Plasmalemmal voltage-activated K(+) currents in protoplasts from tobacco BY-2 cells: possible regulation by actin microfilaments?

Plasmalemmal ionic currents from enzymatically isolated protoplasts of suspension-cultured tobacco 'Bright Yellow-2' cells were investigated by whole-cell patch-clamp techniques. In all protoplasts, delayed rectifier outward K(+) currents having sigmoidal activation kinetics, no inactivation, and very slow deactivation kinetics were activated by step depolarization. Tail current reversal potentials were close to equilibrium potential E(K) when external [K(+)] was either 6 or 60 mM. Several channel blockers, including external Ba(2+), niflumic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, inhibited this outward K(+) current. Among the monovalent cations tested (NH(4)(+), Rb(+), Li(+), Na(+)), only Rb(+) had appreciable permeation (P(Rb)/P(K) (=) 0.7). In addition, in 60 mM K(+) solutions, a hyperpolarization-activated, time-dependent, inwardly rectifying K(+) current was observed in most protoplasts. This inward current activated very slowly, did not inactivate, and deactivated quickly upon repolarization. The tail current reversal potential was very close to E(K), and other monovalent cations (NH(4)(+), Rb(+), Li(+), Na(+)) were not permeant. The inward current was blocked by external Ba(2+) and niflumic acid. External Cs(+) reversibly blocked the inward current without affecting the outward current. The amplitude of the inward rectifier K(+) current was generally small compared to the amplitude of the outward K(+) current in the same cell, although this was highly variable. Similar amplitudes for both currents occurred in only 4% of the protoplasts in control conditions. Microfilament-depolymerizing drugs shifted this proportion to about 12%, suggesting that microfilaments participate in the regulation of K(+) currents in tobacco 'Bright Yellow-2' cells.     

Potassium-selective block of barium permeation through single KcsA channels

Ba(2+), a doubly charged analogue of K(+), specifically blocks K(+) channels by virtue of electrostatic stabilization in the permeation pathway. Ba(2+) block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K(+), the block-time distribution is double exponential, marking at least two Ba(2+) sites in the selectivity filter, in accord with a Ba(2+)-containing crystal structure of KcsA. By analyzing block as a function of extracellular K(+), we determined the equilibrium dissociation constant of K(+) and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb(+) (3 μM) > Cs(+) (23 μM) > K(+) (29 μM) > NH(4)(+) (440 μM) > Na(+) and Li(+) (>1 M). This represents an unusually high selectivity for K(+) over Na(+), with |ΔΔG(0)| of at least 7 kcal mol(-1). These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.     

Stabilization of RNA tertiary structure by monovalent cations

The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.     

7Li-NMR and FTIR studies of lithium, potassium, rubidium, and cesium complexes with ionophore lasalocid in solution

Lasalocid metal salts were combined with 1 : 1 lithium and 2:2 potassium, rubidium, and cesium to form complexes. The nature of the lasolocid salt complexes was studied in a solid and chloroform by FTIR spectroscopy in the middle and far IR regions. The process of the complexation of lithium was also studied by (7)Li-NMR. In chloroform a 1 : 1 complex of lasalocid and Li(+) ions was formed. Continuous absorption was observed in the far FTIR spectrum of this complex. It indicated large Li(+) polarizability, which was due to fast fluctuations of the Li(+) ions in the multiminima potentials, in the monomeric structure. In the lasalocid salt with the other monovalent cations (K(+), Rb(+), Cs(+)) 2:2 complexes were formed in which the cations showed cation polarizability, which strongly depended on the mass and the radius of the cations.     

Study of substrate-enzyme interaction between immobilized pyridoxamine and recombinant porcine pyridoxal kinase using surface plasmon resonance biosensor

Pyridoxal kinase (PK) is an important enzyme involved in bioactivation of vitamin B(6). Binding of PK with its substrate is the prerequisite step for the subsequent catalytic phosphorylation of the substrate. In the present study, a surface plasmon resonance biosensor (BIAcore) was employed to characterize the binding interaction between wild-type porcine PK and an immobilized substrate, pyridoxamine. Pyridoxamine was modified with 11-mercaptoundecanic acid and immobilized on a sensor chip through the formation of a self-assembled monolayer. The binding of PK to the immobilized pyridoxamine was followed in real time and the kinetic parameters were derived from non-linear analysis of the sensorgram. The effects of buffer pH, monovalent cations (Na(+), K(+)) and divalent cations (Mn(2+), Zn(2+), Mg(2+)) on the binding kinetics were determined. Optimal pH for PK-pyridoxamine interaction in the absence of divalent ions is at around 7.4. While K(+) increased and Na(+) decreased the binding affinity (K(A)) of PK to immobilized pyridoxamine, all divalent cations increased the K(A) of PK for pyridoxamine. Solution phase affinity measurement based on a competitive binding assay was used to determine the affinities of PK for different vitamin B(6) analogues. The order of affinity of PK for different analogues is: pyridoxal-oxime>pyridoxine>pyridoxamine>pyridoxal>pyridoxal phosphate. This is the first study to demonstrate that buffer conditions such as pH and concentration of monovalent and/or divalent ions can directly alter the binding of PK for its substrates. The quantitative kinetic and thermodynamic parameters obtained by SPR measurement provide the insight information into the catalytic activity of this enzyme.     

