CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

Kringle 4 from human plasminogen:1H-nuclear magnetic resonance study of the interactions between ω-amino acid ligands and aromatic residues at the lysine-binding site

The interactions of theω-amino acid ligandsε-aminocaproic acid andp-benzylaminesulphonic acid with the isolated kringle 4 domain from human plasminogen have been investigated by¹H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Overall, the data indicate that binding either ligand does not cause the kringle to undergo significant conformational changes. When p-benzylaminesulphonic acid is in excess relative to the kringles, progressive exchange-broadening and high field chemical shifts are observed for the proton resonances of the ligand. The largest effect is seen at the amino end of the molecule, which indicates that the — NH ₃ ⁺ group of the ligand penetrates deeper into the binding site than does the — SO ₃ ^- . Ligand-binding causes signals from the ring-current shifted Leu⁴⁶ CH ₃ ^δ .δ groups and from a number of aromatic side-chains to shift. Depending on the ligand, the latter include Tyr-II (Tyr⁵⁰), Tyr-V (an immobile ring), His-II and His-III imidazole groups and the three Trp indole groups present in kringle 4. In particular,p-benzylaminesulphonic acid-binding induces large high field shifts on the Trp-II H6 triplet and the Trp-III (Trp⁷²) H2 singlet. On the other hand,ε-aminocaproic acid bound to kringle 4 exhibits large chemical shifts of its CH₂ proton resonances, which indicates that the lysine-binding site is rich in aromatic side chains. Overhauser experiments centered on thep-benzylaminesulphonic acid H2,6 and H3,5 aromatic transitions as well as on the shifted Trp-II and Trp-III signals reveal efficient cross-relaxation between these two indole side chains and thep-benzylaminesulphonic acid ring. These experiments also show that the side chains from Phe⁶⁴, Tyr-II (Tyr⁵⁰), Tyr-IV, and His-II (His³¹) interact with the ligand. In combination with reported chemical modification experiments that show requirement of Asp⁵⁷, Arg⁷¹ and Trp⁷² integrity for ligand-binding, our study underscores the relevance of the Cys⁵¹-Cys⁷⁵ loop in defining the kringles’ lysine-binding site. Furthermore, the Cys²²-Cys⁶³ loop is folded so as to place His³¹, His³³, Tyr⁴¹ and Leu⁴⁶ in proximity to the binding site. The involvement of residues within the Cys⁵¹-Cys⁷⁵ loop in ligand-binding suggests that Trp-II and Tyr-IV may correspond to Trp⁶² and Tyr⁷⁴, respectively. As shown by Overhauser experiments, these two residues are in close contact with each other. From these studies and from the shielding and deshielding effects caused byp-benzylaminesulphonic acid, we suggest that the ligand is sandwiched between the indole rings of Trp-II and Trp-III, which form part of the hydrophobic binding site.     

Response of the electrochromic dye,merocyanine 540, to membrane potential in rat liver mitochondria

Summary We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca^2–] _a -dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca^2–-channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca²⁺-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K^–] _a , nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba²⁺] _a . All these data are consistent with the concept that PTX may act on Ca^2– channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca²⁺ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca²⁺ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX-treated cells. Our measurements of whole cell inward Ca²⁺ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca²⁺ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca²⁺-channel conductance (6 nS/cell at 2.6mm [Ca²⁺] _a ). PTX evoked the activation of a new class of Ca²⁺-selective channels (5 pS in 25mm [Ca²⁺]_pipet), which are rather insensitive to membrane potential. We have termed theseG-type calcium channels. These data suggest that treatment with PTX not only increases the probability of L-type Ca²⁺-channel activation at more negative potentials, but also increases the probability of opening of an entirely new, voltage-independent, Ca²⁺ channel. These actions of PTX should promote Ca²⁺ entry and might explain the stimulation by the toxin of CA secretion from medullary chromaffin cells in culture.     

