Diabetes Downregulates Large-Conductance Ca 2+ -Activated Potassium β1 Channel Subunit in Retinal Arteriolar Smooth Muscle

Retinal vasoconstriction and reduced retinal blood flow precede the onset of diabetic retinopathy. The pathophysiological mechanisms that underlie increased retinal arteriolar tone during diabetes remain unclear. Normally, local Ca(2+) release events (Ca(2+)-sparks), trigger the activation of large-conductance Ca(2+)-activated K(+)(BK)-channels which hyperpolarize and relax vascular smooth muscle cells, thereby causing vasodilatation. In the present study, we examined BK channel function in retinal vascular smooth muscle cells from streptozotocin-induced diabetic rats. The BK channel inhibitor, Penitrem A, constricted nondiabetic retinal arterioles (pressurized to 70mmHg) by 28%. The BK current evoked by caffeine was dramatically reduced in retinal arterioles from diabetic animals even though caffeine-evoked [Ca(2+)](i) release was unaffected. Spontaneous BK currents were smaller in diabetic cells, but the amplitude of Ca(2+)-sparks was larger. The amplitudes of BK currents elicited by depolarizing voltage steps were similar in control and diabetic arterioles and mRNA expression of the pore-forming BKalpha subunit was unchanged. The Ca(2+)-sensitivity of single BK channels from diabetic retinal vascular smooth muscle cells was markedly reduced. The BKbeta1 subunit confers Ca(2+)-sensitivity to BK channel complexes and both transcript and protein levels for BKbeta1 were appreciably lower in diabetic retinal arterioles. The mean open times and the sensitivity of BK channels to tamoxifen were decreased in diabetic cells, consistent with a downregulation of BKbeta1 subunits. The potency of blockade by Pen A was lower for BK channels from diabetic animals. Thus, changes in the molecular composition of BK channels could account for retinal hypoperfusion in early diabetes, an idea having wider implications for the pathogenesis of diabetic hypertension.

Human monocytes kill M-CSF-expressing glioma cells by BK channel activation

In this study, human monocytes/macrophages were observed to kill human U251 glioma cells expressing membrane macrophage colony-stimulating factor (mM-CSF) via a swelling and vacuolization process called paraptosis. Human monocytes responded to the mM-CSF-transduced U251 glioma cells, but not to viral vector control U251 glioma cells (U251-VV), by producing a respiratory burst within 20 min. Using patch clamp techniques, functional big potassium (BK) channels were observed on the membrane of the U251 glioma cell. It has been previously reported that oxygen indirectly regulates BK channel function. In this study, it was demonstrated that prolonged BK channel activation in response to the respiratory burst induced by monocytes initiates paraptosis in selected glioma cells. Forced BK channel opening within the glioma cells by BK channel activators (phloretin or pimaric acid) induced U251 glioma cell swelling and vacuolization occurred within 30 min. U251 glioma cell cytotoxicity, induced by using BK channel activators, required between 8 and 12 h. Swelling and vacuolization induced by phloretin and pimaric acid was prevented by iberiotoxin, a specific BK channel inhibitor. Confocal fluorescence microscopy demonstrated BK channels co-localized with the endoplasmic reticulum and mitochondria, the two targeted organelles affected in paraptosis. Iberiotoxin prevented monocytes from producing death in mM-CSF-expressing U251glioma cells in a 24 h assay. This study demonstrates a novel mechanism whereby monocytes can induce paraptosis via the disruption of internal potassium ion homeostasis.

Acute antihypertensive response to tempol in the SHR depends on ATP‐sensitive potassium channel (K ATP ), but not large‐conductance Ca2+‐activated potassium (BK) channels

Acute intravenous (IV) administrations of the piperidine nitroxides reduce BP and heart rate (HR) in SHR in proportion to their superoxide dismutase (SOD) mimetic activity. Blockade of nitric oxide synthase (NOS) blunted 40% of the fall in MAP with IV tempol whereas blockade of ganglionic sympathetic neurotransmission with hexamethonium blunted 40% of the fall in MAP, and combined blockade blunted about 80% of the fall in BP. Central administration of tempol was ineffective. Here, we tested the hypothesis that the tempol activates potassium conductances in vivo. Tempol caused dose-dependent reductions in MAP and HR maximal at 174 μmol/kg (ΔMAP −56 ± 4 mmHg and ΔHR −34 ± 5 min−1, n=7). Activation of KATP channel with cromakalim (50 μg iv bolus and 50 μg /hr, iv), with coinfusion of norepinephrine to restore BP toward control level significantly reduced tempol antihypertensive response by 40% (at 174 μmol/kg, ΔMAP −33 ± 5 mmHg, p < 0.01), whereas blockade of KATP channels with glibenclamide (15 mg/kg) significantly reduced tempol antihypertensive response by 31% (ΔMAP −46 ± 10 vs. control −67 ± 8 mmHg, n=6; p<0.01). In contrast, inhibition of BK channels with iberiotoxin (100 and 300 μg/kg, n=6) did not affect tempol’s antihypertensive effect. In conclusion, the acute antihypertensive response to IV tempol is mediated via potentiation of NO, reduction in sympathetic tone and activation of KATP, but not BK channels.

Role of BK Channels in Testosterone-Induced Relaxation of the Aorta in Spontaneously Hypertensive Rats

The previous data indicated that the testosterone (Tes)-induced relaxation of thoracic aorta is greater in spontaneously hypertensive rats (SHR) than in normotensive rats (Wistar-Kyoto rats; WKY) and that there were differences between SHR and WKY in the functions of K(ATP), K(v), and K(Ca) channels. The present study was carried out to ascertain the mechanisms of the Tes-induced relaxation. Indomethacin (30 muM) pretreatment suppressed the Tes-induced relaxation. Following noradrenalin (NA)-induced vasoconstriction, the relaxation induced by Tes was significantly attenuated by endothelium removal in SHR (not in WKY), but the dilatory effect of Tes following KCl-induced vasoconstriction was not attenuated by endothelium removal. After tetraethylammonium (K(Ca) channel inhibitor) or iberiotoxin (large conductance, Ca(2+) activated BK channel inhibitor) pretreatment, the Tes-induced relaxation was attenuated in SHR, but not in WKY. This attenuation in SHR was not observed after endothelium removal. The above results suggest that the relaxation induced by Tes following NA-induced vasoconstriction in SHR results from hyperpolarization due to BK channel opening.