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+, Ca2+ 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

Knockdown of the small conductance Ca2+-activated K+ channels is potently cytotoxic in breast cancer cell lines

BACKGROUND AND PURPOSE Small conductance calcium-activated potassium (KCa2) channels have a widely accepted canonical function in regulating cellular excitability. In this study we address a potential non-canonical function of KCa2 channels in breast cancer cell survival using in vitro models. EXPERIMENTAL APPROACH The expression of all KCa2 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 KCa2 channels on these cell lines we utilized MTS proliferation assays and in conjunction followed the corresponding expression of apoptotic markers. KEY RESULTS All of the breast cancer cell lines, regardless of their lineage or endocrine responsiveness, were exquisitely sensitive to KCa2 channel blockade. UCL1684 caused cytotoxicity with an LD50 in the low nanomolar range in all cell lines. The role of KCa2 was confirmed using pharmacological inhibition and siRNA-mediated knockdown, this not only caused a reduction in cell viability, but also a reduced expression of Bcl-2 and increased expression of active caspase-7 and 9. Complementary to these results we also show a variety of cell lines can be protected from the effects of the apoptosis inducer staurosporine using the KCa2 channel activator CyPPA. CONCLUSIONS AND IMPLICATIONS These data clearly illustrate that in addition to a well-established role for KCa2 in migration, KCa2 channel blockade is potently cytotoxic in breast cancer cell lines and points to KCa2 channel modulation as a potential therapeutic avenue in breast cancer

Evidence for serca and BKCa activation in BNP protection of reperfused myocardium [Abstract]

B-type natriuretic peptide (BNP) given at the time of reperfusion, limits reperfusion-induced myocardial injury. Studies examining the endogenous components of this protection, have explored KATP channel activation, but no studies have focused on the sarcoplasmic reticulum Ca2+-ATPase (SERCA), or the large conductance K+(BKCa) channel. We hypothesised that BNP induced protection of reperfused myocardium involves SERCA and BKCa channel activation. Male rat hearts were Langendorff-perfused, and subjected to 35 min left coronary artery occlusion and 120 min reperfusion. Hearts were randomised to 15 min treatments, commencing 5 min prior to reperfusion with BNP, the SERCA inhibitors thapsigargin (TSG) and cyclopiazonic acid (CPA) (Study 1), or the BKCa channel activator NS1619 and inhibitor paxilline (PAX) (Study 2). Infarct size was measured using triphenyltetrazolium staining. These findings provide evidence for the involvement of two new post-receptor mechanisms, SERCA and BKCa activation, which are involved in BNP's protection of reperfused myocardium

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 °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% (K1/2=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 ?-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

Characterisation of large-conductance calcium-activated potassium channels (BK(Ca)) in human NT2-N cells

Large-conductance calcium-activated potassium (BKCa) channels were studied in inside-out patches of human NTERA2 neuronal cells (NT2-N). In symmetrical (140 mM) K+ the channel mean conductance was 265 pS, the current reversing at 0 mV. It was selective (PK / PNa = 20:1) and blocked by internal paxilline and TEA. The open probability–voltage relationship for BKCa was fitted with a Boltzmann function, the V½ being 76.3 mV, 33.6 mV and ? 14.1 mV at 0.1 ?M, 3.3 ?M and 10 ?M [Ca2+]i, respectively. The relationship between open probability and [Ca2+]i was fitted by the Hill equation (Hill coefficient 2.7, half maximal activation at 2.0 ?M [Ca2+]i). Open and closed dwell time histograms were fitted by the sum of two and three voltage-dependent exponentials, respectively. Increasing [Ca2+]i produced both an increase in the longer open time constant and a decrease in the longest closed time constant, so increasing mean open time. “Intracellular” ATP evoked a concentration-dependent increase in NT2-N BKCa activity. At + 40 mV half-maximum activation occurred at an [ATP]i of 3 mM (30 nM [Ca2+]i). ADP and GTP were less potent, and AMP-PNP was inactive. This is the first characterisation of a potassium channel in NT2-N cells showing that it is similar to the BKCa channel of other preparations

