Here, we show that IKSper in human sperm is activated weakly by pHi and more strongly by Ca2+. Correspondingly, Vm is strongly regulated by Ca2+ and less so by pHi. We find that inhibitors of Slo3 suppress human IKSper, and we identify the Slo3 protein in the flagellum of human sperm. Moreover, heterologously expressed human Slo3, but not mouse Slo3, is activated by Ca2+ rather than by alkaline pHi; current–voltage relations of human Slo3 and human IKSper are similar. We conclude that Slo3 represents the principal K+ channel in human sperm that carries the Ca2+-activated IKSper current. We propose that, in human sperm, the progesterone-evoked Ca2+ influx carried by voltage-gated CatSper channels is limited by Ca2+-controlled hyperpolarization via Slo3.Sperm are equipped with a unique set of ion channels that orchestrate fertilization. In mouse sperm, the principal KDOI:http://dx.doi.org/10.7554/eLife.01438.001 A sperm that has been ejaculated into the female reproductive tract must complete a number of tasks to pass on its genes to the next generation. First it must travel along a meandering route to encounter an egg, before pushing through a jelly-like coating that surrounds the egg and then, finally, fusing with the egg’s surface membrane. In order to complete these steps and fertilise the egg, a sperm must undergo a process called ‘capacitation’. This process, and a variety of other sperm functions, involves the controlled flux of positive ions into and out of the sperm via specific ion channels that are located in the cell membrane.The properties of the ion channels that allow protons and calcium ions to move into and out of human sperm are well understood, but less is known about the channels that control the movement of potassium ions. In mice, a channel called Slo3 allows potassium ions to flow out of the sperm and makes the membrane voltage of these cells more negative. Also, in mice, this channel is essential for the sperm to function correctly, and for fertilization. However, in humans, it is unclear if the Slo3 channel is present in sperm and if it performs the same role.Now, Brenker et al. have shown that the flow of potassium ions out of human sperm occurs via the Slo3 channel, and that human Slo3 is responsible for setting the membrane voltage of these cells. However, whereas the mouse Slo3 channel is opened in response to a decrease in the concentration of protons within the sperm , human Slo3 is largely controlled by changes in the levels of calcium ions. An increase in the calcium concentration within the cell opens the human Slo3 channel, more than a decrease in the proton concentration does.Altogether, Brenker et al. identify Slo3 as the principal potassium channel in human sperm and reveal more fundamental differences between human sperm and mouse sperm. Thereby, this work further stresses the need to be cautious about using mice as a model of male fertility in humans.DOI:http://dx.doi.org/10.7554/eLife.01438.002 These c[Ca2+]i) .2+ channel is activated by progesterone in humans   (+ channels and not by Cl− channels or CatSper (+ concentration ([K+]o) from 150 to 5 mM shifted the reversal potential (Vrev) from 9.2 ± 1.5 mV to −16.5 ± 10 mV (n = 5)  . Several CatSper : lowerin (n = 5) . At low  further , showing≤ 100 mV . However CatSper . MonovalSper   at pipette pH 6.2    . Moreovee pH 6.2 , but note pH 6.2 . Thus, h5 to 7.5 . In moust manner . Therefo (n = 4)  left; ra (n = 4)  left. Athange Vm  left. Ofgh [K+]o  left. In+ current, we tested whether Ca2+ controls IKSper in human sperm. To compare current–voltage (I–V) relations at low and high [Ca2+]i in the same cell, we studied voltage activation of IKSper before and after rapid photorelease of Ca2+ from caged Ca2+ (DMNP-EDTA)  , while msolution . Photoretentials .10.7554/2+ dependence of IKSper was quantified by recording the I-V relation at 2.5-1000 µM Ca2+ in the pipette  was 3.5 ± 3 pA; increasing Ca2+ to 40 µM and 1 mM Ca2+ increased the amplitude to 14 ± 6 pA and 32 ± 17 pA, respectively (n = 3)   . For  in the pipette, respectively; 30 µM and ≥ 70 µM [Ca2+]i changed Vm to −43 ± 4 mV and about −60 mV, respectively (n = 3-4)  , right. gh [K+]o . In concKSper, although modest, is reminiscent of the IKSper current in mouse sperm carried by Slo3 channels, whereas the Ca2+ sensitivity is reminiscent of the prototypical Ca2+-activated K+ channel Slo1  where CatSper currents are negligibly small  , we idenoteomics , which iw) of about 120 kDa -tag in chinese hamster ovary (CHO) cells . In West 120 kDa . The pre ∼80 kDa ; in both ∼80 kDa . Finally ∼80 kDa , consistse sperm . Togethe2+ .Considering that mouse Slo3 is insensitive to Ca2+ , the Ca2i 7  curves generated from tail currents illustrate the activation of hSlo3 at pHi 7 and pHi 8 in the presence of 0, 60, and 300 μM Ca2+ , in contrast to the only 2.2-fold increase evoked by raising pHi from 7 to 9. Thus, Ca2+ activates hSlo3 much more effectively than alkaline pHi.At pH μM Ca2+ . At pHi 2+-activated tail currents, whether at 60 or 300 µM Ca2+, were similar at pHi 7 and pHi 8  were also markedly increased by 60 µM and 300 μM Ca2+  did not enhance currents , moderate- (KD = 9.7 µM), and low-affinity Ca2+ indicators (KD = 90 µM)  . The Ca2 [Ca2+]i . For ind similar , suggestdicators , our resKSper conform with the properties of hSlo3, but not with those of hSlo1 or other members of the Slo family. First, hSlo3 and IKSper are modestly sensitive to pHi ≤ 7.0 and are more strongly activated by Ca2+. Second, the I–V relations of hSlo3 and IKSper are similar, in the absence and presence of intracellular Ca2+. Third, several Slo3, but not Slo1 inhibitors block IKSper. Fourth, we identify the Slo3 protein and its auxiliary subunit LRRC52 in human sperm. Finally, both human IKSper  of 7.5 µM  , and paxilline. Although we do not know the reason for this discrepancy, there are differences in experimental conditions. We tested the inhibitors at 1 mM extracellular [Ca2+] to prevent monovalent CatSper currents and at 1 mM intracellular [Ca2+] to strongly activate IKSper. Mannowetz et al. tested the drugs at 100 µM extracellular [Ca2+] and in the absence of intracellular Ca2+. Under these conditions, sizeable CatSper currents are recorded  , this worecorded , but actKSper-mediated hyperpolarization reinforces Ca2+ influx via CatSper by increasing the electrical driving force   . In neurmembrane . In micrchannels . It need2+, H+, Na+, Cl−, and probably other cytosolic ligands -containing proteins vary markedly within the RCK domain structures, it is not surprising that simple examination of the Slo3 sequence alignments does not reveal obvious determinants of either pH sensitivity in mSlo3 or Ca2+−sensitivity in hSlo3.Channels of the Slo family have been studied as models for allosteric regulation of gating by ligands . Slo cha ligands . Among d ligands . However ligands . Our stuidentity , there iidentity . Given t2+-regulated mSlo1 is 45.7%, the different ligand dependence between hSlo3 and mSlo3 is not so surprising.At first glance, the fact that Slo3 orthologues differ in their ligand-dependence seems highly unusual. Yet, it is well-established that proteins essential for fertilization are rapidly evolving; orthologues often display a low degree of sequence similarity . For a s2+ indicators were purchased from Invitrogen . UPLC grade formic acid, acetonitrile and methanol were purchased from Biosolve . All other reagents were obtained from Sigma-Aldrich .IBTX was purchased from Tocris . DMNP-EDTA and Ca+) containing (in mM): 97.8 NaCl, 4.69 KCl, 0.2 MgSO4, 0.37 KH2PO4, 2.04 CaCl2, 0.33 Na-pyruvate, 21.4 lactic acid, 2.78 glucose, 21 HEPES, and 25 NaHCO3 adjusted between pH 7.3-7.4 with NaOH. After washing, human serum albumin  was added to HTF+ (referring to as HTF++). Sperm were incubated for at least 2 hr in HTF++ at 37°C in a 10% CO2 atmosphere. Under these conditions, sperm undergo capacitation. All recordings were done on capacitated sperm.Samples of human semen were obtained from healthy volunteers with their prior consent. Sperm were prepared as described . In brieg, 30 min, 4°C) and membrane proteins were processed by in-gel digestion, in-solution digestion, or a FASP protocol. Peptides were subjected to 1D-ESI-LC-MS/MS using a nanoAcquity UltraPerformance LC System  coupled to an LTQ Orbitrap Velos or Elite instrument . The resulting tandem MS data were searched using the Sequest algorithms embedded in Proteome Discoverer 1.2 (Thermo) against a SwissProt/UniProtKB human protein sequence database . The mass tolerance for precursor ions was set to ≤10 ppm; the mass tolerance for fragment ions was set to ≤ 1 amu. For search result filtering, the false discovery rate (FDR) was set to < 1% and only peptides with search result rank 1 were accepted for identification. For targeted mass spectrometry, the instrument control software used a list of theoretical tryptic peptide masses for the proteins of interest for subsequent CID fragmentation, that is once a mass from the list was detected in the orbitrap full scan, it was preferred over all other co-eluting masses, independent of its signal intensity.After purification by a ‘swim-up’ procedure, human sperm were lysed by several ‘freeze/thaw’ cycles and sonification steps in buffer containing (in mM): 10 HEPES pH 7.5, 2 EGTA, 1 DTT, protease inhibitor cocktails , and DNaseI . Membranes were sedimented by centrifugation , HA . Secondary antibodies: for ICC, rabbit Cy3-conjugated and rat Cy5-conjugated antibodies ; for WB, rabbit-HRP and rat-HRP .slo3 gene , 62 base pairs (bp) present around the S10 region in BC028701 correspond to an intron left over from incomplete mRNA splicing. The intron sequences were removed by site-directed mutagenesis. In addition, there are at least three polymorphic sites present in the EST clone corresponding to amino-acid positions 192, 739, and 768 of hSlo3. The three sites were changed to match those of the human genomic sequence . This results in 192W, 739R, and 768W. The full length coding sequence of hSlo3 was subcloned into the oocyte expression vector pXMX  were added. The coding sequence harbored an arginine at the polymorphic site at position 768. The DNA was subcloned into a pcDNA3.1(+) vector (Invitrogen); the sequence coding for the neomycin resistance gene was replaced by the coding sequence for either citrine or EGFP. The hLRRC52-mCherry construct described above was also subcloned into the pcDNA3.1(+) vector for expression in CHO cells. All cDNA clones were verified by sequencing.2HPO4, 1.5 KH2PO4, pH 7.4. To block nonspecific binding sites, cells were incubated for 20 min with blocking buffer  in PBS). Primary antibodies were diluted in blocking buffer and cells were incubated for 1 hr at room temperature. After washing with PBS, cells were incubated with fluorescent secondary antibodies in blocking buffer containing 0.5 µg/µl 4’,6-diamidino-2-phenylindole . After washing with PBS, cells were mounted on slides and examined with a confocal microscope .Human sperm and CHO cells expressing hSlo3 were immobilized on glass coverslips and fixed for 3 min with 4% paraformaldehyde in phosphate buffered saline (PBS) containing (in mM): 137 NaCl, 2.7 KCl, 6.5 Nag (4°C) and the supernatant was used for WB analysis. Human sperm (5 × 106) were resuspended in 2x SDS sample buffer containing β-mercaptoethanol. All samples were heated for 5 min at 95°C and separated by 4–12% SDS–polyacrylamide gel electrophoresis. For WB analysis, proteins were transferred onto PVDF membranes, probed with antibodies, and analysed using the LAS-3000 System (Fujifilm).Wild type CHO cells and cells expressing the human Slo3 channel were lysed in a buffer containing 10 mM Tris/HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and mammalian protease inhibitor cocktail  and incubated on ice for 30 min. The suspension  was centrifuged for 5 min at 10,000×4, 2 CaCl2, 5 glucose, 1 Na-pyruvate, 10 lactic acid, and 20 HEPES adjusted to pH 7.4 with NaOH. K-based HS contained (in mM): 135 KCl, 5 NaCl, 1 MgSO4, 2 CaCl2, 5 glucose, 1 Na-pyruvate, 10 lactic acid, and 20 HEPES adjusted to pH 7.4 with KOH. HS with low [Cl−] contained (in mM): 135 Na-aspartate, 5 KCl, 1 MgSO4, 2 CaCl2, 5 glucose, 1 Na-pyruvate, 10 lactic acid, and 20 HEPES adjusted to pH 7.4 with NaOH. The following pipette (10–15 MΩ) solutions were used (in mM): Cs-based IS: 130 Cs-asparate, 5 EGTA, 5 CsCl, and 50 HEPES at pH 7.3 with CsOH; K-based IS 0 Ca2+: 130 K-aspartate, 10 NaCl, 1 EGTA, and 50 HEPES at pH 7.3 with KOH; K-based IS with 100 µM or 1 mM Ca2+: 130 K-aspartate, 7 NaCl, 2 KCl, 100 µM or 1 mM CaCl2, and 50 HEPES at pH 7.3 with KOH; K-based IS with 40 µM Ca2+: 140 K-aspartate, 50 HEPES, 10 NaOH, 5 KCl, 3 NTA, 1.3 CaCl2, pH 7.3 with KOH; K-based IS with 10 µM Ca2+: 130 K-asparate, 10 NaOH, 1 KCl, 3 HEDTA, 2 CaCl2, and 50 HEPES at pH 7.3 with KOH. All voltages were corrected for the liquid junction potential of 16.5 mV. The osmolarity of intracellular and extracellular solutions was ∼120 mOsm.We recorded from sperm in the whole-cell configuration as described . Seals bXenopus oocytes, RNAs for hSlo3 and hLRRC52 were injected at a ratio of 1:1 by weight. Gigaohm seals were formed while oocytes were bathed in frog Ringer (in mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, and 10 HEPES at pH 7.4. Pipette resistance was typically 0.8–2 MΩ after fire-polishing and filling with pipette solution containing (in mM): 140 K-methanesulfonate (MES), 20 KOH, 2 MgCl2, and 10 HEPES at pH 7. The standard solution bathing excised inside-out patches was (in mM): 140 K-MES, 20 KOH, 5 mM EGTA, and 10 HEPES with pH adjusted as indicated. For addition of Ca2+ to bathing solutions, in most cases free [Ca2+] was sufficiently high that buffering was not required . In one test solution, we omitted EGTA so that the estimated free [Ca2+] was defined presumably by the level of contaminant Ca2+ in our water . Osmolarity for solutions was approximately 310 mOsm.For functional studies in + currents were recorded from inside-out patches with an Axopatch 200B amplifier  and low-pass filtered at 10 kHz with an integral four-pole Bessel filter. Signals were digitized with a Digidata 1322A data acquisition system (Molecular Devices) at 100 kHz. Recordings were controlled by the pClamp 9.2 software suite (Molecular Devices). Capacitative component of currents was subtracted using the appropriately scaled trace for a step from −100 mV to 0 mV. All experiments were performed at room temperature (21–24°C).Solutions were applied directly to patches via an SF-77B fast perfusion stepper system . K2, 1.8 CaCl2, 10 D-glucose, and 5 HEPES adjusted to pH 7.4 with NaOH. For the recordings in high K+ solution, NaCl was exchanged by KCl and pH was adjusted to 7.4 with KOH. To study activation of Slo3 by alkalization, the following pipette (4–5 MΩ) solution was used (in mM): 130 K-aspartate, 10 NaCl, 1 EGTA, 5 HEPES, 15 D-glucose, pH was adjusted either to 6.2 or 7.3 using KOH. To study the activation of Slo3 by Ca2+, the following pipette (2–3 MΩ) solutions were used (in mM): divalent-free solution: 130 K-aspartate, 10 NaCl, 1 EGTA, and 20 HEPES adjusted to 7.3 using KOH; 70 µM Ca2+ intracellular solution: 130 K-aspartate, 10 NaCl, 0.5 CaCl2, 1 NTA, and 20 HEPES adjusted to pH 7.3 using KOH, the final Ca2+concentration was confirmed using the Ca2+ dye Mag-Fura 2; 1 mM Ca2+ intracellular solution: 130 K-aspartate, 10 NaCl, 1 CaCl2, and 20 HEPES adjusted to pH 7.3 using KOH. Series resistance and cell capacitance were compensated to 70–85%. Voltages were corrected for the liquid junction potential. All recordings were performed at 20–22°C.CHO cells were co-transfected with hSlo3 and hLRRC52-mCherry constructs. Seals between pipette and cell were performed in standard extracellular solution (ES) containing (in mM): 140 NaCl, 5.4 KCl, 1 MgCl2+]i were measured in 384 multi-well plates in a fluorescence plate reader  at 30°C. Sperm were loaded with the respective fluorescent Ca2+ indicator (10 μM) (Molecular Probes) in the presence of Pluronic F127 (0.1% wt/vol) for 45 min at 37°C. After incubation, excess dye was removed by a centrifugation step . The sperm pellet was resuspended in HTF++ and equilibrated for 5 min at 37°C. Each well was filled with 50 µl of the sperm suspension (1 × 107 sperm/ml); the fluorescence was excited at 480 nm and emission was recorded at 520 nm with bottom optics. Fluorescence was recorded before and after injection of 10 µl (1:6 dilution) of progesterone or ionomycin in HTF++. The solutions were injected into the wells manually with an electronic multichannel pipette.Changes in i, but is less sensitive to pH. These findings, if confirmed, represent a major step forward in our understanding of sperm membrane potential regulation and fertilization. The third conclusion is very well supported by careful and well-controlled analysis using heterologous expression in both Xenopus oocytes and CHO cells, with or without the auxiliary subunit LRRC52. I find that the first two conclusions, though quite interesting, are not convincing at their present form.There are three major conclusions in this paper. First, the human KKsper is potentiated by Ca2+ but not pHi is in 2+, not pH, controls Vm in human sperm, two [Ca2+]i conditions are compared: 0 Ca2+ and 1 mM Ca2+. The 0 Ca2+ pipette had 1 mM EGTA with no added Ca2+, which results a free [Ca2+] of perhaps < 10 nM. The pipette solution contains no ATP or GTP. Such condition may be fine for studying the biophysical properties of a channel with voltage-clamping, but is too “unphysiological” for studying the regulation of membrane potential. It is hard to interpret the apparent lack of large effect on Vm by pHi change under this condition. The authors also cited the paper by Navarro (PNAS 104:7688-7692) to compare the difference between human and mouse sperm in the effects of pHi on Vm. However, there is large difference between the pipette solutions used in the two studies . The authors should at least test several Ca2+ concentrations close to the physiological levels. In addition, all the pipette solutions have 50 mM HEPES. It's not clear whether 10 mM NH4Cl in the bath is able to significantly increase pHi when 50 mM HEPES is included in the pipette, and this is critical to the conclusion that pHi increase doesn't have much effect on Vm. Finally, the numbers of cells  are too small to draw statistically significant conclusions, especially when measuring Vm of primary cells.The major data supporting the conclusion that human IKsper's Ca2+ sensitivity is not that strong, at least not until [Ca2+]i reaches 100 μM (2+] increases can reach the levels sufficient to activate IKsper. The Ca2+ measurements in 2+ affinity) and, more importantly, are from whole sperm. The majority of the signal is likely from sperm head where Slo3 is not localized , the current Brenker data are convincing that human KSper is most likely Slo3.Brenker et al. report that the principal human K channel, like that in mouse, is encoded by Slo3. This contrasts with the recent 2+, but only modestly by alkaline pH. This agrees with Mannowetz et al. The primary disparity between the papers is that Mannowetz et al found that the specific Slo1 blocker, Iberiotoxin, blocked human KSper, while Brenker et al find that it does not. The experimental solutions used in the two studies may underlie this difference.Unlike mouse KSper, the principal K channel in human sperm is primarily activated by internal CaThere are many more data in the current paper characterizing human KSper and this will be of interest to the field. The work is well done and we find no fault with the data. The most interesting new data is that human Slo3 differs from mouse through the Slo3 pore-containing subunit, rather than through association with an LRRC52 accessory subunit.The work presents another interesting example that shows that species-specific regulation might have evolved on this essential ion channel protein in sperm, analogous to the different activation mechanisms previously shown in mouse and human CatSper.The following issues need to be addressed in the revision: 1) The authors presume that the rapid evolution of gamete genes results in gating ring mutations that confer these changes to human Slo3. It would be of interest to readers to show alignment of mouse, intervening species, and humans to narrow in on the changes most likely to mediate these properties. We do not suggest that the authors sort out the changes by mutagenesis, as this would be a separate work in itself and may take several years to complete.2) The mass spec identification supporting the conclusion is not shown. To support the findings, indicators of merit (such as number of peptides and protein coverage etc.) for all the proteins identified and mentioned in the text should be provided as a Table in Supplementary Information.2+ in some internal solutions to maximize the response, many readers will question the validity of this condition, and it will likely be a source of contention. This should be at least addressed fully in the discussion. Better would be to show data at intervening 10, 30, 100 µM Ca2+. However, the reviewers are not worried by the use of 1 mM Ca2+. The inclusion of data with other concentrations is optional.3) Although we understand the reasons for using 1 mM Ca4) Introduction: Navarro et al definitively identified P2X2 channels, not P2X1, in mouse sperm through KO studies. The Brenker study does not identify the P2X channel subtype. After writing a whole paper on the mis-identification of KSper, it is not reassuring that you misidentify P2X in your own summary! [Editors’ note: the author responses to the first round of peer review follow.]eLife. Each manuscript reaches entirely different conclusions regarding the