Direction Of Na Research Articles

A DNA nanodevice for mapping sodium at single-organelle resolution.

Cellular sodium ion (Na+) homeostasis is integral to organism physiology. Our current understanding of Na+ homeostasis is largely limited to Na+ transport at the plasma membrane. Organelles may also contribute to Na+ homeostasis; however, the direction of Na+ flow across organelle membranes is unknown because organellar Na+ cannot be imaged. Here we report a pH-independent, organelle-targetable, ratiometric probe that reports lumenal Na+. It is a DNA nanodevice containing a Na+-sensitive fluorophore, a reference dye and an organelle-targeting domain. By measuring Na+ at single endosome resolution in mammalian cells and Caenorhabditis elegans, we discovered that lumenal Na+ levels in each stage of the endolysosomal pathway exceed cytosolic levels and decrease as endosomes mature. Further, we find that lysosomal Na+ levels in nematodes are modulated by the Na+/H+ exchanger NHX-5 in response to salt stress. The ability to image subcellular Na+ will unveil mechanisms of Na+ homeostasis at an increased level of cellular detail.

The soybean plasma membrane-localized cation/H+ exchanger GmCHX20a plays a negative role under salt stress.

Cation/H+ -exchanger (CHX) perform diverse functions in plants, including being a part of the protective mechanisms to cope with salt stress. GmCHX1 has been previously identified as the causal gene in a major salt-tolerance quantitative trait locus (QTL) in soybean, but little is known about another close paralog, GmCHX20a, found in the same QTL. In this study, GmCHX20a was characterized along with GmCHX1. The expression patterns of the two genes and the direction of Na+ flux directed by overexpression of these two transporters are different, suggesting that they are functionally distinct. The ectopic expression of GmCHX20a led to an increase in salt sensitivity and osmotic tolerance, which was consistent with its role in increasing Na+ uptake into the root. Although this seems counter-intuitive, it may in fact be part of the mechanism by which soybean could counter act the effects of osmotic stress, which is commonly manifested in the initial stage of salinity stress. On the other hand, GmCHX1 from salt-tolerant soybean was shown to protect plants via Na+ exclusion under salt stress. Taken together these results suggest that GmCHX20a and GmCHX1 might work complementally through a concerted effort to address both osmotic stress and ionic stress as a result of elevated salinity.

Molecular Friction-Induced Electroosmotic Phenomena in Thin Neutral Nanotubes.

We reveal by classical molecular dynamics simulations electroosmotic flows in thin neutral carbon (CNT) and boron nitride (BNT) nanotubes filled with ionic solutions of hydrated monovalent atomic ions. We observe that in (12,12) BNTs filled with single ions in an electric field, the net water velocity increases in the order of Na(+) < K(+) < Cl(-), showing that different ions have different power to drag water in thin nanotubes. However, the effect gradually disappears in wider nanotubes. In (12,12) BNTs containing neutral ionic solutions in electric fields, we observe net water velocities going in the direction of Na(+) for (Na(+), Cl(-)) and in the direction of Cl(-) for (K(+), Cl(-)). We hypothesize that the electroosmotic flows are caused by different strengths of friction between ions with different hydration shells and the nanotube walls.

Extracellular Protons Regulate Human ENaC by Modulating Na+ Self-inhibition

The epithelial Na(+) channel, ENaC, is exposed to a wide range of proton concentrations in the kidney, lung, and sweat duct. We, therefore, tested whether pH alters ENaC activity. In Xenopus oocytes expressing human alpha-, beta-, and gammaENaC, amiloride-sensitive current was altered by protons in the physiologically relevant range (pH 8.5-6.0). Compared with pH 7.4, acidic pH increased ENaC current, whereas alkaline pH decreased current (pH(50) = 7.2). Acidic pH also increased ENaC current in H441 epithelia and in human primary airway epithelia. In contrast to human ENaC, pH did not alter rat ENaC current, indicating that there are species differences in ENaC regulation by protons. This resulted predominantly from species differences in gammaENaC. Maneuvers that lock ENaC in a high open-probability state ("DEG" mutation, proteolytic cleavage) abolished the effect of pH on human ENaC, indicating that protons alter ENaC current by modulating channel gating. Previous work showed that ENaC gating is regulated in part by extracellular Na(+) ("Na(+) self-inhibition"). Based on several observations, we conclude that protons regulate ENaC by altering Na(+) self-inhibition. First, protons reduced Na(+) self-inhibition in a dose-dependent manner. Second, ENaC regulation by pH was abolished by removing Na(+) from the extracellular bathing solution. Third, mutations that alter Na(+) self-inhibition produced corresponding changes in ENaC regulation by pH. Together, the data support a model in which protons modulate ENaC gating by relieving Na(+) self-inhibition. We speculate that this may be an important mechanism to facilitate epithelial Na(+) transport under conditions of acidosis.

