Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

外源EBR对NaCl胁迫下燕麦幼苗无机离子吸收、运输和分配的影响

为了探讨外源2,4-表油菜素内酯(2,4-epibrassinolide,EBR)诱导燕麦(Avena sativa L.)幼苗抗盐性的效果及其生理调节机制,以"青引2号"和"加燕2号"燕麦为材料,研究NaCl胁迫下施用外源EBR对燕麦幼苗无机离子吸收、运输和分配的影响。结果表明:100mmol·L-1NaCl胁迫下,"青引2号"和"加燕2号"燕麦幼苗叶片和根系中的Na+、Cl-含量均显著升高,对阳离子的吸收产生了拮抗作用,导致燕麦幼苗叶片和根系中的K+、Ca2+、Mg2+、Mn2+、Fe2+、Zn2+、Cu2+含量显著降低,离子稳态平衡被打破; 100 mmol·L-1NaCl胁迫下,施用0.01μmol·L-1外源EBR后,"青引2号"和"加燕2号"燕麦幼苗叶片和根系中的Na+和Cl-含量显著降低,促进了燕麦幼苗根系对K+、Ca2+、Mg2+、Fe2+、Mn2+、Cu2+和Zn2+的吸收,叶片和根系中K+/Na+、Cl-/Na+、Ca2+/Na+、Mg2+/Na+、Fe2+/Na+、Mn2+/Na+、Cu2+/Na+和Zn2+/Na+显著升高,并且有效调控燕麦幼苗体内无机离子的运输...     

Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria

In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process.     

Structural equilibria in RNA as revealed by 19F NMR

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.     

盐胁迫下柚和橘幼苗体内矿质元素变化的比较研究

采用沙培法,对盐胁迫下坪山柚和福橘幼苗体内矿质元素的变化进行了研究。结果表明,随着NaCl浓度的增加,坪山柚和福橘幼苗根部及地上部Na^＋、Cl-含量增加,且相同浓度下,福橘比坪山柚高。40mmol／L NaCI胁迫下,坪山柚和福橘幼苗地上部的K^＋、Fe含量,根部的Ca^2＋、Mg^2＋、Zn含量显著下降,而根部Fe含量及地上部Zn含量显著增加。随NaCl浓度增大,坪山柚根部K^＋含量,地上部Ca^2＋、Mg^2＋含量变化不明显,而福橘根部、地上部上述离子含量在NaCl浓度≥160mmol／L时均显著下降。因此,根部K^＋含量,地上部Ca^2＋、Mg^2＋含量存在品种问差异,或许可作为耐盐性鉴定指标。NaCl胁迫降低坪山柚和福橘幼苗根部及地上部P、Mn含量,而Cu含量在较高浓度NaCl胁迫下显著增加。NaCl胁迫明显降低坪山柚和福橘幼苗地上部K^＋／Na^＋、Ca^2＋／Na^＋和Mg^2＋／Na^＋值,其中K^＋／Na^＋值的变化可考虑作为柑橘耐盐性鉴定的指标。     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

The calcium current of Helix neuron

Calcium current, Ica, was studied in isolated nerve cell bodies of Helix aspersa after suppression of Na+ and K+ currents. The suction pipette method described in the preceding paper was used. Ica rises to a peak value and then subsides exponentially and has a null potential of 150 mV or more and a relationship with [Ca2+]o that is hyperbolic over a small range of [Ca2+]o's. When [Ca2+]i is increased, Ica is reduced disproportionately, but the effect is not hyperbolic. Ica is blocked by extracellular Ni2+, La3+, Cd2+, and Co2+ and is greater when Ba2+ and Sr2+ carry the current. Saturation and blockage are described by a Langmuir adsorption relationship similar to that found in Balanus. Thus, the calcium conductance probably contains a site which binds the ions referred to. The site also appears to be voltage-dependent. Activation and inactivation of Ica are described by first order kinetics, and there is evidence that the processes are coupled. For example, inactivation is delayed slightly in its onset and tau inactivation depends upon the method of study. However, the currents are described equally well by either a noncoupled Hodgkin-Huxley mh scheme or a coupled reaction. Facilitation of Ica by prepulses was not observed. For times up to 50 ms, currents even at small depolarizations were accounted for by suitable adjustment of the activation and inactivation rate constants.     