molecular identity of the K+ channel underlying IKSper current in human sperm: we conclude that it is Slo3, whereas Mannowetz et al. propose Slo1; both reports cannot be right at the same time.While our manuscript was under review, a competing manuscript by + current is activated by Ca2+.The two groups agree that the primary human sperm K+ current also retains some, albeit modest pH dependence, consistent with the modest pH dependence of heterologously expressed hSlo3; and (2) conflicting pharmacological and biochemical results concerning the molecular identity of the channel.The major points of difference are: (1) we observe that the human sperm KKSper and depolarize human sperm. In contrast, Mannowetz et al. report that Slo1 inhibitors suppress IKSper. Although we do not know the reason for this discrepancy, there are differences in experimental conditions, which we mention in the Discussion of our revised manuscript. Moreover, to ascertain that human Slo3 is insensitive to Slo1 inhibitors, we studied the action of IBTX, CTX, and paxilline on heterologous hSlo3 + LRRC52 channels, see Concerning the pharmacology, we show that Slo3 inhibitors, but not Slo1 inhibitors, suppress Ii of 17 μM, consistent with the inhibition of IKSper in human sperm  Slo1. I find no reason to doubt either study's conclusions and I expect the controversy to be settled with future work.I believe the paper to be appropriate for publication in eLife, especially as the Lishko paper appeared there also.We thank the referee for the positive comments on our manuscript.Reviewer 2:However, mechanistically and conceptually the advances of the current manuscript are limited. The salient role of Slo3 in mouse sperm has recently been highlighted by several groups. It is well documented in the literature that mouse Slo3 is rather insensitive to calcium and that human Slo3 coexpressed with LRRC52 is less sensitive to alkaline pH as compared to mouse. The authors confirm these observation by others by direct electrophysiological recordings in human sperm. However, no information is provided on the molecular basis underlying these functional differences. Thus, the present manuscript still leaves central mechanistic questions unanswered and the paper is confirmatory to quite some extent.+ current in human sperm is regulated by pH, like mouse KSper and Slo3. If that were the case, we would certainly agree that the manuscript contributes little new information. However, the focus of the manuscript is in establishing that the human sperm K+ current is, in fact, regulated by Ca2+, with weaker regulation by pH. The prototypical Ca2+-activated and pH-activated members of this channel family are Slo1 and Slo3, respectively. Thus, the important question is whether the human sperm K+ current is carried by Slo1 or Slo3. Our results demonstrate that human IKSper current arises from Slo3 and not Slo1. Furthermore, we show that heterologously expressed human Slo3 is Ca2+ regulated. These observations are entirely new and it is surprising that two orthologous proteins, mSlo3 and hSlo3, have such different regulation by cytosolic ligands.The referee’s impression seems to be that the main purpose of our manuscript was to establish that the K2+ and H+ is highly interesting, but is beyond the scope of this manuscript. However, we acknowledge that the extensive introductory paragraph on Slo gating rings in our original manuscript might have led the referee astray. Therefore, we shortened this paragraph and moved it to the Discussion.Identification of the site on the hSlo3 gating ring underlying regulation by CaReviewer 3:KSper by Ca2+ and pHi. This is somewhat surprising given that the referee might have also seen the Mannowetz et al. manuscript; at least in the case of Ca2+, this is not a point of contention between the two groups. Moreover, by manifold controls, we scrutinized the reliability of our recordings:This referee raised several concerns about the reliability of our electrical recordings from human sperm, in particular, the activation of Ii-, and Ca2+-activated currents are carried by K+ channels, we determined the reversal potential Vrev of currents at high and low [K+]o and [Cl-]o.To ascertain that the voltage-, pHKSper from monovalent ICatSper, we substituted intracellular K+ by Cs+ and used 1 mM Ca2+ in the extracellular medium.To distinguish experimentally IKSper by alkalization both by superfusion with NH4Cl and by using different pH values in the pipette. We studied IKsper at low and high [Ca2+]i in one and the same cell by photorelease of Ca2+ from caged Ca2+, while monitoring [Ca2+]i with a fluorescent indicator.We show activation of I2+ activation of IKSper was studied at 6 different intracellular Ca2+ concentrations, ranging from 2.5 to 1000 μM.Cai- and Ca2+ activation of IKSper was confirmed by current-clamp experiments.pHWe tested Slo1 inhibitors both in voltage- and current clamp.We tested Slo3 inhibitors both in voltage- and current clamp.We studied both Slo1 and Slo3 expression in human sperm by targeted mass spectrometry.Finally, heterologous expression of Slo3 in both oocytes and CHO cells corroborated our electrophysiological and pharmacological conclusions drawn from sperm recordings.KSper and hSlo3, and the Ca2+-activation of heterologous hSlo3 both in oocytes and CHO cells provide compelling support for our conclusion that the Ca2+-activated IKSper in human sperm arises from hSlo3, but not Slo1.Altogether, the identification of the hSlo3 protein in human sperm by two independent techniques, the pharmacological fingerprint of human IThere are three major conclusions in this paper. First, the human K+current IKsperis activated by intracellular Ca2+, rather than pH increase as previously established in mice. Second, Ca2+increase in human sperm under “physiological” conditions (presumably through Catsper) is sufficient to activate IKsper. Third, heterologously expressed human Slo3 (hSlo3) is activated by high [Ca2+]i, but is less sensitive to pH. These findings, if confirmed, represent a major step forward in our understanding of sperm membrane potential regulation and fertilization. The third conclusion is very well supported by careful and well-controlled analysis using heterologous expression in both Xenopus oocytes and CHO cells, with or without the auxiliary subunit LRRC52. I find that the first two conclusions, though quite interesting, are not convincing at their present form.2+ activates IKSper . Therefore, we disagree with this statement. The issue concerning the activation of IKSper by physiological Ca2+ concentrations is addressed in more detail below.We provide plenty of experimental evidence that CaThe major data supporting the conclusion that human IKsperis potentiated by Ca2+but not pHiis inKSper is exclusively controlled by Ca2+. In fact, we show that at pipette pH 6.2, alkalization by NH4Cl enhances currents by about 2-fold. Similarly, increasing pipette pH from 6.2 to 7.3 enhances human IKSper by about 2-fold.There seems to be a misunderstanding. We do not claim that human IInwhere the authors want to show what Ca2+, not pH, controls Vmin human sperm, two [Ca2+]iconditions are compared: 0 Ca2+and 1 mM Ca2+. The 0 Ca2+pipette had 1 mM EGTA with no added Ca2+, which results a free [Ca2+] of perhaps < 10 nM. The pipette solution contains no ATP or GTP. Such condition may be fine for studying the biophysical properties of a channel with voltage-clamping, but is too “unphysiological” for studying the regulation of membrane potential. It is hard to interpret the apparent lack of large effect on Vmby pHichange under this condition. The authors also cited the paper by Navarro (PNAS 104:7688-7692) to compare the difference between human and mouse sperm in the effects of pHion Vm. However, there is large difference between the pipette solutions used in the two studies . The authors should at least test several Ca2+concentrations close to the physiological levels.2+ and pH do affect human IKSper and alkalization does hyperpolarize sperm; yet, Ca2+ evokes a more pronounced hyperpolarization. To make comparison of current-clamp and voltage-clamp experiments meaningful, we used the same intracellular solution (without ATP/GTP) throughout the manuscript. Of note, Mannowetz et al. used a similar intracellular solution  to study human IKSper. Under these conditions, our current clamp and voltage-clamp data are consistent, e.g., we observe modest pHi control, but stronger Ca2+ control of IKSper and Vm. Current-clamp experiments are always to a certain degree nonphysiological, no matter whether intracellular ATP/GTP is present or not: the cell’s cytoplasm is exchanged by artificial intracellular solutions and, moreover, experiments are hampered by the high input resistance of sperm . Therefore, we have tempered our assertions that Ca2+ rather than pHi controls Vm of human sperm to our recording conditions. Moreover, we now include a paragraph in the discussion stating that it will be an interesting question for future work to determine the relative contributions of cytosolic pH and [Ca2+] to the regulation of human sperm function.Both Ca2+] of either a few nanomolar or 1 mM to study the action of Ca2+. As requested, we now include current clamp data at 1, 30, and 70 μM Ca2+, showing that the effect of 1 μM Ca2+ is miniscule, whereas 30 μM evokes a sizeable hyperpolarization that saturates at ≤ 70 μM. Thus, the concentration dependence of Ca2+ action is similar in current- and voltage-clamp experiments.In the current-clamp experiments, we used a pipette ireaches 100 μM . Based on, the authors conclude that physiologically stimulated [Ca2+] increases can reach the levels sufficient to activate IKsper. The Ca2+measurements inwith high concentration of progesterone (2 μM) do not have calibration (though with indicators of various Ca2+affinity) and, more importantly, are from whole sperm. The majority of the signal is likely from sperm head where Slo3 is not localized . Therefore, it's hard to make meaningful correlation between the Progesterone-induced Ca2+increase and IKsperactivation.2+] (not 100 μM as this referee states) enhances both IKSper and hSlo3 currents. We agree with the referee on potential limitations of the Ca2+ indicator experiments in terms of defining absolute concentrations. However, although 2+]i, it clearly argues  that [Ca2+]i increases globally μM. In fact, it has been shown that progesterone-induced Ca2+ signals originate in the flagellum and propagate in a tail-to-head direction . Therefore, the local [Ca2+] in the flagellum is predicted to be even higher than in the head. However, we state now in the text that the interplay between CatSper and Slo3 requires further experimentation.Unlike mouse KSper, the principal K channel in human sperm is primarily activated by internal Ca2+, but only modestly by alkaline pH. This agrees with Mannowetz et al.