Inward rectifier potassium channels in the HL‐1 cardiomyocyte‐derived cell line

HL‐1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K⁺ channels. Our aim was to identify and characterize inward rectifier K⁺ channels in HL‐1 cells. External Ba²⁺ (100 µM) inhibited 44 ± 0.05% (mean ± s.e.m., n = 11) of inward current in whole‐cell patch‐clamp recordings. The reversal potential of the Ba²⁺‐sensitive current shifted with external [K⁺] as expected for K⁺‐selective channels. The slope conductance of the inward Ba²⁺‐sensitive current increased with external [K⁺]. The apparent Kd for Ba²⁺ was voltage dependent, ranging from 15 µM at ?150 mV to 148 µM at ?75 mV in 120 mM external K⁺. This current was insensitive to 10 µM glybenclamide. A component of whole‐cell current was sensitive to 150 µM 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), although it did not correspond to the Ba²⁺‐sensitive component. The effect of external 1 mM Cs⁺ was similar to that of Ba²⁺. Polymerase chain reaction using HL‐1 cDNA as template and primers specific for the cardiac inward rectifier K_ir2.1 produced a fragment of the expected size that was confirmed to be K_ir2.1 by DNA sequencing. In conclusion, HL‐1 cells express a current that is characteristic of cardiac inward rectifier K⁺ channels, and express K_ir2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype. J. Cell. Physiol. 225: 751–756, 2010. © 2010 Wiley‐Liss, Inc.     

Ca2+-conductances in cultured rat retinal pigment epithelial cells

Membrane conductances for Ca²⁺ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamiland diltiazem-sensitive Ba²⁺ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba²⁺ currents indicating the presence of L-type Ca²⁺ channels. In order primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels. © 1994 Wiley-Liss, Inc.     

Role of N‐ and L‐type calcium channels in depolarization‐induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals

Multiple types of voltage‐activated calcium (Ca²⁺) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca²⁺ influx through N‐ and L‐ type voltage‐activated Ca²⁺ channels in sympathetic neurons to the depolarization‐induced activation of tyrosine hydroxylase (TH), the rate‐limiting enzyme in norepinephrine (NE) synthesis, and the depolarization‐induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both ω‐conotoxin GVIA (1 μM), a specific blocker of N‐type channels, and nimodipine (1 μM), a specific blocker of L‐type Ca²⁺ channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K⁺, indicating that Ca²⁺ influx through both types of channels contributes to enzyme activation. In contrast, K⁺ stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by ω‐conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K⁺ stimulation of NE release from both ganglia and irises was also blocked completely when ω‐conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca²⁺ influx through N‐ and L‐type Ca²⁺ channels. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 137–148, 1999     

Arabidopsis ROP‐interactive CRIB motif‐containing protein 1 (RIC1) positively regulates auxin signalling and negatively regulates abscisic acid (ABA) signalling during root development

Auxin and abscisic acid (ABA) modulate numerous aspects of plant development together, mostly in opposite directions, suggesting that extensive crosstalk occurs between the signalling pathways of the two hormones. However, little is known about the nature of this crosstalk. We demonstrate that ROP‐interactive CRIB motif‐containing protein 1 (RIC1) is involved in the interaction between auxin‐ and ABA‐regulated root growth and lateral root formation. RIC1 expression is highly induced by both hormones, and expressed in the roots of young seedlings. Whereas auxin‐responsive gene induction and the effect of auxin on root growth and lateral root formation were suppressed in the ric1 knockout, ABA‐responsive gene induction and the effect of ABA on seed germination, root growth and lateral root formation were potentiated. Thus, RIC1 positively regulates auxin responses, but negatively regulates ABA responses. Together, our results suggest that RIC1 is a component of the intricate signalling network that underlies auxin and ABA crosstalk.     