Simultaneous recording of cytosolic Ca2+ levels inDidinium andParamecium during aDidinium attack onParamecium

Summary The carnivorous ciliateDidinium nasutum captures prey such asParamecium by discharging extrusomes, known as toxicysts, while the attackedParamecium defensively discharges trichocysts. Several authors have suggested that both discharges, the toxicysts ofDidinium and the trichocysts ofParamecium, are evoked by the rise in cytosolic Ca²⁺ level in each cell. However, these putative increases in cytosolic Ca²⁺ levels have not as yet been recorded simultaneously in these cells during aDidinium attack onParamecium. We injected the fluorescent Ca²⁺ indicator Ca-Green 1 dextran into bothDidinium andParamecium, and simultaneously observed the cytosolic Ca²⁺ levels in these cells asDidinium attackedParamecium. When aParamecium came into contact with theDidinium proboscis, theDidinium showed a significant rise in cytosolic Ca²⁺ in the basal portion of the proboscis. One video frame (33 ms) after the onset of the Ca²⁺ rise inDidinium, theParamecium also showed an increase in cytosolic Ca²⁺. This is the first simultaneous recording of changes in the Ca²⁺ level during a predator-prey interaction in ciliates. The possible roles of these Ca²⁺ increases are discussed in relation to the discharge of toxicysts during theDidinium attack and of trichocysts as a defensive behavior ofParamecium.Abbreviations AED aminoethyldextran - P_i inorganic phosphate - FITC fluorescein isothiocyanate     

Crystal Structure of Bovine Heart Cytochrome c Oxidase at 2.8 Å Resolution

Thirteen different polypeptide subunits, each in one copy, five phosphatidyl ethanolamines and three phosphatidyl glycerols, two hemes A, three Cu ions, one Mg ion, and one Zn ion are detectable in the crystal structure of bovine heart cytochrome c oxidase in the fully oxidized form at 2.8 Å resolution. A propionate of hems a, a peptide unit (–CO–NH–), and an imidazole bound to Cu_A are hydrogen-bonded sequentially, giving a facile electron transfer path from Cu_A to heme a. The O₂ binding and reduction site, heme a ₃, is 4.7 Å apart from Cu_B. Two possible proton transfer paths from the matrix side to the cytosolic side are located in subunit I, including hydrogen bonds and internal cavities likely to contain randomly oriented water molecules. Neither path includes the O₂ reduction site. The O₂ reduction site has a proton transfer path from the matrix side possibly for protons for producing water. The coordination geometry of Cu_B and the location of Tyr²⁴⁴ in subunit I at the end of the scalar proton path suggests a hydroperoxo species as the two electron reduced intermediate in the O₂ reduction process.     

Toxin III of the scorpionAndroctonus australis Hector: Proton nuclear magnetic resonance assignments and secondary structure

Summary ¹H NMR has been applied to a3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpionAndroctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20–28) which is linked by two disulfide bridges to the central strand of a triple-stranded antiparallel -sheet. Turns were identified at residues 15–17, 47–49 and also at residues 51–53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu³⁷, Leu²³, Val⁴⁷, Tyr¹⁴, Trp⁴⁵ and Tyr⁵. The Trp⁴⁵ and Tyr⁵ rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.Thenomenclature used is similar to that described by Wütrich, 1986.     

Homocysteine inhibits store-mediated calcium entry in human endothelial cells: Evidence for involvement of membrane potential and actin cytoskeleton

The role of homocysteine for store-operated calcium influx was investigated in human umbilical cord endothelial cell line. Homocysteine significantly decreased thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization. GSH and DTT prevented homocysteine-induced inhibition of thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; while GSSG had the opposite effect. Homocysteine blocked large conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a concentration-dependent manner and related to the redox status of the endothelial cells. BK_Ca channels opener NS1619 reversed thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; BK_Ca channels inhibitor iberiotoxin had the opposite effect. The findings suggest that homocysteine is involved in store-regulated Ca²⁺ entry through membrane potential-dependent and actin cytoskeleton-dependent mechanisms, redox status of homocysteine and BK_Ca channels may play a regulatory role in it. (Mol Cell Biochem 269: 37–47, 2005)     