ATP regulates calcium efflux and growth in E. coli

Escherichia coli regulates cytosolic free Ca2+ in the micromolar range through influx and efflux. Herein, we show for the first time that ATP is essential for Ca2+ efflux and that ATP levels also affect generation time. A transcriptome analysis identified 110 genes whose expression responded to an increase in cytosolic Ca2+ (41 elevated, 69 depressed). Of these, 3 transport proteins and 4 membrane proteins were identified as potential Ca2+ transport pathways. Expression of a further 943 genes was modified after 1 h in growth medium containing Ca2+ relative to time zero. Based on the microarray results and other predicted possible Ca2+ transporters, the level of cytosolic free Ca2+ 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 FoF1 ATPase, as defective in Ca2+ efflux. Seven other putative Ca2+ transport proteins exhibited normal Ca2+ handling. The defect in the ΔatpD knockout cells could be explained by a 70% reduction in ATP. One millimolar glucose or 1 mM methylglyoxal raised ATP in the ΔatpD knockout cells to that of the wild type and restored Ca2+ efflux. One millimolar 2,4-dinitrophenol lowered the ATP in wild type to that in the ΔatpD cells. Under these conditions, a similar defect in Ca2+ efflux in wild type was observed in ΔatpD cells. Ten millimolar concentration of Ca2+ 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 Ca2+ transporter, atoA, the β subunit of acetate CoA-transferase likely to be involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate kinase, all indicated no defect in Ca2+ efflux. We therefore propose that ATP is most likely to regulate Ca2+ efflux in E. coli through an ATPase

pH and monovalent cations regulate cytosolic free Ca2+ in E. coli

The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca2+ in E. coli through Ca2+ influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca2+ was 0.2–0.5 µM. In the presence of external Ca2+ (1 mM) at alkaline pH this rose to 4 µM, being reduced to 0.9 µM at acid pH. Removal of external Ca2+ caused an immediate decrease in cytosolic free Ca2+ at 50–100nM s− 1. Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca2+/H+exchanger, appeared not to be a major Ca2+-efflux pathway. In the absence of added Na+, but with 1 mM external Ca2+, cytosolic free Ca2+ rose to approximately 10 µM. The addition of Na+(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca2+ 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 Ca2+ efflux. The results were consistent with H+ and monovalent cations competing with Ca2+ for a non-selective ion influx channel. Ca2+ 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 Ca2+ in Escherichia coli. The number of Ca2+ ions calculated to move per cell per second ranged from 6 million ions per second. This raises fundamental questions about the nature and regulation of Ca2+ 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

In vitro patch-clamp studies in skin fibroblasts

We have conducted single-channel patch-clamp experiments in skin fibroblasts maintained in culture. Two different cell lines, a mouse 3T3-L1 cell line and a human B17 cell line, were selected for these pilot studies. Recordings were made from both cell-attached and excised inside-out patches at room temperature. In the case of the 3T3-L1 cells, the success rate in obtaining good seals (> 1GΩ) was low, and channel openings in either cell-attached or excised patches were rare. We have, however, identified a channel in a cell-attached configuration with a slope conductance of 39 pS in symmetrical K+ solutions. In the case of the human B17 cells, good quality seals were more readily obtained. One principal type of channel opening was identified. In cell-attached patches, the prevalent type of channel in symmetrical K+ solutions had a conductance of 187 pS. This channel was activated by strong depolarization, and there was usually more than one active channel in the patch. It was blocked by extracellular tetraethylammonium (20 mM), and persisted when external Cl− was replaced by aspartate. In excised inside-out patches bathed in symmetrical K+, this channel was activated by an increase in Ca+ applied to the intracellular face. A large conductance channel (175 pS) was also observed in excised inside-out patches, with a reverse physiological K+ gradient. This channel had a reversal potential > 40 mV and appeared not to be voltage-dependent under these recording conditions (2 mM Ca2+i). We conclude that the channel we have identified in these cells belongs to the maxi-K+ channel class