A new quaternary photoluminescence enhancement system of Eu−N‐(o‐vanillin)‐1,8‐diaminonaphthalene−1,10‐phenanthroline−Zn and its application in determining trace amounts of europium and zinc

A new sensitive quaternary photoluminescence enhancement system has been successfully developed to determine trace amounts of Eu³⁺ and Zn²⁺. The photoluminescence intensity of Eu ? N‐(o‐vanilin)‐1,8‐diaminonaphthalene systems was greatly increased by the addition of specific concentrations of 1, 10‐phenanthroline and Zn²⁺. The excitation and emission wavelengths were 274 and 617 nm, respectively. Under optimal system conditions, the photoluminescence intensity showed a linear response toward Eu³⁺ in the range of 5.0 × 10^–6 ~ 2.0 × 10^–5 M with a limit of detection (= 2.2 × 10^–9 M) and the photoluminescence intensity of the system decreased linearly by increasing the Zn²⁺ concentration in the range of 5.0 × 10^–8 ~ 1.0 × 10^–6 M with a limit of detection (= 8.8 × 10^–11 M). This system was successfully applied for the determination of trace amounts of Eu³⁺ in a high purity La₂O₃ matrix and in the synthetic rare earth oxide mixture, and of Zn²⁺ in a high purity Mg(NO₃)₂ · 6H₂O matrix and in synthetic coexisting ionic matrixes. The energy transfer mechanism, photoluminescence enhancement of the system and interference of other lanthanide ions and common coexisting ions were also studied in detail. Copyright © 2013 John Wiley & Sons, Ltd.     

α1‐adrenergic stimulation mediates Ca2+‐dependent inositol phosphate formation through the α1B‐like adrenoceptor subtype in adult rat cardiac myocytes

We studied the effects of increased Ca²⁺ influx on α₁‐adrenoceptor‐stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific α₁‐adrenoceptor subtype. [³H]InsP responses to adrenaline were dependent on extracellular Ca²⁺ concentration, from 0.1 μM to 2 mM, and were completely blocked by Ca²⁺ removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca²⁺ concentrations higher than 1 μM had no effect on adrenaline‐stimulated [³H]InsP formation. Taken together these results suggest that [³H]InsP formation induced by α₁‐adrenergic stimulation is in part mediated by increased Ca²⁺ influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [³H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the α_1B‐adrenoceptor alkylating agent, CEC, [³H]InsP formation remained unaffected by increased Ca²⁺ concentrations, a pattern similar to that observed when intracellular Ca²⁺ was chelated with BAPTA. In contrast, addition of the α_1A‐subtype antagonist, 5′‐methyl urapidil, did not affect the Ca²⁺ dependence of [³H]InsP formation. Neither nifedipine, a voltage‐dependent Ca²⁺ channel blocker nor the inorganic Ca²⁺ channel blockers, Ni²⁺ and Co²⁺, had any effect on adrenaline stimulated [³H]InsP, at concentrations that inhibit Ca²⁺ channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein‐mediated response, α₁‐adrenergic‐stimulated [³H]InsP formation is activated by increased Ca²⁺ influx mediated by the α_1B‐subtype. J. Cell. Biochem. 84: 201–210, 2002. © 2001 Wiley‐Liss, Inc.     

Baclofen and Adenosine Inhibition of Synaptic Transmission at CA3-CA1 Synapses Display Differential Sensitivity to K<Superscript>+</Superscript> Channel Blockade

The metabotropic GABA_B and adenosine A₁ receptors mediate presynaptic inhibition through regulation of voltage-dependent Ca²⁺ channels, whereas K⁺ channel regulation is believed to have no role at the CA3-CA1 synapse. We show here that the inhibitory effect of baclofen (20 μM) and adenosine (300 μM) on field EPSPs are differentially sensitive to Cs⁺ (3.5 mM) and Ba²⁺ (200 μM), but not 4-aminopyridine (100 μM). Barium had no effect on paired-pulse facilitation (PPF) in itself, but gave significant reduction (14 ± 5%) when applied in the presence of baclofen, but not adenosine, suggesting that the effect is presynaptic and selective on the GABA_B receptor-mediated response. The effect of Ba²⁺ on PPF was not mimicked by tertiapin (30 nM), indicating that the underlying mechanism does not involve GIRK channels. Barium did not affect PPF in slices from young rats (P7–P8), suggesting developmental regulation. The above effects of Ba²⁺ on adult tissue were reproduced when measuring evoked whole-cell EPSCs from CA1 pyramidal neurons: PPF was reduced by 22 ± 3% in the presence of baclofen and unaltered in adenosine. In contrast, Ba²⁺ caused no significant change in frequency or amplitude of miniature EPSCs. The Ba²⁺-induced reduction of PPF was antagonized by LY341495, suggesting metabotropic glutamate receptor involvement. We propose that these novel effects of Ba²⁺ and Cs⁺ are exerted through blockade of inwardly rectifying K⁺ channels in glial cells, which are functionally interacting with the GABA_B receptor-dependent glutamate release that generates heterosynaptic depression.     