Mass transfer and replacement of minerals in the course of infiltration metasomatism

The direction of Na, K, Ca, Mg, and Fe transfer was determined in the granite-chloride solution system during the metasomatic replacement of granite in response to a temperature and pressure change. The analysis of modeled metasomatic zoning (metasomatic columns) indicates that metasomatic processes of alkaline/acidic and basification/debasification types are controlled by differences in the chemical potentials of components in the solution and rock and by the addition/removal of material due to its excess/deficit in the solution. The former are manifested in the mineralogy of the rear zones of metasomatic columns, while the latter control the total variations in the contents of components in all zones. One of the consequences of this difference is the occurrence of extrema in the distribution of components in zones. Information on the chemical composition of rocks in any given zone does not provide a clue to either the character of the metasomatic processes or the proportions of component concentrations in the solution. In contrast to isobaric-isothermal columns, their thermogradient analogues are characterized by the continuous replacement of minerals and mass transfer in all zones. This predetermines the possibility of the identification of the gradient character of certain columns.

How many sodium ions does it take to turn an exchanger?

How many sodium ions does it take to turn an exchanger?

Effect of Na(+) flow on Cd(2+) block of tetrodotoxin-resistant Na(+) channels.

Tetrodotoxin-resistant (TTX-R) Na+ channels are 1,000-fold less sensitive to TTX than TTX-sensitive (TTX-S) Na+ channels. On the other hand, TTX-R channels are much more susceptible to external Cd2+ block than TTX-S channels. A cysteine (or serine) residue situated just next to the aspartate residue of the presumable selectivity filter “DEKA” ring of the TTX-R channel has been identified as the key ligand determining the binding affinity of both TTX and Cd2+. In this study we demonstrate that the binding affinity of Cd2+ to the TTX-R channels in neurons from dorsal root ganglia has little intrinsic voltage dependence, but is significantly influenced by the direction of Na+ current flow. In the presence of inward Na+ current, the apparent dissociation constant of Cd2+ (∼200 μM) is ∼9 times smaller than that in the presence of outward Na+ current. The Na+ flow–dependent binding affinity change of Cd2+ block is true no matter whether the direction of Na+ current is secured by asymmetrical chemical gradient (e.g., 150 mM Na+ vs. 150 mM Cs+ on different sides of the membrane, 0 mV) or by asymmetrical electrical gradient (e.g., 150 mM Na+ on both sides of the membrane, −20 mV vs. 20 mV). These findings suggest that Cd2+ is a pore blocker of TTX-R channels with its binding site located in a multiion, single-file region near the external pore mouth. Quantitative analysis of the flow dependence with the flux-coupling equation reveals that at least two Na+ ions coexist with the blocking Cd2+ ion in this pore region in the presence of 150 mM ambient Na+. Thus, the selectivity filter of the TTX-R Na+ channels in dorsal root ganglion neurons might be located in or close to a multiion single-file pore segment connected externally to a wide vestibule, a molecular feature probably shared by other voltage-gated cationic channels, such as some Ca2+ and K+ channels.

Endogenous electric current is associated with normal development of the vertebrate limb.