盐胁迫下盐地碱蓬体内无机离子含量分布特点的研究

用不同浓度NaCl溶液处理盐地碱蓬（Suaeda salsa)植株后,测定并比较老叶、幼叶及根部的无机离子含量和对K的选择性,叶片及根部的Na^ 、Cl^-含量随盐度的增加而升高,且累积趋势相似,盐胁迫下根部Na^ 、Cl^-及总离子含量（K^ 、Na^ 、Ca^2 ,NO3^-,Cl^-)明显低于叶片,说明盐地碱蓬地盐胁迫下,以叶片优先积累大量离子（如Na^ ,Cl^-) 为其适应特征。NaCl处理下,叶片的K^ ,Ca^2 含量低于对照,但随盐度的增加保持相对稳定,而根部K^ 含量,K/Na比、对K的选择性则高于叶片,这对盐胁迫下地上部的K^ 亏缺有一定补偿作用。低盐度处理（100mmol/LNaCl)促进NO3^-的吸收,另外随盐度的增加,叶片渗透势下降,渗透调节能力增强,幼叶渗透势低于老叶,但渗透调节能力相同。     

氯化钠胁迫对不同品种南瓜幼苗阳离子含量的影响

选择19个不同类型南瓜品种,研究了300 mmol·L^-1 NaCl胁迫条件下,幼苗地上部和根系Na⁺、K⁺、Ca²⁺含量、Na⁺/K⁺、Na⁺/Ca²⁺、钠-钾和钠-钙运输选择性系数(S_Na⁺,K⁺和S_Na⁺,Ca²⁺值)的变化.结果表明：NaCl胁迫处理8 d后,不同品种南瓜幼苗Na⁺含量均明显增加,而K⁺含量下降,离子平衡被打破.青栗（Q₁）南瓜幼苗根系Na⁺含量、地上部Na⁺/K⁺、Na⁺/Ca²⁺、S_Na⁺,K⁺和S_Na⁺,Ca²⁺值均明显高于黑蜜南瓜（H₂）和黑籽南瓜（H₃）.不同品种南瓜幼苗体内Na⁺含量、地上部Na⁺/K⁺和Na⁺/Ca²⁺、S_Na⁺,K⁺和S_Na⁺,Ca²⁺值变化趋势与NaCI胁迫下不同品种南瓜幼苗盐害指数的结果基本一致,进一步验证了Q₁耐盐性强与NaCl胁迫下地上部Na⁺/K⁺、Na⁺/Ca²⁺、S_Na⁺,K⁺和S_Na⁺,Ca²⁺值较低以及K⁺、Ca²⁺含量较高有关;而H₂和H₃对盐敏感与NaCl胁迫下地上部Na⁺/K⁺、Na⁺/Ca²⁺、S_Na⁺,K⁺和S_Na⁺,Ca²⁺值较高,以及K⁺、Ca²⁺含量较低有关.     

Complexation and ionophoric properties of taxol and colchicine: complex formation and transport of sodium, potassium, magnesium and calcium ions across a liquid membrane

We report the activities of taxol (an anticancer drug) and colchicine, which are inhibitors of microtubule organization, on the complexation and transport of Na+, K+, Mg2+ and Ca2+ ions across a liquid membrane, using a spectrophotometric procedure. Taxol, a diterpenoid compound, that has been demonstrated to possess a potent antitumour activity, is shown to extract Na+, K+, Mg2+ and Ca2+ ions from the aqueous solution to the organic phase with preference for Ca2+ ions. A kinetic study of the transport and complexation of Na+, K+, Mg2+ and Ca2+ ions through a liquid membrane revealed that the K+ ion is more rapidly transported and the Ca2+ ion is more rapidly complexed than other ions. However, colchicine, another alkaloid compound, extracted and transported only the divalent ions tested, Mg2+ and Ca2+. In both complexation and transport, the flux of the ions increases with the concentration of taxol or colchicine. Complexation and ionophoric properties of taxol and colchicine sheds new lights on therapeutic properties of these drugs. The treatment of disease states by the administration of these drugs to alter membrane permeability will prove to be a valuable therapeutic concept.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. I. An increase in potassium conductance is responsible for both the hyperpolarization and the decrease in intracellular free calcium produced by somatostatin