Diethylstilbestrol regulates mouse gubernaculum testis cell proliferation via PLC‐Ca2+‐CREB pathway

Recent evidence suggested a positive correlation between environmental estrogens (EEs) and high incidence of abnormalities in male urogenital system, but the mechanism remains unclear. Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen that disrupts the morphology and proliferation of gubernaculum testis cells, but the underlying mechanism is unclear. In this study, mouse gubernaculum testis cells were pretreated with phospholipase C (PLC) inhibitor U‐73122 and then treated with DES. The results demonstrated that U‐73122 impaired DES‐evoked intracellular Ca2+ mobilization in gubernaculum testis cells and inhibited DES‐induced proliferation of gubernaculum testis cells. Mechanistically, we found that U‐73122 inhibited DES‐induced activation of cAMP‐response element binding protein (CREB) in gubernaculum testis cells. In conclusion, these data suggest that the effects of DES on mouse gubernaculum testis cells are mediated by PLC‐Ca²⁺‐CREB pathway.

Significance of the study

Environmental estrogens remain a serious threat to male reproductive health, and it is important to understand the mechanism by which EEs affect the male productive system. Here we explore potential mechanisms how the proliferation and contractility of gubernaculum testis cells are regulated by diethylstilbestrol. Our findings provide the first evidence that PLC‐Ca²⁺‐CREB signalling pathway mediates the nongenomic effects of diethylstilbestrol on gubernaculum testis cells. These findings provide new insight into the role of diethylstilbestrol in the aetiology of male reproductive dysfunction and will help develop better approaches for the prevention and therapy of male reproductive malformation.     

Two calcium mobilizing pathways implicated within abscisic acid-induced stomatal closing in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The present study investigated whether, depending on the abscisic acid (ABA) concentration, phospholipase C (PLC) would be implicated within a Ca²⁺ mobilizing pathway that would regulate stomatal aperture under standard watering conditions. Among Al sensitive mutants the als1-1 mutant of Arabidopsis thaliana (L.) Heynh. (Columbia-4 ecotype) was selected for a pharmacological approach of stomatal closing in leaf epidermal peels induced by 3, 20 or 30 μM ABA. Comparison with the wild type (WT) revealed that, exclusively in the als1-1 mutant, the stomatal response to 3 or 20 μM ABA was inhibited by about 40 %, whereas the stomatal response to 30 μM ABA and the wilting response to drought were unaffected. In WT, the Ca²⁺ buffer EGTA and the PLC inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), specifically inhibited by about 70 and 40 %, respectively, the response to 3 or 20 μM ABA, while the Ca²⁺ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) inhibited by about 70 % the response to 3, 20 or 30 μM ABA. EGTA, BAPTA and U73122 did not inhibit the part of the response to 3 or 20 μM ABA that was unaffected by the als1-1 mutation. Together, these results showed that ABA closes the stomata through two different Ca²⁺ mobilizing pathways. Since PLC could be indirectly deactivated in the als1-1 mutant, these results might suggest that, under sufficient water supply, PLC-mediated Ca²⁺ mobilization is needed for the regulation of stomatal aperture by endogenous ABA resting at concentrations below a drought-specific threshold value.     