+ current is activated by Ca2+. The two papers reach different conclusions regarding the role of alkaline pH. Mannowetz et al. claim that human IKSper is completely insensitive to pHi, whereas we and a recent paper by Mansell et al. (Mol. Hum. Reprod. 2014) find that human IKSper is enhanced by alkalization.We trust that the reviewer means that both studies show that human sperm KThe primary disparity between the papers is that Mannowetz et al found that the specific Slo1 blocker, Iberiotoxin, blocked human KSper, while Brenker et al find that it does not. The experimental solutions used in the two studies may underlie this difference.2+] used in the pharmacological experiments differ. Overall, our pharmacological profile is consistent with the biochemical evidence for Slo3, the single-channel fingerprint, and the progesterone sensitivity of human IKSper.Although the pharmacological differences between the two studies remain difficult to explain, we note that the intracellular and extracellular i. In KSper at 2.5, 10, 40, 100, and 1,000 µM [Ca2+]i. Moreover, in 2+]i. We feel this data should address the concerns of the reviewer.Please note that, in 2+]i was used:There are three experiments where only 1 mM i, Vm is independent of [Cl-]o,2) to show that at high [Ca2+]i, IKSper is still potentiated by alkalization.3) to show that at high [Ca2+]i in the results section and in the discussion. For experiments 2 and 3, it is also advisable if not required to use high [Ca2+]i. Accordingly, we do not expect the high concentrations a potential source of contention. Therefore, we prefer to leave the Discussion as it is.For experiment 1, we provide the rationale for using 1 mM [Ca4) Introduction: Navarro et al definitively identified P2X2 channels, not P2X1, in mouse sperm through KO studies. The Brenker study does not identify the P2X channel subtype. After writing a whole paper on the mis-identification of KSper, it is not reassuring that you misidentify P2X in your own summary!Although we assume that the reviewer made this comment in jest, our work was not about mis-identification of KSper, but simply reports our experiments showing that human KSper is regulated by both cytosolic calcium and alkalization, and that properties of heterologously expressed hSlo3 are consistent with human KSper. Moreover, we thank the referee pointing out the typo regarding P2X channels; we have made some clarifying changes to the respective sentence: “purinergic P2X channels are functional in mouse , but not


Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca2+ channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca2+ or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca2+ channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca2+ channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons. Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca Consistent and reproducible generation of functional neurons from iPSC is crucial to the development of cellular models of neurological disease. Although a plethora of protocols are available which might produce such neuronal models, only a few have been driven by robust functional characterisation of neuronal maturation and synaptogenesis -3, whichPluripotent stem cells (PSCs) differentiate into neurons through a program of neuralisation, neuronal-subtype fate specification, cell cycle exit, post-mitotic neuronal differentiation and functional maturation of regulated excitability. Despite significant advances in the development of protocols for PSC neuralisation and neural progenitor fate-specification , the latin vitro; both co-culture with astrocytes, and treatment with astrocyte conditioned medium (ACM), have been shown to enhance neuronal synaptogenesis  from 0.6mM to 1.8mM; 300µM GABA ; 10µM bicuculline ; 2µM nifedipine ; 0.1µM conotoxin ; 0.1µM agatoxin ); 0.1µM SNX482 ; 1µM BayK 8644 .Differentiation medium was supplemented with ACM (1:1), additional CaCl-1, Sigma-Aldrich, Poole, Hants., U.K.) for approximately 30 minutes at 37°C. Digestion was stopped by addition of astrocyte growth medium , 10% fetal bovine serum  and 1% of a cocktail of penicillin, streptomycin and fungizone . A cell suspension was derived by further trituration using a P1000 pipette. Dissociated, cells were washed twice with culture medium and plated onto culture flasks. Cultures were passaged at a 1:6 ratio upon reaching confluency. Confluent cultures at P1 and P2 were used to condition neural differentiation base medium (DMEM:F12 (1:3), 2 % B27, 1% non-essential amino acids; all from Life Technologies, Paisley, Strathclyde, U.K.). ACM was harvested after 72 h, filter sterilised, aliquoted and stored for later use at -80°C. Different batches of ACM were compared using a mouse CCL2 (chemokine C-C motif ligand 2) ELISA  and normalised to a constant final concentration of CCL2 of 1 mg.ml-1. For differentiating neurons, ACM was mixed with differentiation base medium in a 1:1 ratio. ACM was produced from primary mouse astrocyte cultures. Striata were dissected from newborn C57Bl/6J mice. Tissue was dissected into cold Hank’s buffered saline , fragmented by gentle trituration, washed in HBS, and then digested by incubation with accutase  and DNAse1 (0.1mg.mlm). All analyses of currents and Vm were performed off-line using ClampFit 9. For all recordings, pipettes were manufactured using a two-stage electrode puller , were heat-polished using a Narishige Microforge  and had tip resistances of between 4 and 7 MΩ when filled with an the internal solution containing (in mM) 117 KCl, 10 NaCl, 11 EGTA, 2 Na.ATP and 11 HEPES. The control external solution (ESC) contained (in mM) 135 NaCl, 5 KCl, 5 HEPES, 10 glucose, 1.2 MgCl2 and 1.25 CaCl2. Vm and spontaneous action potentials were recorded in fast current clamp mode, where current was clamped at 0mV in ECS. Induced action potentials were recorded in fast current clamp mode during a current step protocol; current was injected to hold the membrane potential at ca. -70 mV and then 100 ms current injection was applied, starting with 0 pA and increasing to 180 pA in 10 pA increments in each successive sweep. Voltage-gated Na+ and K+ currents were measured using a voltage-step protocol; the voltage was held at -70mV and then stepped for 200 ms from -120 mV to 50 mV in 10 mV increments in each successive sweep. Leak currents were subtracted on-line using a P/N = 8 pre-pulse voltage-protocol. ESC was either supplemented with 10 mM tetraethyl ammonium chloride  or NaCl was completely replaced with N-methyl-D-glutamine chloride  to confirm dissect Na+ and K+ current from each other. The voltage-dependence of activation and steady-state inactivation of the Na+ currents were measured using a dual voltage-step protocol. From holding potential of -90 mV, the first 200 ms steps increased in +5mV increments to 0mV (to elicit current activation) and these were followed by a 200 ms step to 0 mV (to assess current inactivation). Following conversion to conductance (G), the G/Gmax values were plotted against the voltage to give activation and inactivation curves; the point of transection of the two curves is the voltage of peak available current, or peak Na+ window.Unless otherwise stated, all reagents for electrophysiology and Ca2+ imaging were purchased from Sigma-Aldrich . Coverslips with attached neurons were transferred to a perfusion bath mounted on the stage of an inverted microscope  with phase contrast and rapid perfusion system capable of 20ms solution changes . Conventional, whole cell patch clamp was performed on differentiating neurons at 1, 2 and 3 weeks post plate-down using an Axopatch 200B amplifier  interfaced with a computer using an Axon Digidata 1320 DAC converter . In voltage-clamp mode, voltage protocols were generated with, and evoked currents were recorded by, the Axon Instruments ClampEx acquisition software in the PClamp 9.2 suite . Similarly, in current clamp mode, ClampEx was used to generate current protocols and to record membrane potentials  solution was used to amplify the voltage gated Ca2+ currents, which contained (in mM) 94.5 NaCl, 5 KCl, 5 HEPES, 10 glucose, 1.2 MgCl2 and 1.25 CaCl2. Nifedipine (2 µM) was employed to determine the contribution that L-type Ca2+channel channels made to the total voltage-activated Ca2+ currents. GABA ligand-gated (GABAA) currents were evoked by a 5 s application of 300 µM GABA (in ECS) at a holding potential of -70 mV or, for generating a GABA-evoked current-voltage relationship, during a voltage-step protocol similar to that used for Na+ and K+ current, as above but with each voltage step lasting for 7 s . Voltage-gated Ca2+ imaging was performed using a monchromator based fluorimeter system . Cells plated on glass coverslips were incubated in 250µl of culture medium with a final concentration of 1µl of fura-2-AM  for 30 minutes at 37°C. Coverslips were then placed in the perfusion chamber mounted on an inverted Olympus IX70 inverted microscope  and continuously perfused using an RSC, rapid perfusion system . Fura-2 was alternately excited through a quartz, oil immersion objective with light at 340 and 380 nm (50-100ms) and re-emission from each wavelength was measured at 505 nm using a CCD camera . 2x binning was applied to each image to increase signal intensity but at the cost of resolution. Off-line, regions of interest were circled and all intensities were background subtracted before the 340:380 emission ratio was calculated. Images were taken every 3 s.Fura-2 Ca+  solution was applied for 10 s at the beginning of each experimental protocol to determine which cells expressed functional, voltage-gated Ca2+ channels and to establish the magnitude of the control response. Ca2+ channel blocking agents were applied in the same high K+ solution. The area under the curve for each high K+ application (or high K+ with agent) was calculated using the sum of the integrals for each data point, subtracting the integral of a line starting just before the application and ending following complete recovery.A high KA responses a 10s high K+ was followed by a 200s rest and a 10s pulse of 300µM GABA in ECS. Any significant Ca2+ influx (in terms of a change in Fura-2 emission intensity ratio (i340/i380) was interpreted as an excitatory GABA response, as the cell depolarised in order to activate voltage activated Ca2+ channels. After 200s rest, a 10s pulse of 300µM GABA in reduced Cl- solution , thus cells functionally expressing GABAA channels and will depolarise the cell and evoke a voltage activated Ca2+ influx. The background subtracted fura-2 intensity ratio traces were visually inspected for each cell, only cells showing a Ca2+ response to both high K+ and GABA with low Cl- solution were included. The included cells were then sub-divided into cells with a Ca2+ response to GABA in ECS, considered to have an excitatory GABA response, and those without a Ca2+ response to GABA in ECS, considered