Knockdown of the small conductance Ca(2+) -activated K(+) channels is potently cytotoxic in breast cancer cell lines.

BACKGROUND AND PURPOSE: Small conductance calcium-activated potassium (KCa 2.x) channels have a widely accepted canonical function in regulating cellular excitability. In this study, we address a potential non-canonical function of KCa 2.x channels in breast cancer cell survival, using in vitro models. EXPERIMENTAL APPROACH: The expression of all KCa 2.x channel isoforms was initially probed using RT-PCR, Western blotting and microarray analysis in five widely studied breast cancer cell lines. In order to assess the effect of pharmacological blockade and siRNA-mediated knockdown of KCa 2.x channels on these cell lines, we utilized MTS proliferation assays and also followed the corresponding expression of apoptotic markers. KEY RESULTS: All of the breast cancer cell lines, regardless of their lineage or endocrine responsiveness, were highly sensitive to KCa 2.x channel blockade. UCL1684 caused cytotoxicity, with LD50 values in the low nanomolar range, in all cell lines. The role of KCa 2.x channels was confirmed using pharmacological inhibition and siRNA-mediated knockdown. This reduced cell viability and also reduced expression of Bcl-2 but increased expression of active caspase-7 and caspase-9. Complementary to these results, a variety of cell lines can be protected from apoptosis induced by staurosporine using the KCa 2.x channel activator CyPPA. CONCLUSIONS AND IMPLICATIONS: In addition to a well-established role for KCa 2.x channels in migration, blockade of these channels was potently cytotoxic in breast cancer cell lines, pointing to modulation of KCa 2.x channels as a potential therapeutic approach to breast cancer.

Natriuretic peptides modulate ATP-sensitive K(+) channels in rat ventricular cardiomyocytes.

B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), and (Cys-18)-atrial natriuretic factor (4-23) amide (C-ANF), are cytoprotective under conditions of ischemia-reperfusion, limiting infarct size. ATP-sensitive K(+) channel (KATP) opening is also cardioprotective, and although the KATP activation is implicated in the regulation of cardiac natriuretic peptide release, no studies have directly examined the effects of natriuretic peptides on cardiac KATP activity. Normoxic cardiomyocytes were patch clamped in the cell-attached configuration to examine sarcolemmal KATP (sKATP) activity. The KATP opener pinacidil (200 µM) increased the open probability of the patch (NPo; values normalized to control) at least twofold above basal value, and this effect was abolished by HMR1098 10 µM, a selective KATP blocker (5.23 ± 1.20 versus 0.89 ± 0.18; P < 0.001). We then examined the effects of BNP, CNP, C-ANF and 8Br-cGMP on the sKATP current. Bath application of BNP (≥10 nM) or CNP (≥0.01 nM) suppressed basal NPo (BNP: 1.00 versus 0.56 ± 0.09 at 10 nM, P < 0.001; CNP: 1.0 versus 0.45 ± 0.16, at 0.01 nM, P < 0.05) and also abolished the pinacidil-activated current at concentrations ≥10 nM. C-ANF (≥10 nM) enhanced KATP activity (1.00 versus 3.85 ± 1.13, at 100 nM, P < 0.05). The cGMP analog 8Br-cGMP 10 nM dampened the pinacidil-activated current (2.92 ± 0.60 versus 1.53 ± 0.32; P < 0.05). Natriuretic peptides modulate sKATP current in ventricular cardiomyocytes. This may be at least partially associated with their ability to augment intracellular cGMP concentrations via NPR-A/B, or their ability to bind NPR-C with high affinity. Although the mechanism of modulation requires elucidation, these preliminary data give new insights into the relationship between natriuretic peptide signaling and sKATP in the myocardium.

Selectivity mechanisms in MscS-like channels: From structure to function.

The E. coli mechanosensitive (MS) channel of small conductance (EcMscS) is the prototype of a diverse family of channels present in all domains of life. While EcMscS has been extensively studied, recent developments show that MscS may display some characteristics not widely conserved in this protein subfamily. With numerous members now electrophysiologically characterized, this subfamily of channels displays a breadth of ion selectivity with both anion and cation selective members. The selectivity of these channels may be relatively weak in comparison to voltage-gated channels but their selectivity mechanisms represent great novelty. Recent studies have identified unexpected residues important for selectivity in these homologs revealing different selectivity mechanisms than those employed by voltage gated K(+), Na(+), Ca(2+) and Cl(-) channels whose selectivity filters are housed within their transmembrane pores. This commentary looks at what is currently known about MscS subfamily selectivity and begins to unravel the potential physiological relevance of these differences.

A large-conductance (BK) potassium channel subtype affects both growth and mineralization of human osteoblasts.