Spatiotemporal calcium signaling in a<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> cell line stably expressing a<Emphasis Type="Italic"> Drosophila</Emphasis> muscarinic acetylcholine receptor

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca²⁺]_i) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca²⁺]_i transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

Rapid effect of light on the K+ channel in the plasmalemma ofNitella

Summary Chlorophyll fluorescence, plasmalemma potential and resistance were measured simultaneously and subjected to a kinetic analysis. It was found that the light-induced changes of all three signals have two time constants in common. The faster one (₄=ca. 20 sec) was assigned to the action of light-induced proton uptake across the thylakoid membrane on the plasmalemma H⁺ pump. The slower one (_5a=40 sec) is related to the light action of an unknown photosynthetic process on the potassium channel. The action on the K⁺ channel was revealed from the reversal potential of the related effect on membrane potential. The comparison of the data with findings of other authors led to the hypothesis that the unknown photosynthetic mechanism is the depletion of NADP⁺, which stimulates the uptake of Ca²⁺ from the cytosol, which is required for the NAD-kinase. The resulting change in cytosolic Ca²⁺ modulates the number of open K⁺ channels.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca²⁺ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca²⁺ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca²⁺ overload. However, exposure of yeast mitochondria to 50–100 μM Ca²⁺ in the presence of the Ca²⁺ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca²⁺/nH⁺-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca²⁺- ETH129-induced activation of the Ca²⁺/H⁺-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca²⁺ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Calcium binding to the subunit c ofE. coli ATP-synthase and possible functional implications in energy coupling

The 8-kDa subunit c of theE. coli F₀ ATP-synthase proton channel was tested for Ca⁺⁺ binding activity using a⁴⁵Ca⁺⁺ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds⁴⁵Ca⁺⁺ strongly with Ca⁺⁺ binding properties very similar to those of the 8-kDa CF₀ subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca⁺⁺ binding capacity, shown by loss of Ca⁺⁺ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca⁺⁺ binding, shown by Ca⁺⁺ binding being retained in twoE. coli mutants, Asp61Asn and Asp61Gly. The Ca⁺⁺ binding is pH dependent in both theE. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca⁺⁺ binding affinity to its F₀ binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca⁺⁺ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca⁺⁺ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80–100 nM was obtained using an EGTA-Ca⁺⁺ buffer system to control free Ca⁺⁺ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca⁺⁺ bound to the periplasimic side of theE. coli F₀ proton channel can block H⁺ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca⁺⁺ binding site is on the lumen side of the thylakoid, where Ca⁺⁺ binding can modulate acid-base jump ATP formation. The Ca⁺⁺ binding to the F₀ and CF₀ complexes is consistent with a pH-dependent gating mechanism for control of H⁺ ion flux across the opening of the H⁺ channel.This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia.     

Substrate Specificity of a Novel Alcohol Resistant Metalloproteinase,Vimelysin, from Vibrio sp. T1800

Vimelysin is a novel alcohol resistant metalloproteinase from Vibrio sp. T1800. The substrate specificity of vimelysin was studied by using natural and furylacryloyl dipeptide substrates. Vimelysin cleaved mainly Pro⁷-Phe⁸ bond and slightly Tyr⁴-Ile⁵ bond in human angiotensin I. Vimelysin also cleaved mainly Phe²⁴-Phe²⁵ and Tyr¹⁶-Leu¹⁷ bonds, and slightly His⁵-Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴-Leu¹⁵, and Gly²³-Phe²⁴ bonds in oxidized insulin B-chain. The substrate specificity of vimelysin, by using furylacryloyl (Fua) dipeptides were also studied. The ratio of k_cat/K_m for Fua-Gly-Phe-NH₂/Fua-Gly-Leu-NH₂, Fua-Phe-Leu-NH₂/Fua-Gly-Leu-NH₂, and Fua-Phe-Phe-NH₂/Fua-Gly-Leu-NH₂ were 15.9, 27.8, and 59.0, respectively. These results indicate that vimelysin easily recognizes phenylalanine in P1′ positions, which is different from thermolysin.     