The neuropeptide somatostatin causes membrane hyperpolarization and reduces the intracellular free calcium ion concentration ([Ca2+]i) in GH pituitary cells. In this study, we have used the fluorescent dyes bisoxonol (bis,-(1,3-diethylthiobarbiturate)-trimethineoxonol) and quin2 to elucidate the mechanisms by which these ionic effects are triggered. Addition of 100 nM somatostatin to GH4C1 cells caused a 3.4 mV hyperpolarization and a 26% decrease in [Ca2+]i within 30 s. These effects were not accompanied by changes in intracellular cAMP concentrations and occurred in cells containing either basal or maximally elevated cAMP levels. To determine which of the major permeant ions were involved in these actions of somatostatin, we examined its ability to elicit changes in the membrane potential and the [Ca2+]i when the transmembrane concentration gradients for Na+, Cl-, Ca2+, and K+ were individually altered. Substitution of impermeant organic ions for Na+ or Cl- did not block either the hyperpolarization or the decrease in [Ca2+]i induced by somatostatin. Decreasing extracellular Ca2+ from 1 mM to 250 nM abolished the reduction in [Ca2+]i but did not prevent the hyperpolarization response. These results show that hyperpolarization was not primarily due to changes in the conductances of Na+, Cl-, or Ca2+. Although the somatostatin-induced decrease in [Ca2+]i did require Ca2+ influx, it was independent of changes in Na+ or Cl- conductance. In contrast, elevating the extracellular [K+] from 4.6 to 50 mM completely blocked both the somatostatin-induced hyperpolarization and the reduction in [Ca2+]i. Furthermore, hyperpolarization of the cells with gramicidin mimicked the effect of somatostatin to decrease the [Ca2+]i and prevented any additional effect by the hormone. These results indicate that somatostatin increases a K+ conductance, which hyperpolarizes GH4C1 cells, and thereby secondarily decreases Ca2+ influx. Since the somatostatin-induced decrease in [Ca2+]i is independent of changes in intracellular cAMP levels, it may be responsible for somatostatin inhibition of hormone secretion by its cAMP-independent mechanism.     

Alterations in Ca2+-binding on plasma membrane after adaptation to salt stress of tobacco cells in suspension

Electrophoresis was used to study effects of salinity on the characteristics of Ca2+ binding to the outer surface of plasma membrane (PM) of protoplasts isolated from two types of tobacco (Nicotiana tabacum L., cv. Bright Yellow) cultured cells that were adapted (tolerant) and unadapted (sensitive) to 50 mM NaCl stress. Electrophoretic analysis of salt-sensitive NaCl-unadapted cells shows that Na+ induced an appreciably higher degree of reduction in the amount of Ca2+ bound to PM compared with K+ with increasing concentration from 0.1 to 30 mM. In salt-tolerant NaCl-adapted cells, however, both Na+ and K+ ions induced almost the same degree of reduction in the amount of Ca2+ bound to PM in the physiological concentration range of Ca2+ in the medium between 2 and 4 mM. These results suggest that, under the physiological conditions, PM of salt-sensitive NaCl-unadapted cells has an appreciable amount of PM-bound Ca2+ that is desorbed much easier by Na+ than K+, whereas PM of salt-tolerant NaCl-adapted cells has the PM-bound Ca2+ that can be equally desorbed by Na+ and K+.     

