Cu(II) inhibition of the proton translocation machinery of the influenza A virus M2 protein.
(1/3605)
The homotetrameric M2 integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M2 transmembrane domain is believed to play an important role in the conduction mechanism of this channel. Also, this residue is believed to form hydrogen-bonded interactions with the ammonium group of the anti-viral compound, amantadine. A molecular model of this channel suggests that the imidazole side chains of His-37 from symmetry-related monomers of the homotetrameric pore converge to form a coordination site for transition metals. Thus, membrane currents of oocytes of Xenopus laevis expressing the M2 protein were recorded when the solution bathing the oocytes contained various transition metals. Membrane currents were strongly and reversibly inhibited by Cu2+ with biphasic reaction kinetics. The biphasic inhibition curves may be explained by a two-site model involving a fast-binding peripheral site with low specificity for divalent metal ions, as well as a high affinity site (Kdiss approximately 2 microM) that lies deep within the pore and shows rather slow-binding kinetics (kon = 18.6 +/- 0.9 M-1 s-1). The pH dependence of the interaction with the high affinity Cu2+-binding site parallels the pH dependence of inhibition by amantadine, which has previously been ascribed to protonation of His-37. The voltage dependence of the inhibition at the high affinity site indicates that the binding site lies within the transmembrane region of the pore. Furthermore, the inhibition by Cu2+ could be prevented by prior application of the reversible blocker of M2 channel activity, BL-1743, providing further support for the location of the site within the pore region of M2. Finally, substitutions of His-37 by alanine or glycine eliminated the high affinity site and resulted in membrane currents that were only partially inhibited at millimolar concentrations of Cu2+. Binding of Cu2+ to the high affinity site resulted in an approximately equal inhibition of both inward and outward currents. The wild-type protein showed very high specificity for Cu2+ and was only partially inhibited by 1 mM Ni2+, Pt2+, and Zn2+. These data are discussed in terms of the functional role of His-37 in the mechanism of proton translocation through the channel. (+info)
In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons.
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Recent evidence suggests that changes in sodium channel expression and localization may be involved in some pathological pain syndromes. SNS, a tetrodotoxin-resistant (TTX-R) sodium channel, is preferentially expressed in small dorsal root ganglion (DRG) neurons, many of which are nociceptive. TTX-R sodium currents and SNS mRNA expression have been shown to be modulated by nerve growth factor (NGF) in vitro and in vivo. To determine whether SNS expression and TTX-R currents in DRG neurons are affected by reduced levels of systemic NGF, we immunized adult rats with NGF, which causes thermal hypoalgesia in rats with high antibody titers to NGF. DRG neurons cultured from rats with high antibody titers to NGF, which do not bind the isolectin IB4 (IB4(-)) but do express TrkA, were studied with whole cell patch-clamp and in situ hybridization. Mean TTX-R sodium current density was decreased from 504 +/- 77 pA/pF to 307 +/- 61 pA/pF in control versus NGF-deprived neurons, respectively. In comparison, the mean TTX-sensitive sodium current density was not significantly different between control and NGF-deprived neurons. Quantification of SNS mRNA hybridization signal showed a significant decrease in the signal in NGF-deprived neurons compared with the control neurons. The data suggest that NGF has a major role in the maintenance of steady-state levels of TTX-R sodium currents and SNS mRNA in IB4(-) DRG neurons in adult rats in vivo. (+info)
Sodium ion uptake into isolated plasma membrane vesicles: indirect effects of other ions.
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Vesicles derived from plasma membrane of corneal endothelium were agitated to their minimum size distribution. When isotonic salt solutions surrounding the vesicles were changed there were alterations to the vesicle size distribution: the modal point of the logarithmic distribution did not change but the log variance did, indicating that substantial fission and fusion of vesicles occurred depending upon the nature of the surrounding solute. Orientation and total membrane area was conserved in the transformed population of vesicles. Although the ions added to the external isotonic salt solutions in the present series of experiments have no direct effect upon sodium membrane transporters in these membranes, kinetics of sodium accumulation into the vesicles were affected in a way that correlated with changes to the vesicle size distribution. Early-saturating (<1 min) intravesicular concentrations of sodium corresponded with apparently stable populations. Late-saturating (>1 min) intravesicular concentrations of sodium corresponded with significant vesicle distribution shifts and included a few seconds of delay. During the linear accumulation phase, both populations showed similar magnitudes of sodium transport. The significance of these data is discussed. (+info)
Evidence for an anion exchange mechanism for uptake of conjugated bile acid from the rat jejunum.
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Absorption of conjugated bile acids from the small intestine is very efficient. The mechanisms of jejunal absorption are not very well understood. The aim of this study was to clarify the mechanism of absorption of conjugated bile acid at the apical membrane of jejunal epithelial cells. Brush-border membrane vesicles from intestinal epithelial cells of the rat were prepared. Absorption of two taurine-conjugated bile acids that are representative of endogenous bile acids in many variate vertebrate species were studied. In ileal, but not jejunal brush-border membrane vesicles, transport of conjugated bile acids was cis-stimulated by sodium. Transport of conjugated bile acids was trans-stimulated by bicarbonate in the jejunum. Absorption of conjugated dihydroxy-bile acids was almost twice as fast as of trihydroxy-bile acids. Coincubation with other conjugated bile acids, bromosulfophthalein, and DIDS, as well as by incubation in the cold inhibited the transport rate effectively. Absorption of conjugated bile acids in the jejunum from the rat is driven by anion exchange and is most likely an antiport transport. (+info)
The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force.