Factors controlling the K+ conductance inChara

Summary Previous current/voltage (I/V) investigations of theChara K⁺ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La³⁺. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K⁺ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K⁺, 20mm Na⁺, more than 10mm Ca²⁺ or 5mm tetraethylammonium (TEA). The K⁺ channels are therefore blocked by TEA, closed by low [K⁺] _o or high [Ca²⁺] _o and are highly selective to K⁺ over Na⁺. With the K⁺ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca²⁺] _o , sometimes displayed a slight shift to more positive PD's with increasing [K⁺] _o and were unaffected by TEA or changes in [Na⁺] _o . The slope of the linear part of theI/V profile was steeper in low [K⁺] _o than in TEA or high [Na⁺] _o (indicating participation of K⁺, but not Na⁺, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K⁺ channels were also strongly affected.     

Delta endotoxin inhibits Rb+ uptake,lowers cytoplasmic pH and inhibits a K+-ATPase inManduca sexta CHE cells

Summary Delta endotoxin, a 68 kilodalton protein isolated fromBacillus thuringiensis spp.Kurstaki, is a potent entomocidal agent that alters a K⁺ current across midgut tissue of many phytophagous insects. This toxin completely inhibited the vanadate-sensitive⁸⁶Rb⁺ uptake and mimicked the vanadate-induced decrease in cytosolic pH in a cell line (CHE) originating fromManduca sexta embryonic tissue. The toxin also inhibited a K⁺-sensitive-ATPase in the plasma membranes isolated from these cells. Using the K⁺-sensitive-ATPase substratp-nitrophenyl phosphate, delta endotoxin was found to have aK _i of 0.4 m. These data suggest that the toxin inhibits a K⁺-ATPase responsible for⁸⁶Pb⁺ uptake in the CHE cells. The relationship between the toxin inhibition of K⁺-ATPase and toxin-altered K⁺ current is discussed.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Cytosolic calcium regulates a potassium current in corn (Zea mays) protoplasts

Summary The voltage- and time-dependent K⁺ current,I _K ⁺ _out , elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La³⁺) to the extracellular medium. These results suggested that the influx of external Ca²⁺ was necessary for K⁺ current activation. The IC₅₀, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 m at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca²⁺ actuated the K⁺ current, we altered either the Ca²⁺ buffering capacity or the free Ca²⁺ concentration of the intracellular medium (pipette filling solution). By these means,I _K ⁺ _out could be varied over a 10-fold range. Increasing the free Ca²⁺ concentration from 40 to 400nm also shifted the activation of the K⁺ current toward more negative potentials. Maintaining cytoplasmic Ca²⁺ at 500nm with 40nm EGTA resulted in a more rapid activation of the K⁺ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca²⁺ on depolarization. Increasing intracellular Ca²⁺ to 500nm or 1 m also led to inactivation of the K⁺ current within a few minutes. It is concluded thatI _K ⁺ _out is regulated by cytosolic Ca²⁺, which is in turn controlled by Ca²⁺ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.     

Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

Ca<Superscript>2+</Superscript> increases the specific coenzyme Q<Subscript>10</Subscript> content in <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Of various metal ions (Ca²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Zn²⁺) added to the culture medium of Agrobacterium tumefaciens at 1 mM, only Ca²⁺ increased Coenzyme Q10 (CoQ₁₀) content in cells without the inhibition of cell growth. In a pH-stat fed-batch culture, supplementation with 40 mM of CaCO₃ increased the specific CoQ₁₀ content and oxidative stress by 22.4 and 48%, respectively. Also, the effect of Ca²⁺ on the increase of CoQ₁₀ content was successfully verified in a pilot-scale (300 L) fermentor. In this study, the increased oxidative stress in A. tumefaciens culture by the supplementation of Ca²⁺ is hypothesized to stimulate the increase of specific CoQ₁₀ content in order to protect the membrane against lipid peroxidation. Our results improve the understanding of Ca²⁺ effect on CoQ₁₀ biosynthesis in A. tumefaciens and should contribute to better industrial production of CoQ₁₀ by biological processes.