to have an inhibitory GABA response.To examine GABA2 tests. Statistics were reported as means or proportions followed by the sample size (number of cells) and standard error for continuous data, the probability value, test statistic and degrees of freedom (if relevant) were included for any statistical test performed. Statistics and graphical representations of the data were performed using either R (x64) 2.14.0 with the default installed libraries or Graphpad Prism version 5.01 .For continuous data-sets where 2 means were compared the data were checked for an approximate fit to a Gaussian distribution. Assuming an approximately Gaussian distribution a two sample unpaired, two tailed, T-test was performed. If the data distribution was significantly distinct from a Gaussian distribution then data transformations were considered in order to apply a T-test. However, if there was no obvious data transformation which resulted in an approximately Gaussian distribution then the medians were compared using a Mann-Whitney U test. For binomial data sets comparing two proportions we used two-tailed Chim and rhythmic bursting behaviour  was almost zero at week 1 . By weekehaviour . Such becurrents . Taken t2 test) in the proportion of cells expressing either voltage-dependent Na+  and larger K+ at week 3 ), these small differences did not apparently affect the ability of cells to generate iAPs  upon current injection , left beate iAPs . These date iAPs , it beca+ and K+ channels, both Na+ current availability and resting Vm are crucial to spontaneous activity. At all weeks, but most strikingly at week 3, cells in ACM expressed significantly hyperpolarised Vm  vs. ACM = -37.7 ± 0.7 (n = 21), P < 0.01) at week 1. However, at later time points, where the differences in spontaneous activity were most marked, ACM evoked no significant changes in peak Na+ current voltage (week 2: control = -42.2 ± 0.7 (n = 7) vs. ACM = -42.6 ± 0.7 (n = 21); week 3: control = -42.5 ± 0.7 (n = 10) vs. ACM = -40 ±1.1 (n = 10)). Consistent with there being few differences in peak Na+ current voltages, the rate of change of voltage (dV/dt) versus voltage plots showed that ACM evoked no significant changes in the threshold for iAPs  at each time point were larger in the cells treated with ACM (control week 1 = 0 ± 0 pA.pF-1 (n = 6) vs. ACM week1 = -3.2 ± 0.6 pA.pF-1 ; control week 2 = -1.9 ± 0.4pA.pF-1 (n = 5) vs. ACM week 2 = -8.7 ± 1.9 pA.pF-1 ; control week 3 = -5.6 ± 1.3 pA.pF-1 (n = 8) vs ACM week 3 = -9.2 ± 1.0 pA.pF- 1 . To dissect the channel subtypes responsible for carrying the Ca2+ current at the early time points, fura-2 based Ca2+ imaging was employed. 50mM KCl (high K+) solution was used to activate all voltage-gated Ca2+ channels in the absence and presence of 10µM Nifedipine (L-type blockade), 100nM conotoxin (N-type blockade), 100nM agatoxin (P/Q-type blockade) or 100nM SNX482 (R-type blockade) (+ application (+ with a mean AUC of 2.8 ± 0.4 (n = 19), whereas 100 % of cells grown in ACM responded with a mean AUC of 6.1 ± 0.7, . Furthermore, there were significant differences in the proportion of cells in each culture condition which were sensitive to nifedipine (31.3 % in control medium (n = 19) vs. 91.3 % in ACM , conotoxin (57.9 % in control medium versus 95.7 % in ACM) and SNX482 , conotoxin-sensitive AUC went from 22.2 ± 10.1 % to 70.2 ± 5.1 (P < 0.001) and SNX482-sensitive AUC went from 5.2 ± 11.7 % to 38 ± 10.4 % (P < 0.001). In conclusion, ACM not only promoted a greater proportion of the cells to express Ca2+ channels, but those channels also made an increased functional contribution to each neuron, again indicative of a more mature neuronal phenotype.Neuronal voltage-gated Caectively . Ca2+ chturation . Therefolockade) . Consistlication , but sigchannels . These dblockers . Thus, n- equilibrium potential, a parameter which is itself developmentally controlled via differential expression of several Cl- transporters [per se. The magnitude of 300 μM GABA-evoked inward currents increased dramatically from week 1 to 2 in ACM (week 1 = -24.8 ± 6.8 pA.pF-1 (n = 8) to week 2 = -87.8 ± 12.5 pA.pF-1 (n = 20)) with smaller increases observed in control medium (week 1 = -22.1 ± 8.2 pA.pF-1 (n = 6) to week 2 = -49.4 ± 14.9 pA.pF-1 (n = 7)). From weeks 2 to 3 no significant increases were observed in the median GABA current densities in either culture condition (control = -79.1 ± 20.7 (n = 11) vs. ACM = -44.9 ± 11.4 (n = 7); A are normally excitatory or inhibitory. Therefore, Ca2+ influx in response to GABA was measured by fura-2-based Ca2+ imaging. Where the GABA response was excitatory, Ca2+ influx could be observed as a consequence of GABA-evoked depolarization to cause activation of voltage gated Ca2+ channels. If no response to GABA was observed, this could be the result of either GABA acting as an inhibitory neurotransmitter (to produce a hyperpolarization), or lack of expression of GABAA receptors and/or Ca2+ channels. To distinguish between these possibilities, GABA was also applied in a low Cl- extracellular solution; in this 9.9 mM Cl- solution, GABAA receptor activation can only be depolarising, and must cause Ca2+ influx if Ca2+ channels are expressed. Ca2+ channel expression was confirmed by application of high K+ to evoke Ca2+ influx directly by depolarisation. Using these approaches, cells that switched from an excitatory (immature) to inhibitory (mature) phenotype between week 1 and 2 were identified, but ACM did not significantly affect this switch  or specific Ca2+ channel blockers. Remarkably, blockade of GABAA receptors, L-type, R-type or N-type Ca2+ channels all significantly depolarised the neurons (from -50.8 ± 2.8 mV (n = 19) in the ACM to -27.1 ± 1.2 mV (n = 10) with 10 µM bicuculline, -21.0 ± 2.5mV (n = 9) with 2 µM nifedipine (L-type Ca2+ channel block), -27.3 ± 3.9 mV (n = 9) with 100 nM conotoxin (N-type Ca2+ channel block), and -25.5 ± 2.4 mV (n = 10) with 100 nM SNX482 (R-type Ca2+ channel block), 2+ influx was an important determinant of neuronal maturation, extracellular Ca2+ concentration was increased in the control medium from 0.6 mM to 1.8 mM and both resting Vm and spontaneous activity measured using whole cell patch-clamp. The increased Ca2+ concentration resulted in an enhancement in the proportion of cells generating spontaneous activity, which became significant at week 2, from 29 % (n = 21) in 0.6 mM Ca2+ to 56 % (n = 16) in 1.8 mM Ca2+  in 0.6mM Ca2+ to -42 ± 4.5 mV (n=16), P<0.0001, m of both groups had stabilized at this hyperpolarised level , to zero (n = 19), P<0.0001) and significantly depolarised Vm (from -41.4 ± 3.2 mV (n = 16) to -19.5 ± 2.7 mV (n = 19), P<0.0001). Moreover, addition of 300 µM GABA was as effective as the increased extracellular Ca2+ at week 1 in augmenting spontaneous activity (from 0% (n = 25) in control to 25 % (n = 8), P < 0.01, m (from -19.8 ± 2.9 mV (n = 25) to -42 ± 3.3 mV (n = 8), P < 0.001, T = 4.3, df = 31, 2+ are both capable of mimicking the ACM-evoked maturation of differentiating neurons. However, beyond week 2, the differential effects of high Ca2+ or GABA are lost and both the Vm and rates of spontaneous activity return to levels closer to those observed in control medium. This might have been attributed to a loss of excitatory GABA stimuli. Indeed, only half of the cells in the high Ca2+ cultures showed an excitatory GABA response (control = 100 % (n = 17) vs. high Ca2+ = 53 % (n = 17), P < 0.001).The early up-regulation of voltage-gated Caed level . These duculline . Additio2+ might augment the maturation of neurons via GABAA-dependent Ca2+ influx through specific Ca2+ channels. In this separate set of experiments, 2 weeks of differentiation in 1.8 mM Ca2+ resulted in the proportion of cells exhibiting spontaneous activity being increased from 19 % (n = 21) to 56 % (n = 16)  and depolarised the Vm (high Ca2+ = -47.1 ± 5.4 mV (n = 16); bicuculline = -30 ± 2.6 mV (n = 19), P < 0.0001; conotoxin = -21.1 ± 4.6 mV (n = 15), P < 0.0001, and; nifedipine = -21.8 ± 2.8 mV (n = 13), P < 0.0001). In contrast, agatoxin was completely without affect  2 together with markers of neuronal subtype-specific fate determination e.g. ,6 Such machieved , the celachieved . Exceediachieved  or the machieved  of Na+ ad by ACM , implyind by ACM . Anothert week 3  where 74aneously . Therefocurrents . Althoug2+ channels and GABAA receptors was investigated during early stages of differentiation. Strikingly, ACM evoked Ca2+ channel remodelling, with large increases in both the proportion of cells expressing L-, N- and R-type channels and the magnitude of the Ca2+ influx through each sub-type  which underlie ACM-enhanced neuronal maturation, the functional expression of voltage-gated Casub-type . In contsporters ,28,34), me point . 2+ channels in the absence of significant changes in Na+, K+ and GABAA channels, it seemed possible that ACM-evoked changes in functional maturation (hyperpolarized Vm and increased spontaneous activity) might be a result of this early Ca2+ channel remodeling. Indeed, inclusion of specific blockers of L-, N-, or R-type channels in the ACM resulted in depolarized Vm values  to ablate the high Ca release  and thatA receptor-induced depolarization and consequent Ca2+ influx. This mechanism can be partially mimicked in vitro by modestly increasing extracellular Ca2+ or GABA concentrations. In the absence of a complete understanding of the factors which are secreted by astrocytes and their individual modes of action, these simple modifications can now be exploited to augment functional maturation in modified differentiation protocols. However, in contrast to ACM, this GABA-dependent mechanism is susceptible to loss of function when GABA becomes inhibitory. Therefore, in the absence of a full understanding of ACM we suggest application of Ca2+ channel openers offers the possibility to bypass such developmental effects and enhance maturation of PSCs as a one-step process. Thus, ACM enhances neuronal maturation principally by amplification of an endogenous pathway which is dependent upon GABA


