Differential modulation by copper and zinc of P2X2 and P2X4 receptor function.
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Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 microM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 microM ATP were 16.3 +/- 0.9 (SE) microM and 19.6 +/- 1.5 microM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 microM enhanced current activated by 5 microM ATP with an EC50 value of 2.4 +/- 0.2 microM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor. (+info)
Effects of stair-step nutrition and trace mineral supplementation on attainment of puberty in beef heifers of three sire breeds.
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A study was conducted to evaluate the influence of nutrition and sire breed on age at puberty and first lactation milk yield in crossbred beef heifers. After weaning, 208 heifers sired by Hereford, Limousin, or Piedmontese bulls were assigned to either a control (CG) or stair-step gain (SSG) dietary regimen plus a mineral supplement with or without Cu, Zn, and Mn top-dressed onto the feed. Heifers on the SSG regimen were fed a diet intended to supply energy to support gains at a rate of 120% of the CG diet for 55 d and then were switched to a diet formulated to produce an ADG at 70% of the rate of the CG diet for 84 d. They then switched back to the 120% diet for the last 30 d before breeding. Total weight gain and overall rate of gain did not differ among dietary treatments. Hereford- and Limousin-sired heifers gained at similar rates, and Piedmontese-sired heifers gained an average of .10 kg/d slower than the other two sire breed groups. During one period, Piedmontese-sired heifers on the CG diet gained .19 kg/d faster ( P < . 01) when supplemented with mineral than when not. During that same period, there was no influence of mineral supplementation on weight gains for Hereford- or Piedmontese-sired heifers on the high SSG diet, but Limousin-sired heifers tended (P = .07) to gain faster (1.00 vs .85 kg/d) when supplemented with Cu, Zn, and Mn than when not. Piedmontese-sired heifers reached puberty at the earliest age (P = .03), followed by Hereford- and then Limousin-sired heifers. There were no treatment effects on milk yield at an average of 70 d of lactation. However, at approximately 120 d of lactation, Piedmontese-sired heifers were producing less milk (P < .05) than Limousin- but not Hereford-sired heifers. Hereford-sired heifers had lower (P < .05) plasma Cu concentrations than Piedmontese-sired heifers. There were no treatment effects on plasma Zn concentrations. Heifers sired by bulls of breeds that differ in potential muscularity differed in growth, reproduction, milk yield, and plasma mineral concentrations, but dietary treatments resulted in little to no differences in these variables. (+info)
Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase. Enzymatic activity and effect of pterin.
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Recently, we obtained x-ray crystallographic data showing the presence of a ZnS4 center in the structure of Escherichia coli-expressed bovine endothelial nitric-oxide synthase (eNOS) and rat neuronal nitric-oxide synthase (nNOS). The zinc atom is coordinated by two CXXXXC motifs, one motif being contributed by each NOS monomer (cysteine 326 through cysteine 331 in rat nNOS). Mutation of the nNOS cysteine 331 to alanine (C331A) results in the loss of NO. synthetic activity and also results in an inability to bind zinc efficiently. Although prolonged incubation of the C331A mutant of nNOS with high concentrations of L-arginine results in a catalytically active enzyme, zinc binding is not restored. In this study, we investigate the zinc stoichiometry in wild-type nNOS and eNOS, as well as in the C331A-mutated nNOS, using a chelation assay and electrothermal vaporization-inductively coupled plasma-mass spectrometry. The data reveal an approximate 2:1 stoichiometry of heme to zinc in (6R)-5,6,7,8-tetrahydro-L-biopterin-replete, wild-type nNOS and eNOS and show that the reactivated C331A mutant of nNOS has a limited ability to bind zinc. The present study substantiates that the zinc in NOS is structural rather than catalytic and is important for maintaining optimally functional, enzymatically active, constitutive NOSs. (+info)
Copper and zinc ions differentially block asialoglycoprotein receptor-mediated endocytosis in isolated rat hepatocytes.
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Asialoglycoprotein receptors on hepatocytes lose endocytic and ligand binding activity when hepatocytes are exposed to iron ions. Here, we report the effects of zinc and copper ions on the endocytic and ligand binding activity of asialoglycoprotein receptors on isolated rat hepatocytes. Treatment of cells at 37 degrees C for 2 h with ZnCl2 (0-220 microM) or CuCl2 (0-225 microM) reversibly blocked sustained endocytosis of 125I-asialoorosomucoid by up to 93% (t1/2 = 62 min) and 99% (t1/2 = 54 min), respectively. Cells remained viable during such treatments. Zinc- and copper-treated cells lost approximately 50% of their surface asialoglycoprotein receptor ligand binding activity; zinc-treated cells accumulated inactive asialoglycoprotein receptors intracellularly, whereas copper-treated cells accumulated inactive receptors on their surfaces. Cells treated at 4 degrees C with metal did not lose surface asialoglycoprotein receptor activity. Exposure of cells to copper ions, but not to zinc ions, blocked internalization of prebound 125I-asialoorosomucoid, but degradation of internalized ligand and pinocytosis of the fluid-phase marker Lucifer Yellow were not blocked by metal treatment. Zinc ions reduced diferric transferrin binding and endocytosis on hepatocytes by approximately 33%; copper ions had no inhibitory effects. These findings are the first demonstration of a specific inhibition of receptor-mediated endocytosis by non-iron transition metals. (+info)
Cationic currents induced by Clostridium perfringens type A enterotoxin in human intestinal CaCO-2 cells.
