Coupling Ion Research Articles

Ion chromatography with inductively coupled plasma mass spectrometry, a powerful analytical tool for complex matrices: Estimation of Pt and Pd in environmental samples

A new method for palladium and platinum direct determination in environmental samples is proposed by coupling ion chromatography with quadrupole inductively coupled plasma MS. In order to optimise Pd and Pt separation and to minimise interference from matrix in real samples, several anionic and cationic stationary phases have been compared at different mobile phase compositions. In particular, the effect of acidity and of the addition of oxalic acid to the eluent on separation and detection performance has been studied, and the anion-exchange column AG11 turned out to be more suitable. After chromatographic and mass spectrometer parameter optimisation, several potential interferences and the main quality parameters of the method, according to the Eurachem-CITAC recommendations, were evaluated: the detection limit for Pt was 5 ng l −1 while the value for Pd was 230 ng l −1. The method was successfully employed in the determination of platinum group elements in urban road dust and atmospheric particulates and the complete absence of matrix spectral interferences was demonstrated.

Torque–speed Relationship of the Na +-driven Flagellar Motor of Vibrio alginolyticus

The torque–speed relationship of the Na +-driven flagellar motor of Vibrio alginolyticus was investigated. The rotation rate of the motor was measured by following the position of a bead, attached to a flagellar filament, using optical nanometry. In the presence of 50 mM NaCl, the generated torque was relatively constant (∼3800 pN nm) at lower speeds (speeds up to ∼300 Hz) and then decreased steeply, similar to the H +-driven flagellar motor of Escherichia coli. When the external NaCl concentration was varied, the generated torque of the flagellar motor was changed over a wide range of speeds. This result could be reproduced using a simple kinetic model, which takes into consideration the association and dissociation of Na + onto the motor. These results imply that for a complete understanding of the mechanism of flagellar rotation it is essential to consider both the electrochemical gradient and the absolute concentration of the coupling ion.

The dynamics of electron–ion coupling in the shock transition region

In the shock transition region the process of electron–ion coupling under Buneman instability between inflow electrons and reflected ions is investigated. Buneman instability often evolves into a highly nonlinear state with strong wave–particle interaction like velocity phase hole (vortex) dynamics. Previous studies have shown that these electron holes contribute to the strong electron heating and acceleration. In this study, how electron holes affect the ions and interact with them is examined. Under the condition of Buneman instability dominant, the electron holes preserve their coherent large-amplitude structure for a sufficiently long period of time to modify the ion population electrostatically. Electron holes resonate with the ion population through the ion-acoustic branch, and then some of the inflow ions are rapidly decelerated and reflected by the electric field at the electron holes. As a result of gaining additional energy from the inflow ion population, the amplitude of the electric field of the hole becomes 40% larger than what was estimated in some previous studies, which did not take the coupling ions into account. Although the electron hole–ion coupling state is seen in such a small scale as electron inertia length (c/ωpe), this large electric field of the electron hole leads to a strong disturbance and heating of the plasma. This study may provide a first indication of what regulates strong electron heating in the shock transition region through the coupling process between the electrons and ions .

Sodium ion cycling mediates energy coupling between complex I and ATP synthase.

We show here sodium ion cycling between complex I from Klebsiella pneumoniae and the F(1)F(0) ATP synthase from Ilyobacter tartaricus in a reconstituted proteoliposome system. In the course of NADH oxidation by complex I, an electrochemical sodium ion gradient was established and served as a driving force for the synthesis of ATP from ADP and phosphate. In the opposite direction, the deltamu(Na(+)) generated by ATP hydrolysis could be coupled to NADH formation by reversed electron transfer from ubiquinol to NAD. For reverse electron transfer, a transmembrane voltage larger than 30 mV was obligatory. No NADH-driven proton transport into the lumen of proteoliposomes was detected. We conclude that Na(+) is used as the exclusive coupling ion by the enterobacterial complex I.

Experimental verification of a sequence-based prediction: F(1)F(0)-type ATPase of Vibrio cholerae transports protons, not Na(+) ions.