There is wide-spread human exposure to bisphenol A (BPA), a ubiquitous estrogenic endocrine disruptor that has been implicated as having potentially harmful effects on human heart health. Higher urine BPA concentrations have been shown to be associated with cardiovascular diseases in humans. However, neither the nature nor the mechanism(s) of BPA action on the heart are understood.−9 M) rapidly induced arrhythmogenic triggered activities. The effects of BPA were particularly pronounced when combined with estradiol. Under conditions of catecholamine stimulation, E2 and BPA promoted ventricular arrhythmias in female, but not male, hearts. The cellular mechanism of the female-specific pro-arrhythmic effects of BPA and E2 were investigated. Exposure to E2 and/or BPA rapidly altered myocyte Ca2+ handling; in particular, estrogens markedly increased sarcoplasmic reticulum (SR) Ca2+ leak, and increased SR Ca2+ load. Ryanodine (10−7 M) inhibition of SR Ca2+ leak suppressed estrogen-induced triggered activities. The rapid response of female myocytes to estrogens was abolished in an estrogen receptor (ER) β knockout mouse model.The rapid (<7 min) effects of BPA and 17β-estradiol (E2) in the heart and ventricular myocytes from rodents were investigated in the present study. In isolated ventricular myocytes from young adult females, but not males, physiological concentrations of BPA or E2 (102+ handling, particularly increases in SR Ca2+ leak. Our study provides the first experimental evidence suggesting that exposure to estrogenic endocrine disrupting chemicals and the unique sensitivity of female hearts to estrogens may play a role in arrhythmogenesis in the female heart.Physiologically-relevant concentrations of BPA and E2 promote arrhythmias in a female-specific manner in rat hearts; the pro-arrhythmic actions of estrogens are mediated by ERβ-signaling through alterations of myocyte Ca Estrogenic endocrine disrupting chemicals (EDCs) are a structurally diverse group of compounds that mimic, or antagonize the effects of endogenous estrogens. A particularly significant example of estrogenic EDCs to human health is the nearly ubiquitous xenoestrogen bisphenol A (BPA). BPA is structurally similar to the potent non-steroidal synthetic estrogen diethylstilbestrol. As one of the highest produced synthetic chemicals worldwide, BPA is used extensively in the production of polycarbonate plastic and epoxy resins that are found in a wide range of consumer products such as food containers, food cans, water bottles, baby bottles, dental sealants and water pipes. Bioactive BPA is released from epoxy resins and polycarbonate food and beverage containers, especially after exposure to elevated temperatures There is significant public, scientific and regulatory interest in elucidating the impact of BPA exposure on human health. Experimental evidence has demonstrated potential links between BPA exposure and cancer, obesity, diabetes, and disorders of the reproductive, neuroendocrine and immune systems in a variety of cellular and animal models 2+ handling, and excitation-contraction coupling Cardiovascular disease is a leading cause of mortality in developed countries, for both men and women. Estrogen has strong influences on the sexually-dimorphic baseline physiology of the heart, and on myocardial responses associated with various cardiac pathophysiological conditions including hypertrophy, failure, ischemic injury, and arrhythmias The aims of the present study were to investigate the rapid, sex-specific effects of physiologically-relevant concentrations of BPA and E2 on rodent cardiac myocytes and hearts, with a focus on the arrhythmogenic effect of estrogens, and to determine the underlying cellular mechanism of the rapid actions of estrogens.−9 M) resulted in detectable after-contractions in 21% and 18% of the myocytes, respectively. By comparison, only 1% of myocytes displayed after-contraction under control conditions. Exposure to a mixture of 10−9 M BPA and 10−9 M E2 significantly increased the percentage of cells with after-contractions to 42%. Measurements of triggered activity as spontaneous Ca2+ transients following pacing yielded similar results (−9 M) induced spontaneous Ca2+ transients in 27% and 23% of female rat myocytes, respectively, and in 45% of myocytes when added as 10−9 M equal molar mixture.Triggered activities are abnormal excitations of cardiac myocytes “triggered” by preceding impulses, and are a key arrhythmogenic mechanism in the heart  results . BPA or −9 M to 2×10−9 M did not increase the induced responses in female rat myocytes , 10−8 M], exposure to BPA and E2 markedly increased the frequency of ectopic ventricular beats from 5.8±2.2 to 20±8 per 20 min  was 2.3 in control myocytes, and 3.5 and 3.6 in myocytes exposed to BPA or E2, respectively. An additional increase in peak Ca2+ transient amplitude to 4.8 was observed in myocytes exposed to BPA and E2. The decay rate of the Ca2+ transients was also increased   . Consistat basis . The decaffected . The fraxposures . Howeverd by BPA . As a wh2+ release was further assessed by analysis of Ca2+ sparks. Ca2+ sparks, which represent local and quantal release of SR Ca2+ through clusters of RyRs −9 M BPA significantly increased the average Ca2+ spark frequency in female rat myocytes from 3.2 to 4.9 sparks/100 µm/s . Therefore, the observed increase in Ca2+ release is most likely the result of increased RyR opening. The role of increased spontaneous SR Ca2+ release, or “Ca2+ leak”, in the development of estrogen-induced triggered activities was examined using ryanodine −7 M ryanodine did not affect field-stimulation elicited Ca2+ transients  in the development of cardiac arrhythmias in the female heart.Endogenous estrogens are well recognized to play important roles in the regulation and maintenance of sex-specific differences in cardiac physiology and pathophysiology; however, little is known about the cardiac effects of environmental estrogenic EDCs, such as the ubiquitous BPA, and their interactions with endogenous estrogens. In the present study, we show that physiologically-relevant concentrations of BPA and E2 have female-specific pro-arrhythmic effects in rodent cardiac myocytes and whole hearts. The effects of BPA and E2 are mediated by ERβ-signaling, through rapid alteration of myocyte Ca−12 M, with 10−9 M being the most efficacious (data not shown). Therefore, based on experimental considerations, estimated human exposure levels, and BPA's pharmacodynamic profile, 10−9 M was selected for further analysis. We demonstrated here that in ventricular myocytes from female but not male rats, exposure to 1 nM BPA or E2, and particularly BPA combined with E2, markedly increased the percentage of myocytes with spontaneous excitations following repeated pacing. These triggered activities are indicative of delayed after-depolarizations, or DADs, a myocyte phenomenon that is recognized as a key non-reentrant arrhythmogenic mechanism 2+ overload condition and are the result of diastolic spontaneous Ca2+ release from the SR. Such spontaneous SR Ca2+ release triggers Ca2+ extrusion by the Na+/Ca2+ exchanger (NCX); the activity of the electrogenic NCX generates a depolarizing current, leading to excitation of the myocyte during the diastole. These ectopic excitations can propagate through the myocardium and initiate arrhythmia events 2+ load, and abnormal SR Ca2+ release . In particular, aberrant RyR opening and diastolic SR leak have been shown to be a central factor in the development of DADs and lethal ventricular arrhythmias under disease conditions such as heart failure 2+ reuptake, SR load, and the fraction of SR Ca2+ release on a beat-to-beat basis. Further, E2 and BPA significantly increased the frequency of spontaneous Ca2+ sparks, likely as a result of increasing RyR open probability. Supporting a key role of abnormal SR leak in the pro-arrhythmic effect of BPA in female myocytes, we showed that suppression of RyR release by ryanodine, while not affecting normal Ca2+ transients, abolished DADs triggered by BPA alone or BPA combined with E2.When assessed using representative urine samples, BPA was detected in over 90% of the US population with mean concentration in the low ng/mL, or low-nanomolar range 2+ influx through the L-type Ca2+ channels, SR reuptake (and consequently SR Ca2+ load), and RyR activities 2+ release, and likely potentiate the pro-arrhythmic actions of these estrogens. Our results suggest that BPA exposure may become a particularly significant factor for arrhythmogenesis in females under stress conditions, and possibly target females with existing cardiac pathophysiological conditions that provide the substrate for arrhythmogenesis.Unlike the robust response of female myocytes to BPA and E2, neither compound induced arrhythmias under baseline conditions. However, with β-adrenergic receptor stimulation, BPA combined with E2 resulted in a marked increase in ventricular arrhythmic events, including premature ventricular contractions and ventricular tachycardia in female rat hearts. The discrepancy between the effects of estrogens at the myocyte and whole heart levels likely reflects the fact that only a fraction of the single myocyte-level abnormal electrical activities may propagate and result in arrhythmic events. How myocyte-level triggered activities lead to whole-myocardium arrhythmias, particularly sustained arrhythmias, is complex and not fully understood. Catecholamines affect myocyte mechanics by enhancing Ca2+ handling appeared to be amplified in the presence of E2. This intriguing experimental effect raises the possibility that the pro-arrhythmic effect of BPA may be more pronounced in females with higher levels of endogenous estrogens, such as during pregnancy. Bisphenol A has been shown to have additive or synergistic effects with E2 or xenoestrogens in various cell types or systems in other studies The rapid effects of BPA on DAD development and Ca2+ handling Bisphenol A elicits its biological responses through activation of ER or ER-like receptors. Both ERα and ERβ, including those localized to the membrane, have been shown to be expressed in cardiomyocytes from species including rat and human Despite the perception that female hormones protect women from cardiovascular disease, disease of the cardiovascular system is the leading cause of mortality for women in the US. According to the American Heart Associate statistics, since 1984 more women than men have died of cardiovascular disease every year in the US; 53% of total cardiovascular disease deaths occur in women. There is well-recognized sexually dimorphism in cardiovascular disease, including cardiac arrhythmias. Compared with men, women have a lower incidence of sudden cardiac death owning to the protective effect of estrogens on coronary artery disease, and have lower risk of atrial fibrillation; however, women have higher rates of long QT syndrome, sudden cardiac death in the absence of coronary artery disease, and ventricular arrhythmias post myocardial infarction Reagents and solvents used were of the highest purity available. All aqueous solutions were prepared using BPA-free water . Dimethyl sulfoxide , bisphenol A (BPA), CAS 80-05-7, >99%, 23,965-8; lot Cl03105ES, isoproterenol hydrochloride, CAS 5984-95-2 and BSA, CAS 9048-46-8, >98% (batch no. 078K0730) were from Sigma-Aldrich . 1,3,5 (10)-estratriene-3,17β-diol  was from Steraloids (Newport RI). Other chemicals were from Sigma-Aldrich unless otherwise stated.All animal procedures were done in accordance with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee and followed recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.