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7β‐hydroxy‐cholesterol (7β‐OH) and cholesterol‐5β,6β‐epoxide (β‐epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca²⁺) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca²⁺ changes on apoptosis induced by 7β‐OH and β‐epoxide. Ca²⁺ responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura‐2. Over 15‐min exposure of differentiated U937 cells to 30 μM of 7β‐OH induced a slow but significant rise in fura‐2 ratio. The Ca²⁺ channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca²⁺. Moreover, dihydropyridine (DHP) binding sites identified with BODIPY‐FLX‐DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca²⁺ occurred through L‐type channels. However, following long‐term incubation with 7β‐OH, elevated levels of cytoplasmic Ca²⁺ were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca²⁺ may be an initial trigger of 7β‐OH–induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca²⁺ on apoptotic cell death appears to be less significant. In contrast, Ca²⁺ did not appear to be involved in β‐epoxide–induced apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:324–332, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20295     

Ca2+‐regulated and diurnal rhythm‐regulated Na+/Ca2+ exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis

Calcium (Ca²⁺) is vital for plant growth, development, hormone response and adaptation to environmental stresses, yet the mechanisms regulating plant cytosolic Ca²⁺ homeostasis are not fully understood. Here, we characterize an Arabidopsis Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger AtNCL that regulates Ca²⁺ and multiple physiological processes. AtNCL was localized to the tonoplast in yeast and plant cells. AtNCL appeared to mediate sodium (Na⁺) vacuolar sequestration and meanwhile Ca²⁺ release. The EF‐hand domains within AtNCL regulated Ca²⁺ binding and transport of Ca²⁺ and Na⁺. Plants with diminished AtNCL expression were more tolerant to high CaCl₂ but more sensitive to both NaCl and auxin; heightened expression of AtNCL rendered plants more sensitive to CaCl₂ but tolerant to NaCl. AtNCL expression appeared to be regulated by the diurnal rhythm and suppressed by auxin. DR5::GUS expression and root responses to auxin were altered in AtNCL mutants. The auxin‐induced suppression of AtNCL was attenuated in SLR/IAA14 and ARF6/8 mutants. The mutants with altered AtNCL expression also altered flowering time and FT and CO expression; FT may mediate AtNCL‐regulated flowering time change. Therefore, AtNCL is a vacuolar Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger that regulates auxin responses and flowering time.     

Identification of low Ca2+ stress‐induced embryo apoptosis response genes in Arachis hypogaea by SSH‐associated library lift (SSHaLL)

Calcium is a universal signal in the regulation of wide aspects in biology, but few are known about the function of calcium in the control of early embryo development. Ca²⁺ deficiency in soil induces early embryo abortion in peanut, producing empty pods, which is a general problem; however, the underlying mechanism remains unclear. In this study, embryo abortion was characterized to be caused by apoptosis marked with cell wall degradation. Using a method of SSH cDNA libraries associated with library lift (SSHaLL), 62 differentially expressed genes were isolated from young peanut embryos. These genes were classified to be stress responses, catabolic process, carbohydrate and lipid metabolism, embryo morphogenesis, regulation, etc. The cell retardation with cell wall degradation was caused by up‐regulated cell wall hydrolases and down‐regulated cellular synthases genes. HsfA4a, which was characterized to be important to embryo development, was significantly down‐regulated under Ca²⁺‐deficient conditions from 15 days after pegging (DAP) to 30 DAP. Two AhCYP707A4 genes, encoding abscisic acid (ABA) 8′‐hydroxylases, key enzymes for ABA catabolism, were up‐regulated by 21‐fold under Ca²⁺‐deficient conditions upstream of HsfA4a, reducing the ABA level in early embryos. Over‐expression of AhCYP707A4 in Nicotiana benthamiana showed a phenotype of low ABA content with high numbers of aborted embryos, small pods and less seeds, which confirms that AhCYP707A4 is a key player in regulation of Ca²⁺ deficiency‐induced embryo abortion via ABA‐mediated apoptosis. The results elucidated the mechanism of low Ca²⁺‐induced embryo abortion and described the method for other fields of study.     