Effects of Several Cations on the Neuronal Uptake of Dopamine and the Specific Binding of [3H]GBR 12783: Attempts to Characterize the Na+ Dependence of the Neuronal Transport of Dopamine

We have studied the effects of several cations on (1) the neuronal uptake of [3H]dopamine ([3H]DA) and (2) the specific binding of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-3H]propenyl)piperazi ne ([3H]GBR 12783) to a site associated with the neuronal carrier of DA, in preparations obtained from rat striatum. When studied under the same experimental conditions, both the uptake of [3H]DA and the binding of [3H]GBR 12783 were similarly impaired by the gradual replacement of NaCl by sucrose. In both processes, no convenient substitute for Na+ was found. Furthermore, potential substitutes of Na+ acted as inhibitors of the uptake with a rank order of potency as follows: K+ = Li+ > or = Cs+ > or = Rb+ > choline+ > Tris+ > sucrose, which was somewhat different from that observed in binding studies, i.e., Cs+ > Rb+ > choline+ > or = K+ > Li+ > Tris+ > sucrose. In the presence of either 36 mM or 136 mM Na+, [3H]DA uptake was optimal with 2 mM Mg2+, 1 mM K+, or 1 mM Ca2+. In contrast, higher concentrations of divalent cations competitively blocked the uptake process. K+ concentrations > 50 mM impaired the specific binding, whereas in the millimolar range of concentrations, K+ noncompetitively inhibited the uptake. Decreasing the Na+ concentration increased the inhibitory effect of K+, Ca2+, and Mg2+ on the specific uptake. An increase in NaCl concentration from 0 to 120 mM elicited a significant decline in the affinity of some substrates for the [3H]GBR 12783 binding site. An uptake study performed using optimal experimental conditions defined in the present study revealed that decreasing Na+ concentration reduces the affinity of DA for the neuronal transport. We propose a hypothetical model for the neuronal transport of DA in which both Na+ and K+ membrane gradients are involved.     

Assembly of the enveloped bacteriophage phi 6 in environments which perturb the host cell membranes.

The effects of known membrane-perturbing agents (pH, Na+, Ca2+, and a small lipid-soluble molecule) on the enveloped bacteriophage phi 6 host cell system were investigated at the levels of cellular growth, virus assembly and stability, and the physical and chemical properties of host cell membranes. Spin-label probes of cellular membranes indicate that growth in high levels of Na+ or the small spherical hydrophobic molecule adamantanone results in membranes having increased "fluidity," while growth in high levels of Ca2+ results in slightly greater rigidity of the membranes. In addition, the phospholipid composition of the cellular membranes is dependent on the NaCl concentration in the growth medium. None of these membrane alterations, however, prevent the production of infectious phi 6 virus particles.     

Sodium and calcium binding to Panulirus interruptus hemocyanin as studied by 23Na nuclear magnetic resonance.

Addition of Panulirus hemocyanin to NaCl solutions produces marked changes in the 23Na relaxation parameters; they show that sodium ions interact with binding sites on the protein and exchange rapidly with the bulk. The observed non-lorentzian lineshapes and the non-exponential decay of the transverse magnetization indicate that non-extreme narrowing conditions apply and give information on the dynamics of the interaction. Panulirus hemocyanin has at least two classes of Na+ binding sites; the binding constant of the more strongly bound sodium ions is in the order of 1 X 10(2) M-1. Competition between Na+ and Ca2+ for protein binding sites is demonstrated by the effect of Ca2+ on the 23Na relaxation parameters. However, only the more strongly bound Na+ are displaced by Ca2+. The number of Ca2+ needed to displace these sodium ions is 3--5 per oxygen binding site. The 23Na relaxation parameters are influenced also by the state of oxygenation of the protein, indicating a linkage between Na+ and oxygen binding. The simplest interpretation of the data is that sodium ions bind more strongly to oxyhemocyanin in agreement with oxygen equilibrium experiments.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