Effect of divalent cations on ion fluxes and leaf photochemistry in salinized barley leaves

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)>Zn(2+)=Ca(2+)>Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Formation and study of single metal ion-phospholipid complexes in biphasic electrospray ionization mass spectrometry

Single metal ion-phospholipid complexes are observed in biphasic electrospray ionization mass spectrometry (BESI-MS) using a dual-channel microsprayer. Such a microsprayer makes it possible to put into contact two immiscible liquids within the Taylor cone. Thus, L-α-dipalmitoyl phosphatidylcholine (DPPC) dissolved in 1,2-dichloroethane (DCE) reacts with aqueous metal cations (M = Na(+), K(+), Ca(2+), Cu(2+), La(3+)) yielding the formation of [M-DPPC(n)](z+) complexes. The number of phospholipid molecules ranges from 1 to 4 for monovalent ions, to 8 for divalent and to more than 10 for trivalent ions respectively. The large number of ligands observed involves the formation of solvent free single ion-phospholipid complexes.     

Monovalent cation size and DNA conformational stability

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ～ Na(+) ～ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+). Isothermal studies indicate that the decrease in the hairpin melting temperature with increasing cation hydrophobicity is not due to saturable, site-specific binding of the cation to the random coil conformation, but to the concomitant increase in cation size with increasing hydrophobicity. Larger cations are less effective at shielding the charged phosphate residues in B-form DNA because they cannot approach the DNA backbone as closely as smaller cations. By contrast, larger cations are relatively more effective at shielding the phosphate charges in the random coil conformation, where the phosphate-phosphate distance more closely matches cation size. Hydrophobic interactions between alkylammonium ions interacting electrostatically with the phosphate residues in the coil may amplify the effect of cation size on DNA thermal stability.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Ion specificity and ionic strength dependence of the osmoregulatory ABC transporter OpuA

The ATPase subunit of the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis has a C-terminal extension, the tandem cystathionine beta-synthase (CBS) domain, which constitutes the sensor that allows the transporter to sense and respond to osmotic stress (Biemans-Oldehinkel, E., Mahmood, N. A. B. N., and Poolman, B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 10624-10629). C-terminal of the tandem CBS domain is an 18-residue anionic tail (DIPDEDEVEEIEKEEENK). To investigate the ion specificity of the full transporter, we probed the activity of inside-out reconstituted wild-type OpuA and the anionic tail deletion mutant OpuADelta12; these molecules have the tandem CBS domains facing the external medium. At a mole fraction of 40% of anionic lipids in the membrane, the threshold ionic strength for activation of OpuA was approximately 0.15, irrespective of the electrolyte composition of the medium. At equivalent concentrations, bivalent cations (Mg(2+) and Ba(2+)) were more effective in activating OpuA than NH(4)(+), K(+), Na(+), or Li(+), consistent with an ionic strength-based sensing mechanism. Surprisingly, Rb(+) and Cs(+) were potent inhibitors of wild-type OpuA, and 0.1 mM RbCl was sufficient to completely inhibit the transporter even in the presence of 0.2 M KCl. Rb(+) and Cs(+) were no longer inhibitory in OpuADelta12, indicating that the anionic C-terminal tail participates in the formation of a binding site for large alkali metal ions. Compared with OpuADelta12, wild-type OpuA required substantially less potassium ions (the dominant ion under physiological conditions) for activation. Our data lend new support for the contention that the CBS module in OpuA constitutes the ionic strength sensor whose activity is modulated by the C-terminal anionic tail.     