-/-; Jackson Laboratory; Bar Harbor, ME) were utilized as non-surviving sources of ventricular tissue and hearts. Animals were anesthetized with sodium pentobarbital  and heart rapidly dissected for myocyte dissociation. The ERβ-/- mice and age matched wild type littermates were reared at the University of Cincinnati laboratory animal facility and used at 6–8 weeks of age. This ensured that the hearts were free of abnormality associated with systemic effects of ERβ ablation that set in much later, at 5 months of age Adult Sprague-Dawley rats  and ERβ (Esr2) knockout mice (ERβad libitum Teklad diet 2020 (Harlan Laboratories Inc.) which lacks soybean meal, alfalfa or animal products that might complicate the study by introduction of uncontrolled levels of estrogenic compounds. Sterile drinking water was generated by a dedicated water purification system (Millipore Rios 16 with ELIX UV/Progard 2) that reduces oxidizable organics to less than 1% of source levels. Drinking water was dispensed from glass water bottles. Concentrations of BPA in drinking water and all experimental reagents were confirmed below the level of detection of a BPA-specific and extremely sensitive ELISA based assay with a minimum quantitative detection limit of 0.05 ng/ml Animals were maintained on a 14 h light, 10 h dark light cycle in standard polycarbonate caging with Sani-chip bedding  to eliminate possible corn-based mycoestrogen exposure. All animals are fed 2PO4 0.33, MgCl2 2, glucose 10 (pH = 7.4) and containing 0.7 mg/ml or 1 mg/ml type II collagenase  for rat and mouse hearts, respectively. For rat myocyte dissociation, the solution also contained 1 mg/ml BSA, 0.2 mg/ml hyaluronidase, and 0.025 mM CaCl2. Isolated ventricular myocytes were suspended in 1.0 mM Ca2+-Tyrode solution and allowed to sediment by gravity. Supernatant was then removed and myocytes were resuspended. This process was repeated 2–3 times to enrich rod-shaped live myocytes and remove dead cells and non-myocytes. Isolated rat myocytes were suspended in a Medium-199 based solution , plated on laminin -coated glass coverslips at a density of approximately 5×104 cells/cm2 in 35×10 mm polystyrene culture dishes, and allowed to adhere for 10 minutes at 37°C. Dead myocytes did not adhere to the cover slips and were rinsed off with additional medium. Myocytes were cultured in 2 ml medium/dish in 5% CO2 at 37°C in a humidified incubator for 24 hours prior to analysis. Resulting cultures typically contained >95% myocytes.Ventricular myocytes from rodent hearts were enzymatically dissociated using Langendorff perfusion with a Tyrode solution composed of (mM) NaCl 118, KCl 5.4, HEPES 10, NaH2) containing either vehicle or treatment drug, in a custom made glass-plexiglass recording chamber at room temperature (24°C). Analysis of Ca2+ transients and sparks was made between 2 to 7 minutes following treatment. Ca2+ sparks were recorded from quiescent, unstimulated myocytes. For Ca2+ after-transient recording, myocytes were paced with field stimulation  for 8 seconds at a rate of 2 Hz with 2 ms 1.5x threshold pulses. The presence of any spontaneous Ca2+ after-transients following pacing was recorded. Fluorescence images were recorded using a Zeiss LSM 510 inverted confocal microscope through a 40x water-immersion objective lens with excitation wavelength of 488 nm. Fluorescence signals were measured at >515 nm with line-scan imaging at 3.07 ms intervals, with each line comprising 512 pixels spaced at 0.056 µm. Image processing and data analysis were performed using IDL software . For analysis of the amplitude and kinetics of field-stimulated or caffeine-induced Ca2+ transients, fluorescence signals were measured from fluo-4 loaded myocytes using a Nikon TE 2000 microscope and an InCyt Standard PM photometry system . Field stimulated Ca2+ transients were elicited with 2 ms 1.5x threshold pulses at a rate of 0.5 Hz. For recording of caffeine-induced Ca2+ transients, myocytes were field stimulated at 0.5 Hz for 20 seconds, and were exposed to solution containing 10 mM caffeine immediately following cessation of pacing.Isolated ventricular myocytes were loaded with fluo-4 acetoxymethyl ester  for 15 min at room temperature, and then washed with Tyrode's solution. Myocytes were placed in Tyrode solution . Electrocardiograph was continuously measured from the surface of the heart, with electrodes positioned at the base and apex of the heart. Data collection and analysis were performed using the Powlab 4/30 data acquisition system and LabChart 7 software .Following dissection, hearts were quickly cannulated via the aorta, and retrograde perfused on a Langendorff apparatus with 37°C Krebs-Henseleit solution containing (mM) NaCl 118, KCl 4.7, MgSO2-Tyrode solution at room temperature (24°C). The solution also contained either vehicle or various treatment drugs. Myocyte contraction was recorded between 2 to 7 minutes following treatment. Myocytes were excited with field stimulation  with 2 ms 1.5x threshold pulses at a rate of 0.5 Hz. Steady state myocyte shortening was imaged with a CCD camera and examined using a video-edge detector . Data were sampled through an Axon Digidata 1322A board using the PCLAMP 9 software .Myocytes were placed in a plexiglass cell chamber filled with 1.8 mM CaCl2+ current was recorded at room temperature (24°C) using the whole-cell patch clamp technique with an Axonpatch-200B amplifier . Extracellular solution contained (in mM) TEA-Cl 137, CsCl 5.4, CaCl2 2, MgCl2 1, HEPES 5, glucose 10, and 4-aminopyridine 3 (pH = 7.4). Glass pipettes were filled with solution containing (in mM) Cs-aspartate 115, CsCl 20, EGTA 11, HEPES 10, MgCl2 2.5, Mg-ATP 2 and Na-GTP 0.1 (pH = 7.2), and had a resistance of 1.5–2.5 MΩ. After the membrane was ruptured, cells were clamped at –50 mV for 5 minutes to allow dialysis of the intracellular solution and stabilization of the Ca2+ currents before measurement began. Data collection and analysis were performed using PCLAMP 9 software.The L-type Ca2) test. Minimal level of statistical significance for differences in values is considered to be p<0.05 (*). Data was analyzed with SigmaPlot 11.0, Excel and GraphPad Prism® version 5.0.All experiments were independently repeated using myocytes isolated from at least 4 hearts. Statistical analysis was conducted using a paired or unpaired t-test, or one-way or 2- way (e.g. sex vs. treatment) analysis of variance (ANOVA) with differences between treatment groups assessed using a multiple comparison post-test. Frequency of events  was analyzed using a chi-square (χ


A DNA microarray analysis revealed that the expression of several genes, including metal-related genes , endoplasmic reticulum (ER)-stress related genes , and the calcium (Ca)-related gene Arc (activity-related cytoskeleton protein), were affected after Zn exposure. The co-existence of carnosine or His inhibited the expression of GADD34, p8, and Arc, although they did not influence the expression of the metal-related genes. Therefore, ER-stress and the disruption of Ca homeostasis may underlie the mechanisms of Zn-induced neurotoxicity, and carnosine might be a possible drug candidate for the treatment of VD.Zinc (Zn) is an essential trace element that is abundantly present in the brain. Despite its importance in normal brain functions, excess Zn is neurotoxic and causes neurodegeneration following transient global ischemia and plays a crucial role in the pathogenesis of vascular-type dementia (VD). We have investigated the molecular mechanisms of Zn-induced neurotoxicity using immortalized hypothalamic neurons (GT1–7 cells) and found that carnosine  and histidine (His) inhibited Zn Finally, Ca2+ dyshomeostasis, which involves the entry of large quantities of Ca2+ into glutamate-responsive neurons, triggers the delayed death of vulnerable populations of neurons, such as pyramidal neurons in the hippocampus—an area associated with learning and memory. Thereafter, the development of an infarct and subsequent cognitive dysfunction mark the pathogenesis of VD in elderly people. Approximately 30% of stroke patients show symptoms of dementia within 3 months of the initial stroke i), namely, Zn translocation, occurs in vulnerable neurons in the CA1 or CA3 regions of the hippocampus prior to the onset of delayed neuronal death after transient global ischemia [et al. have observed temporal changes in [Zn2+]i in cultured cortical neurons with a Zn-sensitive fluorescent dye; those results revealed that at least 3 major routes of Zn2+ entry have been identified: Voltage-gated Ca2+ channels (VGLG), NMDA-type glutamate receptors, and AMPA/kainate-type glutamate receptors (A/K-R). Although the NMDA-type glutamate receptors are present in most neurons, the permeability of Zn2+ and Ca2+ through AMPA/kainate channels is greater than that of NMDA-type glutamate receptor channels , or those of inhibitory neurotransmitters  attenuated the viability of GT1–7 cells after Zn exposure . Our fin3+ and Gd3+ significantly inhibited Zn-induced neuronal death. Moreover, the overloading of Ca2+ and Mg2+ inhibited the Zn-induced death of GT1–7 cells, and Zn protected GT1–7 cells from the neurotoxicity induced by Ca2+ overload and vice versa -Bay K 8466 [2+ entry through voltage-gated Ca2+ channels [2+]i after exposure to Zn [2+]i among GT1–7 cells after 3–30 min of exposure [2+]i revealed that the pretreatment of Al3+ significantly blocked the Zn-induced [Ca2+]i increases. Thus, it is possible that Al3+, a known blocker of various types of Ca2+ channels, might attenuate Zn-induced neurotoxicity by blocking Zn-induced increases in [Ca2+]i.Based on these findings, we investigated the molecular mechanisms underlying Zn-induced neurotoxicity. Our results suggested that Ca dyshomeostasis may be involved in the mechanisms of Zn-induced neurotoxicity. It has also been reported that Zn neurotoxicity in PC12 cells is attenuated by an y K 8466 . Additiochannels . In addire to Zn . Zn causexposure . A detai2.4.MT)-1, MT-2, and the Zn transporter 1 (ZnT-1) were induced by Zn exposure. Metallothioneins are intracellular polypeptides with a remarkable ability to bind metallic ions. MT1 and MT2 play roles in the detoxification of heavy metals. Zn-specific membrane transporter proteins (Zn transporters) also control Zn homeostasis; they facilitate Zn influx during deficiencies and efflux during Zn excess. Thus, these proteins are upregulated by increases in [Zn2+]i. (see Zn in GADD)34, GADD45, and p8) and the Ca2+-related gene Arc (activity-related cytoskeleton protein) [2+ homeostasis in Zn-induced neurotoxicity. It is widely accepted that the ER regulates the levels of [Ca2+]i and that ER stress causes the apoptotic death of various cells by the accumulation of unfolded or misfolded proteins [GADD34 and GADD45 are the genes of the sensor proteins of ER stress, and they are induced by DNA damage and have been implicated in DNA repair and tumorigenesis [p8 mRNA is induced in response to divergent stress, and it might be involved in tumorigenesis [Arc is a gene that encodes a protein that exists in dendrites and plays crucial roles in synaptic plasticity and memory consolidation. Arc expression is induced by the increased neuronal activity that occurs in response to learning and by brain-derived neurotrophic factor (BDNF) [Moreover, we investigated the alterations in gene expression during Zn-induced neurotoxicity with a DNA microarray and RT-PCR . The expprotein) . These fproteins . Emerginproteins ,45. In pigenesis . The p8 igenesis . In addir (BDNF) .MT1, MT2, or ZnT-1 during Zn-induced neurotoxicity , dietary supplementation of carnosine might be an effective strategy for the prevention or treatment of neurodegenerative diseases, such as ischemia, VD, AD, and prion diseases. Corona del mice . We havedel mice . Therefodel mice  and for In the brain, carnosine also exists in the olfactory bulb . We have