Zn2+ enhances the molecular chaperone function and stability of alpha-crystallin

Alpha-crystallin, the major eye lens protein, is a molecular chaperone that plays a crucial role in the suppression of protein aggregation and thus in the long-term maintenance of lens transparency. Zinc is a micronutrient of the eye, but its molecular interaction with alpha-crystallin has not been studied in detail. In this paper, we present results of in vitro experiments that show bivalent zinc specifically interacts with alpha-crystallin with a dissociation constant in the submillimolar range (Kd approximately 0.2-0.4 mM). We compared the effect of Zn2+ with those of Ca2+, Cu2+, Mg2+, Cd2+, Pb2+, Ni2+, Fe2+, and Co2+ at 1 mM on the structure and chaperoning ability of alpha-crystallin. An insulin aggregation assay showed that among the bivalent metal ions, only 1 mM Zn2+ improved the chaperone function of alpha-crystallin by 30% compared to that in the absence of bivalent metal ions. Addition of 1 mM Zn2+ increased the yield of alpha-crystallin-assisted refolding of urea-treated LDH to its native state from 33 to 38%, but other bivalent ions had little effect. The surface hydrophobicity of alpha-crystallin was increased by 50% due to the binding of Zn2+. In the presence of 1 mM Zn2+, the stability of alpha-crystallin was enhanced by 36 kJ/mol, and it became more resistant to tryptic cleavage. The implications of enhanced stability and molecular chaperone activity of alpha-crystallin in the presence of Zn2+ are discussed in terms of its role in the long-term maintenance of lens transparency and cataract formation.     

Further characterization of the mechanisms mediating the rise in cytosolic free Na+ in thrombin-stimulated platelets. Evidence for inhibition of the Na+,K(+)-ATPase and for Na+ entry via a Ca2+ influx pathway.

Human platelets were loaded with the fluorescent Na(+)-sensitive dye sodium-binding benzofuran isophtalate (SBFI), and changes in the fluorescence excited at 345 and 385 nm were analyzed after manipulations that evoked predictable changes in the cytosolic Na+ concentration ([Na+]i). Raising [Na+]i by either gramicidin D or monensin specifically increased the fluorescence excited at 345 nm and decreased that excited at 385 nm. Hence, calculation of changes in the 345/385 nm excitation ratio yields an estimate of actual changes in [Na+]i. A transient activation of Na+/H+ exchange evoked by addition of acidified platelets to buffer, pH 7.4, evoked a transient rise in [Na+]i. The re-establishment of basal [Na+]i could be prevented by ouabain, indicating an involvement of the Na+,K(+)-ATPase. Upon stimulation by 0.5 unit/ml of thrombin, [Na+]i immediately increased by 16 +/- 4 mM and this rise continued for at least 60 min after addition of agonist, albeit at a lower rate. This latter sustained rise could not be curtailed by scavenging thrombin by means of hirudin. Addition of ouabain or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced a comparable slow rise in the 345/385 excitation ratio. This may indicate a protein kinase C-mediated inhibition by thrombin of the Na+,K(+)-ATPase. In the absence of extracellular Ca2+ (Ca2+o), the [Na+]i gain was augmented to 38 +/- 9 mM. This additional uptake of Na+ was prevented by (i) Mn2+ ions, (ii) La3+ ions, (iii) the blocker of receptor-mediated Ca2+ entry (1-[beta[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-im ida zole hydrochloride), and (iv) by hirudin which reversed receptor occupancy by thrombin. These findings suggest that the additional thrombin-induced [Na+]i gain in the absence of Ca2+o is due to Na+ influx through a Ca2+ entry pathway. The increase in [Na+]i in the presence of Ca2+o results from Na+ influx via Na+/H+ exchange.     

Regulation of connexin hemichannels by monovalent cations

Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.     

Divalent ion trapping inside potassium channels of human T lymphocytes

Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca2+, Ba2+, K+, Rb+, and internal Ca2+ on the rate of K+ channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca2+ or Ba2+, or reducing external K+, accelerated the rate of the K+ current decay during a depolarizing voltage pulse. External Ba2+ also produced a use-dependent block of the K+ channels by entering the open channel and becoming trapped inside. Raising internal Ca2+ accelerated inactivation at lower concentrations than external Ca2+, but increasing the Ca2+ buffering with BAPTA did not affect inactivation. Raising [K+]o or adding Rb+ slowed inactivation by competing with divalent ions. External Rb+ also produced a use-dependent removal of block of K+ channels loaded with Ba2+ or Ca2+. From the removal of this block we found that under normal conditions approximately 25% of the channels were loaded with Ca2+, whereas under conditions with 10 microM internal Ca2+ the proportion of channels loaded with Ca2+ increased to approximately 50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a non-selective, voltage-independent conductance. We conclude that Ca2+ ions from the outside or the inside can bind to a site at the K+ channel and thereby block the channel or accelerate inactivation.