A steady ionic current is driven out of both developing and regenerating amphibian limbs. In the developing limbs of anurans and urodeles, focal outwardly directed current (0.5-2 microA/cm(2)) predicts the location of mesenchyme accumulations producing the early bud. Here, we report measurements of a similar outwardly directed ionic current associated with the development of the limb bud in the mouse and chick embryo by using a noninvasive, self-referencing electrode for the measurement of extracellular current. In both the mouse and chick embryo, flank currents were usually inwardly directed - the direction of Na(+) uptake by ectoderm. Outward currents associated with the mouse limb bud ranged from 0.04-10.8 microA/cm(2). Mouse limb bud and flank currents were similar to those measured in amphibian larvae, because they were reversibly collapsed and/or reversed by application of 30 microM amiloride, a Na(+) channel blocker. Unlike the amphibian embryos, flank ectoderm adjacent to the mouse limb bud in the anterior/posterior axis was usually associated with outwardly directed ionic current. This raises the possibility of a different, or changing, gradient of extracellular voltage experienced by mesenchyme cells in this plane of development than that observed in other regions of the limb bud. In the chick flank caudal to the somites, a striking reversal of the inwardly directed flank currents to very large ( approximately 100 microA/cm(2)) outwardly directed currents occurred three developmental stages before limb bud formation. We tested the relevance of this outwardly directed ionic current to limb formation in the chick embryo by reversing it by using an artificially applied "countercurrent" pulled through a microelectrode inserted just beneath the caudal ectoderm of the embryo. This application was performed for approximately 6 hr 2.5-3 developmental stages before hindlimb bud formation. This method resulted in abnormal limb formation by the tenth day of gestation in some embryos, whereas all control embryos developed normally. These data suggest an early physiological control of limb development.

Osmomechanics of the Propionigenium modestum F(o) motor.

In Propionigenium modestum, ATP is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of Na+ ions. The P. modestum ATP synthase is a clear member of the family of F-type ATP synthases and the only major distinction is an extension of the coupling ion specificity to H+, Li+, or Na+, depending on the conditions. The use of Na+ as a coupling ion offers unique experimental options to decipher the ion-translocation mechanism and the osmotic and mechanical behavior of the enzyme. The single a subunit and the oligomer of c subunits are part of the stator and rotor, respectively, and operate together in the ion-translocation mechanism. During ATP synthesis, Na+ diffuses from the periplasm through the a subunit channel onto the Na+ binding site on a c subunit. From there it dissociates into the cytoplasm after the site has rotated out of the interface with subunit a. In the absence of a membrane potential, the rotor performs Brownian motions into either direction and Na+ ions are exchanged between the two compartments separated by the membrane. Upon applying voltage, however, the direction of Na+ flux and of rotation is biased by the potential. The motor generates torque to drive the rotation of the gamma subunit, thereby releasing tightly bound ATP from catalytic sites in F(1). Hence, the membrane potential plays a pivotal role in the torque-generating mechanism. This is corroborated by the fact that for ATP synthesis, at physiological rates, the membrane potential is indispensable. We propose a catalytic mechanism for torque generation by the F(o) motor that is in accord with all experimental data and is in quantitative agreement with the requirement for ATP synthesis.

Use of a channel biosensor for the assay of paralytic shellfish toxins

Gonyautoxin (GTX), saxitoxin (STX) and tetrodotoxin (TTX), also known as paralytic shellfish poisons (PSP), block Na + channels, including those in the frog bladder membrane. A tissue biosensor has been developed, consisting of a Na + electrode covered with a frog bladder membrane integrated within a flow cell. The direction of Na + transfer, investigated in the absence of Na + channel blockers, established that active transport of Na + occurs across the frogs bladder membrane from the internal to the external face. Transfer was shown to be TTX sensitive. The tissue sensor response to each of the different PSP was recorded and the results compared with toxicities determined by the standard mouse bio-assay. Using high concentrations of TTX from the puffer fish Takifugu niphobles, a linear correlation was found between the results from the two assay systems. However, the tissue biosensor system was also able to detect very low concentrations of TTX in samples from two species of puffer fish ( Takifugu niphobles and Takifugu pardalis) at concentrations below the detection limit of the mouse bio-assay.

Effect of stimulation and veratrine on total cellular calcium in rat and guinea-pig ventricular myocytes.