The pharmacology of the large-conductance K(+) (BK) channel in human osteoblasts is not well defined, and its role in bone is speculative. Here we assess BK channel properties in MG63 cells and primary human osteoblasts and determine whether pharmacological modulation affects cell function. We used RT-PCR and patch-clamp methods to determine the expression of BK channel subunits and cell number assays in the absence and presence of BK channel modulators. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3, and KCNMB4 subunits. The BK channel was voltage dependent, with a mean unitary conductance of 228.8 pS (n = 10) in cell-attached patches (140 mM K(+)/140 mM K(+)) and a conductance of 142.5 pS (n = 16) in excised outside-out and 155 pS (n = 6) in inside-out patches in 3 mM K(+)/140 mM K(+). The selectivity ratio (ratio of K(+) to Na(+) permeability) was 15:1. The channel was blocked by tetraethylammonium (TEA, 0.3 mM), iberiotoxin (5-60 nM), tetrandrine (5-30 microM), and paxilline (10 microM) and activated by isopimaric acid (20 microM). BK channel modulators affected MG63 cell numbers: TEA and tetrandrine significantly increased cell numbers at low concentrations (3 mM and 3 microM, respectively) and reduced cell numbers at higher concentrations (>10 mM and >10 microM, respectively). Neither iberiotoxin (20-300 nM) nor slotoxin (300 nM) affected cell numbers. The increase in cell numbers by TEA was blocked by isopimaric acid. TEA (0.1-3.0 mM) significantly increased mineralization in primary osteoblasts. In conclusion, the BK channel has a distinctive pharmacology and is thus a target for therapeutic strategies aimed at modulating osteoblast proliferation and function.

ATP regulates calcium efflux and growth in E. coli.

Escherichia coli regulates cytosolic free Ca(2+) in the micromolar range through influx and efflux. Herein, we show for the first time that ATP is essential for Ca(2+) efflux and that ATP levels also affect generation time. A transcriptome analysis identified 110 genes whose expression responded to an increase in cytosolic Ca(2+) (41 elevated, 69 depressed). Of these, 3 transport proteins and 4 membrane proteins were identified as potential Ca(2+) transport pathways. Expression of a further 943 genes was modified after 1 h in growth medium containing Ca(2+) relative to time zero. Based on the microarray results and other predicted possible Ca(2+) transporters, the level of cytosolic free Ca(2+) was measured in selected mutants from the Keio knockout collection using intracellular aequorin. In this way, we identified a knockout of atpD, coding for a component of the F(o)F(1) ATPase, as defective in Ca(2+) efflux. Seven other putative Ca(2+) transport proteins exhibited normal Ca(2+) handling. The defect in the DeltaatpD knockout cells could be explained by a 70% reduction in ATP. One millimolar glucose or 1 mM methylglyoxal raised ATP in the DeltaatpD knockout cells to that of the wild type and restored Ca(2+) efflux. One millimolar 2,4-dinitrophenol lowered the ATP in wild type to that in the DeltaatpD cells. Under these conditions, a similar defect in Ca(2+) efflux in wild type was observed in DeltaatpD cells. Ten millimolar concentration of Ca(2+) resulted in a 30% elevation in ATP in wild type and was accompanied by a 10% reduction in generation time under these conditions. Knockouts of pitB, a potential Ca(2+) transporter, atoA, the beta subunit of acetate CoA-transferase likely to be involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate kinase, all indicated no defect in Ca(2+) efflux. We therefore propose that ATP is most likely to regulate Ca(2+) efflux in E. coli through an ATPase.

pH and monovalent cations regulate cytosolic free Ca(2+) in E. coli.

The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca(2+) in E. coli through Ca(2+) influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca(2+) was 0.2-0.5 microM. In the presence of external Ca(2+) (1 mM) at alkaline pH this rose to 4 microM, being reduced to 0.9 microM at acid pH. Removal of external Ca(2+) caused an immediate decrease in cytosolic free Ca(2+) at 50-100 nM s(-1). Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca(2+)/H(+)exchanger, appeared not to be a major Ca(2+)-efflux pathway. In the absence of added Na(+), but with 1 mM external Ca(2+), cytosolic free Ca(2+) rose to approximately 10 microM. The addition of Na(+)(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca(2+) efflux. However, this effect was not specific, since K(+) also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca(2+) efflux. The results were consistent with H(+) and monovalent cations competing with Ca(2+) for a non-selective ion influx channel. Ca(2+) entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca(2+) in Escherichia coli. The number of Ca(2+) ions calculated to move per cell per second ranged from <1 to 100, depending on conditions. Yet a single eukaryote Ca(2+) channel, conductance 100 pS, should conduct >6 million ions per second. This raises fundamental questions about the nature and regulation of Ca(2+) transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.