The membrane energetics of the intestinal pathogen Vibrio cholerae involves both H(+) and Na(+) as coupling ions. The sequence of the c subunit of V. cholerae F(0)F(1) ATPase suggested that this enzyme is H(+) specific, in contrast to the results of previous studies on the Na(+)-dependent ATP synthesis in closely related Vibrio spp. Measurements of the pH gradient and membrane potential in membrane vesicles isolated from wild-type and DeltaatpE mutant V. cholerae show that the F(1)F(0) ATPase of V. cholerae is an H(+), not Na(+), pump, confirming the bioinformatics assignments that were based on the Na(+)-binding model of S. Rahlfs and V. Müller (FEBS Lett. 404:269-271, 1999). Application of this model to the AtpE sequences from other bacteria and archaea indicates that Na(+)-specific F(1)F(0) ATPases are present in a number of important bacterial pathogens.

An Environmental Focus Using Inductively Coupled Plasma-Optical Emission Spectrometry and Ion Chromatography

The Western Carolina University chemistry faculty have developed an environmental focus to their curriculum. Inductively coupled plasma-optical emission spectrometry (ICP-OES) and ion chromatography (IC) have been shown to be useful tools for the determination of elements and ions, respectively. Several novel experiments have been developed monitoring these analytes in environmental samples, including water, pressure-treated wood, and nutritional supplements. In addition, ICP-OES and IC have been used to teach seniors the principles of analytical method development. Lastly, this equipment has been employed extensively in a vigorous research program.

Redox Coupled Ion Exchange in Copolymers of Aniline with Aminobenzoic Acids

Copolymers of aniline and 2-aminobenzoic acid are prepared by chemical copolymerization. The copolymers are soluble in basic media and thin films are prepared, onto glassy carbon electrodes, by evaporation of the solutions. The modified electrodes show a voltammetric response, with two peaks, similar to polyaniline with two peaks. The dependence of both peak potentials on pH is similar to that of PANI, suggesting that proton expulsion is the mechanism of charge compensation on oxidation. The mechanism is confirmed by Probe Beam Deflection, which shows proton expulsion on oxidation. However, while polyaniline films exchange mainly anions at pH>0, poly(aniline-co-(2-aminobenzoic acid)) (p2ABA) film exchange protons up to pH 4.

Glutamate 172, essential for modulation of L247T alpha7 ACh receptors by Ca2+, lines the extracellular vestibule.

Neuronal alpha7 nicotinic ACh receptors (nAChRs) are permeable to and modulated by Ca2+, Ba2+, and Sr2+. These permeant divalent cations interact with slowly desensitizing L247T alpha7 nAChRs to increase the potency and maximal efficacy of ACh, increase the efficacy of dihydro-beta-erythroidine (DHbetaE), and increase agonist-independent activity. Mutation of glutamate 172 (E172) to glutamine or cysteine eliminated these effects of permeant divalent cations. 2-(Trimethylammonium)ethyl methanethiosulfonate (MTSET), a cysteine-modifying reagent directed at water-accessible thiols, inhibited ACh-evoked currents of E172C/L247T alpha7 nAChRs by >90%, demonstrating that E172 was accessible to permeant ions. The data are consistent with a model of alpha7 receptors, derived from the crystal structure of the ACh binding protein (AChBP) from Lymnaea stagnalis, in which E172 projects toward the lumen of the extracellular vestibule. The observations that E172 was essential for divalent cation modulation of L247T alpha7 nAChRs and was accessible to permeating ions suggest that this residue participates in coupling ion permeation with modulation of receptor activity.

Membrane embedded location of Na+ or H+ binding sites on the rotor ring of F1F0 ATP synthases.

Recent crosslinking studies indicated the localization of the coupling ion binding site in the Na+-translocating F1F0 ATP synthase of Ilyobacter tartaricus within the hydrophobic part of the bilayer. Similarly, a membrane embedded H+-binding site is accepted for the H+-translocating F1F0 ATP synthase of Escherichia coli. For a more definite analysis, we performed parallax analysis of fluorescence quenching with ATP synthases from both I. tartaricus and E. coli. Both ATP synthases were specifically labelled at their c subunit sites with N-cyclohexyl-N'-(1-pyrenyl)carbodiimide, a fluorescent analogue of dicyclohexylcarbodiimide and the enzymes were reconstituted into proteoliposomes. Using either soluble quenchers or spinlabelled phospholipids, we observed a deeply membrane embedded binding site, which was quantitatively determined for I. tartaricus and E. coli to be 1.3 +/- 2.4 A and 1.8 +/- 2.8 A from the bilayer center apart, respectively. These data show a conserved topology among enzymes of different species. We further demonstrated the direct accessibility for Na+ ions to the binding sites in the reconstituted I. tartaricus c11 oligomer in the absence of any other subunits, pointing to intrinsic rotor channels. The common membrane embedded location of the binding site of ATP synthases suggest a common mechanism for ion transfer across the membrane.