In rat cardiac myocytes, calcium efflux by Na+/Ca(2+)-exchange is expected only during ventricular systole following initial action potential repolarization. In contrast, in guinea-pigs, calcium influx via Na+/Ca(2+)-exchange is expected only during the initial portion of the action potential. Thus electrical stimulation is expected to result in reduced intracellular calcium ([Ca2+]i) in rat and an increase in guinea pig. We tested this hypothesis by measuring total cellular calcium ([Ca]tot) using 45Ca following stimulation of isolated rat and guinea-pig ventricular myocytes. Many studies have also emphasized that the rate and the direction of Na+/Ca(2+)-exchange across the sarcolemma are in part dependent on the magnitude of the transsarcolemmal sodium gradient. Thus, increasing intracellular sodium ([Na+]i) is expected to result in an increased [Ca2+]i. This hypothesis was also tested by measuring [Ca]tot following veratrine administration. Enzymatically isolated rat and guinea-pig ventricular myocytes were divided into two groups; non-stimulated and stimulated (1 Hz). The concentration-dependent effects of veratrine (1,10,100 micrograms/ml) on [Ca]tot were determined in both these groups. In the absence of veratrine, non-stimulated rat myocytes had a significantly higher [Ca]tot than did stimulated ones. Non-stimulated guinea-pig myocytes had a significantly lower [Ca]tot when compared with stimulated ones. Veratrine increased [Ca]tot in both species in a concentration-dependent fashion. In addition, following veratrine the difference between [Ca]tot in non-stimulated and stimulated rat myocytes was no longer significant. These results support those of others who have demonstrated that stimulation is associated with a gain of cellular calcium in both rabbit and guinea-pig ventricle and a calcium loss in rat ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Gallamine triethiodide-induced modifications of sodium conductance in Myxicola giant axons.

1. Internal gallamine triethiodide (Flaxedil) modifies Na+ channel kinetics in Myxicola axons but does not alter K+ conductance. The drug has no effect externally. 2. Gallamine initially increases the leakage-conductance, but this effect completely reverses within 30 min despite the maintained presence of drug. 3. During step depolarizations to membrane potentials less than -10 mV, gallamine slows the rate of Na+ inactivation, but all channels which have opened can still inactivate. During depolarizations to more positive potentials, gallamine-modified Na+ currents show a biphasic decline, and at VM greater than -10 mV, Na+ inactivation is incomplete as evidenced by the large Na+ tail currents which follow pulses sufficiently long to have allowed complete inactivation of normal Na+ channels. The tail currents are slower than normal Na+ tails, and exhibit a pronounced hook. With gallamine, the fraction of Na+ channels which do not inactivate increases sigmoidally over the range 0 mV to +80 mV. 4. For VM greater than ENa, gallamine almost completely blocks outward Na+ currents. The block is determined by the direction of Na+ current, rather than the absolute membrane potential. 5. Gallamine has no effect upon the rate of Na+ channel activation, the maximum Na+ conductance, the steady-state Na+ inactivation curve, or the rate of development or removal of inactivation by prepulses. 6. Gallamine eliminated physiological immobilization of intramembrane charge movements (QOFF and QON) and does not itself induce immobilization. Thus, in the presence of gallamine, QOFF following long pulses is the same as QOFF following short pulses.

Effects of certain steroids and other compounds on sodium efflux from erythrocytes.

SUMMARY The effect of hormones and drugs on net sodium (Na+) efflux from human erythrocytes was studied. On cooling, the red cells gain Na+ from the extra-cellular fluid, the excess Na+ being driven out again on incubation at 37° C. This net loss of Na+ from red cells was inhibited by digoxin and much larger concentrations of SC 5233, an aldosterone antagonist. The latter drug's effect was markedly enhanced by deoxycorticosterone (DOC), the combination being able to reverse the direction of Na+ flux. However, DOC alone or combined with digoxin had no influence on net Na+ efflux. Other hormones and drugs tested by the same method had no significant action on net Na+ efflux from erythrocytes. Implications of these findings are discussed.