Methylglyoxal and other carbohydrate metabolites induce lanthanum-sensitive Ca2+ transients and inhibit growth in E. coli.

The results here are the first demonstration of a family of carbohydrate fermentation products opening Ca2+ channels in bacteria. Methylglyoxal, acetoin (acetyl methyl carbinol), diacetyl (2,3 butane dione), and butane 2,3 diol induced Ca2+ transients in Escherichia coli, monitored by aequorin, apparently by opening Ca2+ channels. Methylglyoxal was most potent (K(1/2) = 1 mM, 50 mM for butane 2,3 diol). Ca2+ transients depended on external Ca2+ (0.1-10 mM), and were blocked by La3+ (5 mM). The metabolites affected growth, methylglyoxal being most potent, blocking growth completely up to 5 h without killing the cells. But there was no affect on the number of viable cells after 24 h. These results were consistent with carbohydrate products activating a La3+-sensitive Ca2+ channel, rises in cytosolic Ca2+ possibly protecting against certain toxins. They have important implications in bacterial-host cell signalling, and where numbers of different bacteria compete for the same substrates, e.g., the gut in lactose and food intolerance.

Cytosolic Ca2+ regulates protein expression in E. coli through release from inclusion bodies.

The results here are the first clear demonstration of a physiological role for cytosolic Ca(2+) in Escherichia coli by releasing a Ca(2+) binding protein, apoaequorin, from inclusion bodies. In growth medium LB the cytosolic free Ca(2+) was 0.1-0.3 microM. Addition of EGTA reduced this to <0.1 microM, whereas addition of Ca(2+) (10mM) resulted in a cytosolic free Ca(2+) of 1-2 microM for at least 2h. Ca(2+) caused a 1.5- to 2-fold increase in the level of apoaequorin induced by IPTG. Whereas EGTA induced a 50% decrease. The effect of a Ca(2+) was explained by release of protein from the inclusion bodies, together with a stabilisation of apoaequorin against degradation. Ca(2+) also reduced the generation time by 4-5 min. These results have important implications for unravelling the physiological role of cytosolic Ca(2+) in bacteria, particularly where several species are competing for the same nutrients, such as in the gut.

Downregulation of the HERG (KCNH2) K(+) channel by ceramide: evidence for ubiquitin-mediated lysosomal degradation.

The HERG (KCNH2) potassium channel underlies the rapid component of the delayed rectifier current (I(kr)), a current contributing to the repolarisation of the cardiac action potential. Mutations in HERG can cause the hereditary forms of the short-QT and long-QT syndromes, predisposing to ventricular arrhythmias and sudden cardiac death. HERG is expressed mainly in the cell membrane of cardiac myocytes, but has also been identified in cell membranes of a range of other cells, including smooth muscle and neurones. The mechanisms regulating the surface expression have however not yet been elucidated. Here we show, using stable HERG-expressing HEK 293 cells, that ceramide evokes a time-dependent decrease in HERG current which was not attributable to a change in gating properties of the channel. Surface expression of the HERG channel protein was reduced by ceramide as shown by biotinylation of surface proteins, western blotting and immunocytochemistry. The rapid decline in HERG protein after ceramide stimulation was due to protein ubiquitylation and its association with lysosomes. The results demonstrate that the surface expression of HERG is strictly regulated, and that ceramide modifies HERG currents and targets the protein for lysosomal degradation.

Lactose causes heart arrhythmia in the water flea Daphnia pulex.

The cladoceran Daphnia pulex is well established as a model for ecotoxicology. Here, we show that D. pulex is also useful for investigating the effects of toxins on the heart in situ and the toxic effects in lactose intolerance. The mean heart rate at 10 degrees C was 195.9+/-27.0 beats/min (n=276, range 89.2-249.2, >80% 170-230 beats/min). D. pulex heart responded to caffeine, isoproteronol, adrenaline, propranolol and carbachol in the bathing medium. Lactose (50-200 mM) inhibited the heart rate by 30-100% (K(1/2)=60 mM) and generated severe arrhythmia within 60 min. These effects were fully reversible by 3-4 h. Sucrose (100-200 mM) also inhibited the heart rate, but glucose (100-200 mM) and galactose (100-200 mM) had no effect, suggesting that the inhibition by lactose or sucrose was not simply an osmotic effect. The potent antibiotic ampicillin did not prevent the lactose inhibition, and two diols known to be generated by bacteria under anaerobic conditions were also without effect. The lack of effect of l-ribose (2 mM), a potent inhibitor of beta-galactosidase, supported the hypothesis that lactose and other disaccharides may affect directly ion channels in the heart. The results show that D. pulex is a novel model system for studying effects of agonists and toxins on cell signalling and ion channels in situ.