Coupled ion–interface dynamics and ion transfer across the interface of two immiscible liquids

When an ion moves across the interface of two immiscible electrolytes it moves together with the ion-induced protrusion of one solvent into the other. For an infinitely slow motion of an ion the height of the protrusion, heq, is a function of the position of the ion z. Due to a finite relaxation time the protrusion may not be able to spontaneously follow the motion of the ion, and this will cause slowing down of the ion transfer. The relaxation of the protrusion involves the movements of many solvent molecules and must be considered on the same footing as the motion along the coordinate of the ion. In this paper we develop a theory of such coupled motion which determines the kinetic laws of the ion transfer across the interface. When the equilibrium electrochemical potential for the ion has no barrier, the process of ion transport is purely diffusional and the effective diffusion coefficient may be evaluated as Deff=kBT/{6η[ri+(4/3)(hmax/Λ)2L]}, where η is the average viscosity of the liquids, ri is the Stokes radius of an ion, L and hmax is the lateral size and the maximal height of the protrusion, and Λ is the half width of the function heq(z), which characterizes equilibrium ion–interface coupling. When there is a barrier, the theory recovers, depending on the height of the barrier, the mechanisms of ion transfer considered by Marcus or Gurevich–Kharkats–Schmickler. The effect of the nature of the ion and the solvents in contact is discussed.

Tet(L) and tet(K) tetracycline-divalent metal/H+ antiporters: characterization of multiple catalytic modes and a mutagenesis approach to differences in their efflux substrate and coupling ion preferences.

The Tet(L) protein encoded in the Bacillus subtilis chromosome and the closely related Tet(K) protein from Staphylococcus aureus plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me(2+)) complex that bears a net single positive charge, Na+, and K+. Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H+ as the coupling ion. In this study, competitive cross-inhibition between K+ and other cytoplasmic efflux substrates was demonstrated. Tet(L) and Tet(K) had also been shown to use K+ as an alternate coupling ion in support of Na+ or K+ efflux. Here they were shown to couple TC-Me(2+) efflux to K+ uptake as well, exhibiting greater use of K+ as a coupling ion as the external pH increased. The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K+, both as a cytoplasmic efflux substrate and as an external coupling ion. Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative "antiporter motif," motif C, of Tet proteins would be involved in these characteristic preferences. Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K+ preference both as coupling ion and efflux substrate. A reciprocal S157A mutant of Tet(K) exhibited reduced K+ preference. Competitive inhibition among substrates and the parallel effects of the single mutation upon K+ preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion. However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates.

Guest Editor's Introduction: Functional Assay Systems for Drug Discovery at G-Protein Coupled Receptors and Ion Channels

Guest Editor's Introduction: Functional Assay Systems for Drug Discovery at G-Protein Coupled Receptors and Ion Channels

Cysteine-scanning Mutagenesis Reveals a Conformationally Sensitive Reentrant Pore-Loop in the Glutamate Transporter GLT-1

Removal of glutamate from the synaptic cleft by (Na(+) + K(+))-coupled transporters prevents neurotoxicity due to elevated concentrations of the transmitter. These transporters exhibit an unusual topology, including two reentrant loops. Reentrant loop II plays a pivotal role in coupling ion and glutamate fluxes. Here we used cysteine-scanning mutagenesis of the GLT-1 transporter to test the idea that this loop undergoes conformational changes following sodium and substrate binding. 15 of 22 consecutive single cysteine mutants in the stretch between Gly-422 and Ser-443 exhibited 30-100% of the transport activity of the cysteine-less transporter when expressed in HeLa cells. The transport activity of 11 of the 15 active mutants including five consecutive residues in the ascending limb was inhibited by small hydrophilic methanethiosulfonate reagents. The sensitivity of seven cysteine mutants, including A438C and S440C, to the reagents was significantly reduced by sodium ions, but the opposite was true for A439C. The non-transportable analogue dihydrokainate protected at almost all positions throughout the loop, and at two of the positions, the analogue protected even in the absence of sodium. Our results indicate that reentrant loop II forms part of an aqueous pore, the access of which is blocked by the glutamate analogue dihydrokainate, and that sodium influences the conformation of this pore-loop.

Chemiosmotic energy conservation with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by Acetobacterium woodii.