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Clostridium perfringens type A produces an enterotoxin that induces diarrhoea experimentally in man and animals. The enterotoxin causes increased membrane permeability in susceptible cells which is thought to be due to pore formation in the host cell membrane. The effect of purified C. perfringens enterotoxin on intact intestinal CaCO-2 monolayers was examined in Ussing chambers and on single cells by whole-cell patch clamp. Mucosal application of C. perfringens enterotoxin resulted in prompt increases in short-circuit current coupled with a reduction in transepithelial resistance consistent with movement of sodium and other cations smaller than diethanolamine from mucosa to serosa. These changes were independent of extracellular calcium. Increases in short-circuit current were also observed in the apical membranes of CaCO-2 monolayers permeabilised across the basolateral membrane with nystatin. Currents were blocked by subsequent exposure to mucosal barium and zinc. Zinc also prevented the development of the current increases in apical membranes. Cationic currents were also observed following exposure of single CaCO-2 cells in whole-cell patch clamp recordings. These data indicate that C. perfringens enterotoxin is able to form cation permeant pores in the apical membrane of human intestinal CaCO-2 epithelia and the increases in short-circuit current can be prevented by pre-exposure to zinc ions. (+info)
Paradoxical stimulation of a DEG/ENaC channel by amiloride.
(78/8763)
Extracellular amiloride inhibits all known DEG/ENaC ion channels, including BNC1, a proton-activated human neuronal cation channel. Earlier studies showed that protons cause a conformational change that activates BNC1 and exposes residue 430 to the extracellular solution. Here we demonstrate that, in addition to blocking BNC1, amiloride also exposes residue 430. This result suggested that, like protons, amiloride might be capable of activating the channel. To test this hypothesis, we introduced a mutation in the BNC1 pore that reduces amiloride block, and found that amiloride stimulated these channels. Amiloride inhibition was voltage-dependent, suggesting block within the pore, whereas stimulation was not, suggesting binding to an extracellular site. These data show that amiloride can have two distinct effects on BNC1, and they suggest two different interaction sites. The results suggest that extracellular amiloride binding may have a stimulatory effect similar to that of protons in BNC1 or extracellular ligands in other DEG/ENaC channels. (+info)
Kinetic properties and metal content of the metallo-beta-lactamase CcrA harboring selective amino acid substitutions.
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The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations. (+info)
Zinc and ifenprodil allosterically inhibit two separate polyamine-sensitive sites at N-methyl-D-aspartate receptor complex.
(80/8763)
In this study, we investigated the hypothesis that inhibition of the N-methyl-D-aspartate (NMDA) receptor complex by zinc involves a polyamine-sensitive regulatory site. We found that the specific binding of the open channel ligand [3H]MK-801 to rat hippocampal membranes 1) was inhibited by low concentrations of Zn2+ (IC50 = 5.5 microM) by 65%. 2) This high-affinity component of inhibition was reversed by the polyamine spermine to an extent that could be reconciled with competitive interaction between Zn2+ and spermine. 3) Partial inhibition by Zn2+ was additive with partial inhibition by ifenprodil, an inhibitor of the NMDA receptor complex supposed to act at a polyamine-sensitive regulatory site, and 4) in membranes prepared from several other brain regions, inhibition of [3H]MK-801 binding by Zn2+ and by ifenprodil was either less than additive, or superadditive. Our observation that ifenprodil, at concentrations saturating its high-affinity component of inhibition, prevented spermine from reversing the inhibition by Zn2+ indicates that spermine did not increase [3H]MK-801 binding by competition with Zn2+ but rather via another polyamine regulatory site not sensitive to zinc but sensitive to ifenprodil. We conclude that Zn2+ reduces channel opening of the NMDA receptor complex by allosteric inhibition of a polyamine-sensitive regulatory site different from that inhibited by ifenprodil and that these two allosteric sites influence each other in a manner dependent on the brain region investigated. The different proportions of zinc/ifenprodil inhibition in different regions could reflect different percentages of various NMDA receptor subtypes. (+info)