Effect of rubidium and cesium ions on the dimeric quaduplex formed by the Oxytricha nova telomeric repeat oligonucleotide d(GGGGTTTTGGGG)

The DNA sequence d(GGGGTTTTGGGG) consists of 1.5 units of the repeat in telomeres of Oxytricha nova. It has been shown by NMR and x-ray crystallographic analysis that it is capable to form a dimeric quadruplex structure and that a variety of cations, namely K(+), Na(+), and NH(4)(+), are able to interact with this complex with different affinity, leading to complexes characterized by different local conformations. Thus, in order to improve the knowledge of this kind of molecule, and in particular to provide further insight into the role of monovalent cations in the G-quadruplex folding and conformation, we have investigated by (1)H-NMR the effect of the addition of Rb(+) and Cs(+) to the quadruplex formed by the oligonucleotide d(GGGGTTTTGGGG).     

Osmotic shrinkage of human cervical cancer cells induces an extracellular Cl- -dependent nonselective cation channel,which requires p38 MAPK

This study is to integrate a functional role of nonselective cation (NSC) channels into a model of volume regulation on osmotic shrinkage for human cervical cancer cells. Application of a hypertonic solution (400 mosm kg(-1)) induced cell shrinkage, which was accompanied by a 7-fold increase of inward currents at -80 mV from -4.1 +/- 0.4 pA pF(-1) to -29 +/- 1.1 pA pF(-1) (n = 36, p < 0.001). There is a good correlation of channel activity and cell volume changes. Replacement of bath Na(+) by K(+), Cs(+), Li(+), or Rb(+) did not affect the stimulated inward current significantly, but replacement by Ca(2+), Ba(2+), or the impermeable cation N-methyl-d-glucamine abolished the inward current; this demonstrates that the shrinkage-induced currents discriminate poorly between monovalent cations but are not carried by divalent cations. Replacement of extracellular Cl(-) by gluconate abolished the shrinkage-induced currents in a concentration-dependent manner without changing the reversal potential. Gadolinium (Gd(3+)) inhibited the stimulated current, whereas bumetanide and amiloride had no inhibitory effect. Cell shrinkage triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of MAP/extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (MEK1/2), and p38 kinase. Interference with p38 MAPK by either the specific inhibitor (SB202190), or a dominant-negative mutant profoundly suppressed the activation of the shrinkage-induced NSC channels. In contrast, the regulatory mechanism of shrinkage-induced NSC channels was independent of the volume-responsive MEK1/2 signaling pathway. More importantly, the cell volume response to hypertonicity was inhibited significantly in p38 dominant-negative mutant or by SB202190. Therefore, p38 MAPK is critically involved in the activation of a shrinkage-induced NSC channel, which plays an important role in the volume regulation of human cervical cancer cells.     

Schiff base of gossypol with 3,6,9-trioxa-decylamine complexes with monovalent cations studied by mass spectrometry, (1)H-NMR, FTIR, and PM5 semiempirical methods

A new Schiff base of gossypol with 3,6,9-trioxo-decylamine (GSTB) forms stable complexes with monovalent cations. This process of complex formation was studied by electrospray ionization mass spectrometry, (1)H-NMR and FTIR spectroscopy, and the PM5 (parametric method 5) semiempirical method. It is found that GSTB forms 1 : 1 and 1 : 2 complexes with Li(+) and Na(+) and 1 : 1 complexes with K(+), Rb(+), or Cs(+) cations and exists in all these complexes in the enamine-enamine tautomeric form. Moreover, within these complexes only Li(+) cations can fluctuate between the oxygen atoms of trioxo-alkyl chains. All other cations are strongly localized. In the complex of GSTB with two protons localized on the N atoms of the Schiff base, the imine-imine tautomeric form is realized. The complexes of the Schiff base with K(+), Rb(+), or Cs(+) cations are the 1 : 1 type with the oxygen atoms of the trioxo-alkyl chains, as well as the O(1)H or O(1')H group coordinating the cation. The structures of the complexes are calculated by the PM5 semiempirical method and discussed.     

A kinetic study of the gill (Na+, K+)-ATPase, and its role in ammonia excretion in the intertidal hermit crab, Clibanarius vittatus