Effects of barium and ouabain on electrogenesis in various sites of intact and detubulated skeletal muscle fibers of the frog <Emphasis Type="Italic">R. temporaria</Emphasis>

In loose patch clamp experiments on intact sartorius muscle fibers of the frog Rana temporaria, there have been two types of waveforms of extracellularly recorded action potentials (AP). Responses of the first type (T1AP) consisted of an initial positive phase with a subsequent phase of strong negativity, the latter only in a few cases followed by a weak positive phase. Responses of the second type (T2AP) always had an additional positive phase concluding their waveform. In the detubulated fibers, only T1AP were recorded. Application of Ba²⁺ (10 μM) to the muscle led to a significant increase in the amplitude of the third T2AP phase whereas the T1AP characteristics of both intact and detubulated muscle preparations remained unchanged. In some of the studied intact fibers, after Ba²⁺ or ouabain (50 μM) applied, the latest positive signal phase was replaced by a negative phase. The amplitude of this latest negative phase was increased markedly by highfrequency stimulation. Under the simultaneous action of ouabain and Ba²⁺, there was a summation of their effects. Our results can be tentatively explained by that the T-system of a muscle fiber produces electrical responses substantially differing in their pattern and barium sensitivity from those transmitted across cell membranes. These differences could be resultant from the activity of T-tubular inwardly rectifying potassium channels (K_ir) and that of Na,K-ATPase as they both provide absorption of excessive extracellular potassium.     

n‐3 Fatty Acids Modulate T‐Cell Calcium Signaling in Obese Macrosomic Rats

Objective: We investigated the effects of a diet containing EPAX‐7010, rich in PUFAs such as eicosapentaenoic acid [20:5(n‐3)] and docosahexaenoic acid [22:6(n‐3)], i.e., a PUFA/EPAX regimen, on T‐cell activation in diabetic pregnant rats and their obese pups. Research Methods and Procedures: Mild hyperglycemia in pregnant rats was induced by intraperitoneal injection of streptozotocin on Day 5 of gestation. T‐cell blastogenesis was assayed by using ³H‐thymidine, whereas intracellular free calcium concentrations ([Ca²⁺]i) were measured by using Fura‐2 in diabetic pregnant rats and their obese offspring. Results: Concavalin‐A‐stimulated T‐cell proliferation was decreased in both pregnant diabetic rats and their obese pups as compared with control animals. Feeding the PUFA/EPAX diet restored T‐cell proliferation in both groups of animals. We also employed ionomycin, which at 50 nM opens calcium channels, and thapsigargin (TG), which recruits [Ca²⁺]i from endoplasmic reticulum pool. We observed that ionomycin‐induced increases in [Ca²⁺]i in T‐cells of diabetic mothers and obese offspring were greater than in those of control rats. Furthermore, feeding PUFA/EPAX diet diminished significantly the ionomycin‐evoked rise in [Ca²⁺]i in diabetic and obese animals. TG‐induced increases in [Ca²⁺]i in T‐cells of diabetic pregnant rats and their obese offspring were greater than in those of control rats. The feeding of the experimental diet significantly curtailed the TG‐evoked increases in [Ca²⁺]i in both diabetic and obese rats. Discussion: Together, these observations provide evidence that T‐cell activation and T‐cell calcium signaling are altered during gestational diabetes and macrosomia. Hence, dietary fish oils, particularly eicosapentaenoic acid and docosahexaenoic acid, may restore these T‐cell abnormalities.     

Ectopic expression of wheat TaCIPK14, encoding a calcineurin B‐like protein‐interacting protein kinase,confers salinity and cold tolerance in tobacco

Calcineurin B‐like protein‐interacting protein kinases (CIPKs) are components of Ca²⁺ signaling in responses to abiotic stresses. In this work, the full‐length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT‐PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H₂O₂. Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H₂O₂ and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na⁺ content in the transgenic lines under salt stress. Higher expression of stress‐related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress‐responsive gene in plants.     

Comparison of metal‐bound and unbound structures of aminopeptidase B proteins from Escherichia coli and Yersinia pestis

Protein degradation by aminopeptidases is involved in bacterial responses to stress. Escherichia coli produces two metal‐dependent M17 family leucine aminopeptidases (LAPs), aminopeptidase A (PepA) and aminopeptidase B (PepB). Several structures have been solved for PepA as well as other bacterial M17 peptidases. Herein, we report the first structures of a PepB M17 peptidase. The E. coli PepB protein structure was determined at a resolution of 2.05 and 2.6 Å. One structure has both Zn²⁺ and Mn²⁺, while the second structure has two Zn²⁺ ions bound to the active site. A 2.75 Å apo structure is also reported for PepB from Yersinia pestis. Both proteins form homohexamers, similar to the overall arrangement of PepA and other M17 peptidases. However, the divergent N‐terminal domain in PepB is much larger resulting in a tertiary structure that is more expanded. Modeling of a dipeptide substrate into the C‐terminal LAP domain reveals contacts that account for PepB to uniquely cleave after aspartate.     