Cell suspensions of Acetobacterium woodii prepared from cultures grown on fructose plus caffeate catalyzed caffeate reduction with electrons derived from molecular hydrogen. Hydrogen-dependent caffeate reduction was strictly Na(+) dependent with a K(m) for Na(+) of 0.38 mM; Li(+) could substitute for Na(+). The sodium ionophore ETH2120, but not protonophores, stimulated hydrogen-dependent caffeate reduction by 280%, indicating that caffeate reduction is coupled to the buildup of a membrane potential generated by primary Na(+) extrusion. Caffeate reduction was coupled to the synthesis of ATP, and again, ATP synthesis coupled to hydrogen-dependent caffeate reduction was strictly Na(+) dependent and abolished by ETH2120, but not by protonophores, indicating the involvement of a transmembrane Na(+) gradient in ATP synthesis. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) abolished ATP synthesis, and at the same time, hydrogen-dependent caffeate reduction was inhibited. This inhibition could be relieved by ETH2120. These experiments are fully compatible with a chemiosmotic mechanism of ATP synthesis with Na(+) as the coupling ion during hydrogen-dependent caffeate reduction by A. woodii.

Fabrication of electrostatic micro-actuators for a hard disk drive application

Micro-actuators are developed for a hard disk drive application. The aim is to obtain a high density of data storage by positioning the read and write head element precisely. The desired specifications are a displacement of ±0.5 μm, operating voltage of 20 V and a structural resonance frequency of 15 kHz. We manufactured the metallic actuator by using thick photoresist lithography and nickel electroplating. The electrostatic gap between the mover and the stator has a high aspect ratio, in order to achieve low voltage operation. We deposited a copper layer by electroplating as a vertical sacrificial layer. Then we removed the copper layer at last process step to make the electrostatic gap with high aspect ratio. We utilize a photoresist which has a thickness of 10 μm as the first trial. We completed the process and operated the actuator successfully. We also developed a silicon actuator using the silicon deep etching and deposition technology. We use an Inductive Coupled Plasma-Reactive Ion Etching (ICP-RIE) machine for silicon etching and a Low Pressure Chemical Vapor Deposition (LPCVD) machine for deposition of the vertical sacrificial layer of SiO2 and the mover structure of polySi. We obtained an actuator which had thickness of 100 μm with the electrode gap having a aspect ratio of 50.

Coupling ion mobility separations, collisional activation techniques, and multiple stages of MS for analysis of complex peptide mixtures.

An ion trap/ion mobility/quadrupole/collision cell/time-of-flight mass spectrometer that incorporates a differentially pumped orifice-skimmer cone region at the back of the drift tube has been developed for the analysis of peptide mixtures. The combined approach allows a variety of strategies to be employed for collisionally activating ions, and fragments can be monitored by subsequent stages of mass spectrometry in a parallel fashion, as described previously (Anal. Chem. 2000, 72, 2737). Here, we describe the overall experimental approach in detail. Applications involving different aspects of the initial mobility separation and various collisional activation and parallel sequencing strategies are illustrated by examining several simple peptide mixtures and a mixture of tryptic peptides from beta-casein. Detection limits associated with various experimental configurations and the utility for analysis of complex systems are discussed.

Membrane Topography of the Coupling Ion Binding Site in Na+-translocating F1F0 ATP Synthase

A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na(+)-translocating F(1)F(0) ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na(+) ions but not in its presence, indicating the specific reaction with the Na(+) binding c-Glu(65) residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu(65) was therefore in close contact with phospholipid but does not contact subunit a. Na(+)in/(22)Na(+)out exchange activity of the ATP synthase was not affected by modifying the c-Glu(65) sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na(+) ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospholipases C and A(2) and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu(65) is located within the core of the membrane.

Guest Editor's Introduction: Functional Assay Systems for Drug Discovery at G-Protein Coupled Receptors and Ion Channels

Guest Editor's Introduction: Functional Assay Systems for Drug Discovery at G-Protein Coupled Receptors and Ion Channels

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter.

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.

Using Fermi statistics to create strongly coupled ion plasmas in atom traps.

We investigate the possibility of forming a strongly coupled ion plasma from a cold atomic gas. We show that rapid ion heating occurs as correlations develop from the initial disordered configuration. This heating severely reduces the Coulomb coupling of the final configuration, although the final Coulomb coupling can be maximized by introducing order into the initial atomic gas. We show that such order can be introduced into the initial state by employing a degenerate Fermi gas whereby the Pauli hole mimics the Coulomb hole. The corresponding initial state correlations can enhance the Coulomb coupling in the final state by orders of magnitude.