To better comprehend the role of gill ion regulatory mechanisms, the modulation by Na(+), K(+), NH(4)(+) and ATP of (Na(+), K(+))-ATPase activity was examined in a posterior gill microsomal fraction from the hermit crab, Clibanarius vittatus. Under saturating Mg(2+), Na(+) and K(+) concentrations, two well-defined ATP hydrolyzing sites were revealed. ATP was hydrolyzed at the high-affinity sites at a maximum rate of V=19.1+/-0.8 U mg(-1) and K(0.5)=63.8+/-2.9 nmol L(-1), obeying cooperative kinetics (n(H)=1.9); at the low-affinity sites, hydrolysis obeyed Michaelis-Menten kinetics with K(M)=44.1+/-2.6 mumol L(-1) and V=123.5+/-6.1 U mg(-1). Stimulation by Na(+) (V=149.0+/-7.4 U mg(-1); K(M)=7.4+/-0.4 mmol L(-1)), Mg(2+) (V=132.0+/-5.3 U mg(-1); K(0.5)=0.36+/-0.02 mmol L(-1)), NH(4)(+) (V=245.6+/-9.8 U mg(-1); K(M)=4.5+/-0.2 mmol L(-1)) and K(+) (V=140.0+/-4.9 U mg(-1); K(M)=1.5+/-0.1 mmol L(-1)) followed a single saturation curve and, except for Mg(2+), obeyed Michaelis-Menten kinetics. Under optimal ionic conditions, but in the absence of NH(4)(+), ouabain (K(I)=117.3+/-3.5 mumol L(-1)) and orthovanadate inhibited up to 67% of the ATPase activity. The inhibition studies performed suggest the presence of F(0)F(1), V- and P-ATPases, but not Na(+)-, K(+)- or Ca(2+)-ATPases as contaminants in the gill microsomal preparation. (Na(+), K(+))-ATPase activity was synergistically modulated by NH(4)(+) and K(+). At 20 mmol L(-1) K(+), a maximum rate of V=290.8+/-14.5 U mg(-1) was seen as NH(4)(+) concentration was increased up to 50 mmol L(-1). However, at fixed NH(4)(+) concentrations, no additional stimulation was found for increasing K(+) concentrations (V=135.2+/-4.1 U mg(-1) and V=236.6+/-9.5 U mg(-1) and for 10 and 30 mmol L(-1) NH(4)(+), respectively). This is the first report to detail ionic modulation of gill (Na(+), K(+))-ATPase in C. vittatus, revealing an asymmetrical, synergistic stimulation of the enzyme by K(+) and NH(4)(+), as yet undescribed for other (Na(+), K(+))-ATPases, and should provide a better understanding of NH(4)(+) excretion in pagurid crabs.     

Ca(2+) function in photosynthetic oxygen evolution studied by alkali metal cations substitution

Effects of adding monovalent alkali metal cations to Ca(2+)-depleted photosystem (PS)II membranes on the biochemical and spectroscopic properties of the oxygen-evolving complex were studied. The Ca(2+)-dependent oxygen evolution was competitively inhibited by K(+), Rb(+), and Cs(+), the ionic radii of which are larger than the radius of Ca(2+) but not inhibited significantly by Li(+) and Na(+), the ionic radii of which are smaller than that of Ca(2+). Ca(2+)-depleted membranes without metal cation supplementation showed normal S(2) multiline electron paramagnetic resonance (EPR) signal and an S(2)Q(A)(-) thermoluminescence (TL) band with a normal peak temperature after illumination under conditions for single turnover of PSII. Membranes supplemented with Li(+) or Na(+) showed properties similar to those of the Ca(2+)-depleted membranes, except for a small difference in the TL peak temperatures. The peak temperature of the TL band of membranes supplemented with K(+), Rb(+), or Cs(+) was elevated to approximately 38 degrees C which coincided with that of Y(D)(+)Q(A)(-) TL band, and no S(2) EPR signals were detected. The K(+)-induced high-temperature TL band and the S(2)Q(A)(-) TL band were interconvertible by the addition of K(+) or Ca(2+) in the dark. Both the Ca(2+)-depleted and the K(+)-substituted membranes showed the narrow EPR signal corresponding to the S(2)Y(Z)(+) state at g = 2 by illuminating the membranes under multiple turnover conditions. These results indicate that the ionic radii of the cations occupying Ca(2+)-binding site crucially affect the properties of the manganese cluster.     

The role of glutamic acid-69 in the activation of Citrobacter freundii tyrosine phenol-lyase by monovalent cations

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30-fold by monovalent cations, the most effective being K(+), NH(4)(+), and Rb(+). Previous X-ray crystal structure analysis has demonstrated that the monovalent cation binding site is located at the interface between subunits, with ligands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chain and monodentate ligation by one of the epsilon-oxygens of Glu69 from another chain [Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S. (1993) Biochemistry 32, 4195]. We have studied the effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to determine the role of Glu69 in the activation of TPL. E69Q TPL is activated by K(+), NH(4)(+), and Rb(+), with K(D) values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activation. In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capable of binding, indicating that the geometry of the monovalent cation binding site is critical for activation. Rapid-scanning stopped-flow kinetic studies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold) and of phenol elimination. Similar rapid-scanning stopped-flow results were obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase in the rate of quinonoid intermediate formation with K(+). Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in stopped flow kinetic experiments, suggesting that the activation of TPL by monovalent cations is a slow process. In agreement with this conclusion, a slow increase (k < 0.5 s(-)(1)) in fluorescence intensity (lambda(ex) = 420 nm, lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed with K(+), Rb(+), and NH(4)(+) but not Li(+) or Na(+). E69D TPL shows no change in fluorescence under these conditions. High concentrations (>100 mM) of all monovalent cations result in inhibition of wild-type TPL. This inhibition is probably due to cation binding to the ES complex to form a complex that releases pyruvate slowly.     