The Zn2+ and Ca2+‐binding S100B and S100A1 proteins: beyond the myths

The S100 genes encode a conserved group of 21 vertebrate‐specific EF‐hand calcium‐binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium‐ and zinc‐binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn²⁺. The Ca²⁺ and Zn²⁺‐dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate‐specific proteins and propose a new model for stable interactions of S100B dimers with full‐length target proteins. A chaperone‐associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.     

Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration

This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 (TRPM8) ion channels, to increase intracellular Ca²⁺ concentration, [Ca²⁺]_i, in human glioblastoma cells (DBTRG cells), which resulted in activation of the large-conductance Ca²⁺-activated K⁺ membrane ion channels (BK channels). Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 μM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 μM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol increased [Ca²⁺]_i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca²⁺-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell [Ca²⁺]_i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline reverses menthol-stimulated increase of [Ca²⁺]_i and of cell migration. Thus, BK channels function to maintain elevations in [Ca²⁺]_i needed to sustain increases in DBTRG cell migration.     

Cadmium ion stimulation of growth and versicolorin synthesis in a mutant strain ofAspergillus parasiticus

Cultures ofAspergillus parasiticus produce the polyketide versicolorin A in response to elevation of the Zn²⁺ content of the growth medium. With suboptimal Zn²⁺ (0.8 μM) mycelial growth is about half maximal, and versicolorin synthesis is essentially zero. Inclusion of Cd²⁺ (1–100 μM) in the Zn²⁺-limiting growth medium allows optimal growth and stimulates full versicolorin synthesis. Cd²⁺, like Zn²⁺, will stimulate versicolorin sysnthesis only when added within the first 30 h after conidial inoculation. The transport system for Cd²⁺ uptake may be the same as that for Zn²⁺, as judged byin vivo competition studies. Cd²⁺ is a competitive inhibitor of Zn²⁺ uptake, with K_i = 20 μM.     

Voltage‐dependent κ‐opioid modulation of action potential waveform‐elicited calcium currents in neurohypophysial terminals

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca²⁺ channels results in less calcium influx into the terminals and presumably a reduction in transmitter release. In the neurohypophysis (NH), Ca²⁺ channel kinetics, and the associated Ca²⁺ influx, is primarily controlled by membrane voltage and can be modulated, in a voltage‐dependent manner, by G‐protein subunits interacting with voltage‐gated calcium channels (VGCCs). In this series of experiments we test whether the κ‐ and µ‐opioid inhibition of Ca²⁺ currents in NH terminals is voltage‐dependent. Voltage‐dependent relief of G‐protein inhibition of VGCC can be achieved with either a depolarizing square pre‐pulse or by action potential waveforms. Both protocols were tested in the presence and absence of opioid agonists targeting the κ‐ and µ‐receptors in neurohypophysial terminals. The κ‐opioid VGCC inhibition is relieved by such pre‐pulses, suggesting that this receptor is involved in a voltage‐dependent membrane delimited pathway. In contrast, µ‐opioid inhibition of VGCC is not relieved by such pre‐pulses, indicating a voltage‐independent diffusible second‐messenger signaling pathway. Furthermore, relief of κ‐opioid inhibition during a physiologic action potential (AP) burst stimulation indicates the possibility of activity‐dependent modulation in vivo. Differences in the facilitation of Ca²⁺ channels due to specific G‐protein modulation during a burst of APs may contribute to the fine‐tuning of Ca²⁺‐dependent neuropeptide release in other CNS terminals, as well. J. Cell. Physiol. 225: 223–232, 2010. © 2010 Wiley‐Liss, Inc.