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Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of V. cholerae. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V. cholerae. The energy source of its flagellar motor was investigated. We examined the Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V. cholerae strains and present the evidence that the polar flagellar motor of V. cholerae is driven by an Na+ motive force. (+info)
Homozygous deletion in KVLQT1 associated with Jervell and Lange-Nielsen syndrome.
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BACKGROUND: Long-QT (LQT) syndrome is a cardiac disorder that causes syncope, seizures, and sudden death from ventricular arrhythmias, specifically torsade de pointes. Both autosomal dominant LQT (Romano-Ward syndrome) and autosomal recessive LQT (Jervell and Lange-Nielsen syndrome, JLNS) have been reported. Heterozygous mutations in 3 potassium channel genes, KVLQT1, KCNE1 (minK), and HERG, and the cardiac sodium channel gene SCN5A cause autosomal dominant LQT. Autosomal recessive LQT, which is associated with deafness, has been found to occur with homozygous mutations in KVLQT1 and KCNE1 in JLNS families in which QTc prolongation was inherited as a dominant trait. METHODS AND RESULTS: An Amish family with clinical evidence of JLNS was analyzed for mutations by use of single-strand conformation polymorphism and DNA sequencing analyses for mutations in all known LQT genes. A novel homozygous 2-bp deletion in the S2 transmembrane segment of KVLQT1 was identified in affected members of this Amish family in which both QTc prolongation and deafness were inherited as recessive traits. This deletion represents a new JLNS-associated mutation in KVLQT1 and has deleterious effects on the KVLQT1 potassium channel, causing a frameshift and the truncation of the KVLQT1 protein. In contrast to previous reports in which LQT was inherited as a clear dominant trait, 2 parents in the JLNS family described here have normal QTc intervals (0.43 and 0.44 seconds, respectively). CONCLUSIONS: A novel homozygous KVLQT1 mutation causes JLNS in an Amish family with deafness that is inherited as an autosomal recessive trait. (+info)
Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae.
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The expression of several virulence factors of Vibrio cholerae is coordinately regulated by the ToxT molecule and the membrane proteins TcpP/H and ToxR/S, which are required for toxT transcription. To identify proteins that negatively affect toxT transcription, we screened transposon mutants of V. cholerae carrying a chromosomally integrated toxT::lacZ reporter construct for darker blue colonies on media containing 5-bromo-4-chlor-3-indolyl beta-D galactoside (X-gal). Two mutants had transposon insertions in a region homologous to the nqr gene cluster of Vibrio alginolyticus, encoding a sodium-translocating NADH-ubiquinone oxidoreductase (NQR). In V. alginolyticus, NQR is a respiration-linked Na+ extrusion pump generating a sodium motive force that can be used for solute import, ATP synthesis, and flagella rotation. Inhibition of NQR enzyme function in V. cholerae by the specific inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) resulted in elevated toxT::lacZ activity. Increased toxT::lacZ expression in an nqr mutant strain compared with the parental strain was observed when the TcpP/H molecules alone were strongly expressed, suggesting that the negative effect of the NQR complex on toxT transcription is mediated through TcpP/H. However, the ability of the TcpP/H proteins to activate the toxT::lacZ reporter construct was greatly diminished in the presence of high NaCl concentrations in the growth medium. The flagellar motor of V. cholerae appears to be driven by a sodium motive force, and modulation of flagella rotation by inhibitory drugs, high media viscosity, or specific mutations resulted in increases of toxT::lacZ expression. Thus, the regulation of the main virulence factors of V. cholerae appears to be modulated by endogenous and exogenous sodium levels in a complex way. (+info)
Stimulus-secretion coupling in neurohypophysial nerve endings: a role for intravesicular sodium?
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It is generally accepted that Ca is essentially involved in regulated secretion, but the role of this cation, as well as others such as Na, is not well understood. An illustrative example occurs in neurohypophysial secretion, where an experimentally induced increase in the cytosolic concentration of Na+ can induce continuous neuropeptide release. In contrast, an increase in cytosolic Ca2+ will have only a transient stimulatory effect. The secretion-promoting targets for Ca2+ are not known; they may be cytosolic, as is usually assumed, but they may also be intravesicular, especially in view of evidence that Ca-rich secretory vesicles are preferentially secreted. In the present work, we have investigated the movements of these cations into and out of secretory vesicles during stimulus-secretion coupling. Isolated rat neurohypophysial nerve endings were stimulated by potassium (55 mM) depolarization, and at 6 min (peak secretion) and 20 min after the onset of stimulation, the elemental content of individual secretory vesicles was measured by quantitative x-ray microanalysis. A depolarization-induced transient increase in intravesicular Na+ concentration was found to coincide with the onset of secretion. Moreover, only a predicted small fraction of peripheral vesicles-presumably the docked ones-were Na+-loaded. The low sulfur concentration of Na+-rich vesicles most likely resulted from vesicle swelling. The results suggest that high intravesicular Na+ concentrations in docked vesicles, occurring by Na+/Ca2+ exchange or by transient fusion pore opening, is a proximal event in exocytosis. (+info)