Biophysical properties of menthol-activated cold receptor TRPM8 channels

The temperature-sensitive transient receptor potential channel, TRPM8, was recently cloned and found to be activated by cold and menthol. Whole-cell recordings show that TRPM8 is permeable to multiple cations and exhibits a strong outward rectification. Here, we examine the mechanism underlying menthol-evoked current rectification of TRPM8 transiently expressed in tsA-201 cells at room temperature ( approximately 25 degrees C). Whole-cell currents (ruptured, bath: Na(+), K(+), Ca(2+), or Ba(2+); pipette: KCl) exhibited a strong outward rectification in the presence of menthol, consistent with previous studies. The outward K(+) current was reduced in the presence of external Ca(2+) or Ba(2+). Single-channel recordings (cell-attached) showed that menthol induced brief channel openings with two conducting states in the voltage range between -80 and +60mV. The small current (i(S)) conducted both monovalent and divalent ions, and the large one (i(L)) predominantly monovalent ions. The i-V plot for Ca(2+) was weakly outward rectifying, whereas those for monovalent ions were linear. The i(S) may result in the divalent ion-induced reduction of the whole-cell outward current. The open probability (P(o)) in all ion conditions tested was low at negative voltages and increased with depolarization, accounting for the small inward currents observed at the whole-cell level. In conclusion, our results indicate that menthol induced steep outward rectification of TRPM8 results from the voltage-dependent open channel probability and the permeating ion-dependent modulation of the unitary channel conductance.     

Phenylene-ethynylene trication as an efficient fluorescent signal transducer in an aptasensor for potassium ion

A tricationic phenylene-ethynylene (N(3+)) fluorophore is investigated as a fluorescent transducer in homogeneous aptasensing system for potassium ion (K(+)) assay in aqueous media. The enhancement of the fluorescent signal of N(3+) by three K(+) aptamers consisting of 12, 15, and 21 nucleotides are observed and used for the determination of N(3+)-aptamer binding affinities. The binding affinities increase with the length of the aptameric oligonucleotides and are proven to be important to the sensitivity and selectivity of the aptasensors. The enhanced fluorescent signal of each N(3+)-aptamer solution is selectively quenched by K(+) due to the ability of K(+) in stabilizing the G-quadruplex structure of the aptamer. Among three aptamers, the 15-base aptamer provides optimal sensitivity and selectivity over other ions such as Li(+), Na(+), NH(4)(+), Mg(2+), Ca(2+) and Sr(2+). The sensing system shows the detection limit of 1 μM of K(+) in clean buffered solution and 30 μM of K(+) in the solution containing 4800-fold excess of Na(+), with wide linear dynamic ranges of micro- to millimolar concentration. This label-free fluorescence aptasensor is conveniently and effectively applicable for analysis of K(+) in urine samples.     

Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. lactis

A nucleoside N-deoxyribosyltransferase-homologous gene was detected by homological search in the genomic DNA of Lactococcus lactis subsp. lactis. The gene yejD is composed of 477 nucleotides encoding 159 amino acids with only 25% identity, which is low in comparison to the amino acid sequences of the N-deoxyribosyltransferases from other lactic acid bacteria, i.e. Lactobacillus leichmannii and Lactobacillus helveticus. The residues responsible for catalytic and substrate-binding sites in known enzymes are conserved at Gln49, Asp73, Asp93 (or Asp95), and Glu101, respectively. The recombinant YejD expressed in Escherichia coli shows a 2-deoxyribosyl transfer activity to and from both bases of purine and pyrimidine, showing that YejD should be categorized as a class II N-deoxyribosyltransferase. Interestingly, the base-exchange activity as well as the heat stability of YejD was enhanced by the presence of monovalent cations such as K(+), NH(4)(+), and Rb(+), indicating that the Lactococcus enzyme is a K(+)-activated Type II enzyme. However, divalent cations including Mg(2+) and Ca(2+) significantly inhibit the activity. Whether or not the yejD gene product actually participates in the nucleoside salvage pathway of Lc. lactis remains unclear, but the lactic acid bacterium possesses the gene coding for the nucleoside N-deoxyribosyltransferase activated by K(+) on its genome.