Citrus bergamia Risso  on intracellular Ca2+ in human umbilical vein endothelial cells. Fura-2 fluorescence was used to examine changes in intracellular Ca2+ concentration [Ca2+]i. In the presence of extracellular Ca2+, BEO increased [Ca2+]i, which was partially inhibited by a nonselective Ca2+ channel blocker La3+. In Ca2+-free extracellular solutions, BEO increased [Ca2+]iin a concentration-dependent manner, suggesting that BEO mobilizes intracellular Ca2+. BEO-induced [Ca2+]iincrease was partially inhibited by a Ca2+-induced Ca2+ release inhibitor dantrolene, a phospholipase C inhibitor U73122, and an inositol 1,4,5-triphosphate (IP3)-gated Ca2+ channel blocker, 2-aminoethoxydiphenyl borane (2-APB). BEO also increased [Ca2+]iin the presence of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial Ca2+ uptake. In addition, store-operated Ca2+ entry (SOC) was potentiated by BEO. These results suggest that BEO mobilizes Ca2+ from primary intracellular stores via Ca2+-induced and IP3-mediated Ca2+ release and affect promotion of Ca2+ influx, likely via an SOC mechanism.The purpose of the present study is to examine the effects of essential oil of  Citrus bergamia Risso), a roughly pear-shaped citrus fruit. BEO is widely used in aromatherapy to alleviate symptoms of stress-induced anxiety, mild mood disorders, and cancer pain i via release from intracellular Ca2+ stores and through store-operated Ca2+ entry (SOC).Up till now, the effects of BEO on i was measured using a microfluorometer system consisting of an inverted microscope  and a PTI Filter Scan power illuminator system . Fura-2 AM (2 μM) was added to the bath and the cells were incubated for 25 min at 37°C. The cells were illuminated alternatively at wavelengths of 340 and 380 nm through a chopper wheel (frequency = 50 Hz). Fluorescence was measured at 510 nm, and autofluorescence was subtracted from the signals obtained. The Ca2+-free concentration was calculated from the ratio of the fluorescence signals emitted at each excitation wavelength. The calibration procedure was identical to that described previously i in a concentration-dependent manner in EA cells  was obtained at 0.04 ± 0.01%. DMSO (0.25% v/v) itself did not change intracellular Ca2+ levels. The cells showed no morphological change after treatment with BEO. Then we investigated whether BEO changed [Ca2+]i in the presence of extracellular Ca2+ in EA cells. Application of BEO increased [Ca2+]i to 1.41 ± 0.14 μM, which was partially and reversibly inhibited by the non-selective Ca2+ channel blocker La3+ (1 μM). [Ca2+]i was reduced to 1.03 ± 0.09 μM by La3+  and mitochondria, is affected by BEO in EA cells. Ca2+ release from the ER depends on two mechanisms: Ca2+-induced Ca2+ release (CICR), involving ryanodine receptors, and IP3-induced Ca2+ release (IICR), involving inositol 1,4,5-triphosphate (IP3) receptors i. To determine whether mitochondria participate in the reuptake of BEO-induced Ca2+ release, we examined the effects of BEO on [Ca2+]i in a Ca2+-free solution in the presence of BHQ (an SR/ER Ca2+-ATPase inhibitor) and CCCP . In cells treated with BHQ and/or CCCP, [Ca2+]i transiently increased and then decreased slowly to a steady state, suggesting that an SR/ER Ca2+-ATPase and mitochondrial Ca2+ uptake participate in the regulation of [Ca2+]i under basal conditions  is (are) activated by BEO. Thus, we next examined whether BEO modulated Ca2+ entry via an SOC mechanism. Exposure of EA cells to the BHQ in a Ca2+-free solution induced a transient increase in [Ca2+]i, which then decreased slowly to a steady state (2+ following emptying of intracellular Ca2+ stores with BHQ caused an increase in [Ca2+]i, indicating activation of an SOC mechanism. When BEO was applied after SOC was evoked, SOC was further enhanced, suggesting that BEO also activates Ca2+ influx through an SOC pathway (Considering the inhibitory effect of Lady state . Reappli pathway . However2+ from extracellular and intracellular sources in endothelial cells. Our present results also suggest that BEO increased intracellular Ca2+ level through both mobilization of intracellular Ca2+ stores, ER and mitochondria, and promotion of Ca2+ influx, via an SOC mechanism. These findings will provide insight into the physiological mechanisms involved in Ca2+ regulation in endothelial cells following exposure to BEO.In the present study, we firstly demonstrate that BEO mobilized CaCitrus bergamia contains psoralens which bind to DNA under ultraviolet A light exposure producing mono- and biadducts that are cytotoxic and highly mutagenic i, known as Ca2+ sensitization [2+]i initiates smooth muscle contraction via activating myosin light chain kinase, Ca2+ sensitization mediates smooth muscle contraction by modulating myosin light chain phosphatase [2+]i in endothelial cell plays a role in synthesis and the release of vasoactive compounds such as nitric oxide or prostaglandins [2+ sensitization in smooth muscle cells. It has been reported that linalyl acetate, one of the main components of BEO, induces relaxation of the smooth muscle via partially endothelium-dependent pathway [Contraction and relaxation of smooth muscle are regulated not only by changes in cytoplasmic calcium concentration but also by other important signaling mechanisms, that is, independent of the changes in [Catization . Althougsphatase . The incglandins , thereby pathway . Further2+-elevating pathways enlisted by BEO under pathological conditions.In conclusion, before BEO can be considered for use in treating vascular-related diseases, further studies are necessary to define the Ca


This response is caused by Ca2+ release from intracellular Ca2+ stores, which are comprised of the endoplasmic reticulum and the nuclear envelope. The Ca2+ release leads to the activation of Ca2+ entry from the extracellular space, which is called capacitative, or store-operated Ca2+ entry. These movements of Ca2+ are essential for DNA synthesis during S-phase. Spontaneous Ca2+ oscillations also occur synchronously across the cells. This synchronization is mediated by voltage fluctuations in the membrane potential of the nuclear envelope due to Ca2+ release and the counter movement of K+ ions; the voltage fluctuation induces alternating current (AC), which is transmitted via capacitative electrical coupling to the neighboring cells. The membrane potential across the plasma membrane is stabilized through gap junction coupling by lowering the input resistance. Thus, stored Ca2+ ions are a key player in the maintenance of the cellular activity of neuroepithelial cells.During the embryonic development of the central nervous system, neuroepithelial cells act as neural stem cells. They undergo interkinetic nuclear movements along their apico-basal axis during the cell cycle. The neuroepithelial cell shows robust increases in the nucleoplasmic [Ca During the embryonic development of the central nervous system, cells in the neuroepithelium act as neural stem cells. The neuroepithelium forms the neural tube, from which the central nervous system including the spinal cord, retina, and brain is derived. The neuroepithelial cell has a polarized structure: the apical process faces the ventricle, and the furthest portion of the basal process makes contact with the basement membrane. This contact is necessary for the cell to undergo interkinetic nuclear movement along the apico-basal axis during the cell cycle –4. NeuroTo study ion channel activities in the neuroepithelial cell, the retinal neuroepithelium is a suitable model because the retina can be isolated from an optic cup at early stages of embryonic development. The neural tube evaginates laterally to form two optic vesicles, each of which invaginates to form an optic cup. The neuroepithelium on the inner wall of the optic cup becomes the retina. 2+ concentrations ([Ca2+]). Activation of these GPCRs leads to the production of inositol 1,4,5-trisphosphate (InsP3) from phosphatidylinositol 4,5-bisphosphate (PIP2) via the phospholipase C enzyme. InsP3 activates the InsP3 receptor Ca2+ channel to cause the release of Ca2+ from intracellular Ca2+ stores (Ca2+ mobilization)  occur in the nucleoplasm of S-phase cells, of which somata are localized to the basal region of the developing neuroepithelium. In contrast, Ca2+ mobilization decreases in M-phase cells, which are located in the apical region. In newborn retinal ganglion cells, which migrate to the basal region  show Ca2+ increases.Using confocal fluorescence microscopy and a Call cycle . Increasl region , Ca2+ montiation . It is n2+ response of the neuroepithelial cell also depends upon the developmental stage of the retina. On embryonic day 3 (E3), nearly all the cells in the neural retina arise from the self-renewal of the neuroepithelial cells , which regulates cell cycle progression  in the retinal neuroepithelial cell is higher than that in a mature neuron and the equilibrium potential of Cl− is more positive than the resting membrane potential , which may be caused by Ca2+ influx through voltage-dependent Ca2+ channels during depolarization. Cells expressing BK channels in the plasma membrane show a voltage sag in response to a depolarizing current injection. This is the case in newborn retinal ganglion cells, which do not yet generate a tetrodotoxin-sensitive Na+-dependent action potential [2+] is increased.BK  and by an increase in the luminal [Ca2+] , 40. Sucred Ca2+ .2+ oscillations occur synchronously across the cells in the retinal neuroepithelium, while agonist-induced [Ca2+] rises occur asynchronously [2+ oscillations has been a matter of debate [3 or Ca2+ itself through gap junction channels is unlikely to synchronize a [Ca2+] rise, because an agonist-induced Ca2+ release is not synchronized even with the application of a supramaximal concentration of the agonist  is increased. Thus, stored Ca2+ ions are a key player in the maintenance of the cellular activity of neuroepithelial cells.