Effects of Saxitoxin (STX) and Veratridine on Bacterial Na+-K+ Fluxes: a Prokaryote-Based STX Bioassay

Saxitoxin (STX) is a potent natural sodium channel blocker and represents a significant health concern worldwide. We describe here the antagonistic effects of STX and veratridine (VTD), an Na⁺ channel activator, on three gram-negative bacteria and their application to an STX bioassay. STX reduced the total cellular levels of both Na⁺ and K⁺, as measured by flame photometry, whereas VTD increased the cellular concentrations relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205. Endogenous STX production in toxic cyanobacterial strains of C. raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress. Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5 to 8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. The addition of STX, or its analogue neoSTX, prior to treatment with VTD plus VAN prevented complete lysis in non-STX-producing cyanobacteria. VTD also affected cyanobacterial metabolism, and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan. Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD. Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel blocker, with samples exhibiting resistance to the VTD effect. Our findings demonstrate that STX and VTD influence bacterial Na⁺ and K⁺ fluxes in opposite ways, and these principles can be applied to the development of a prokaryote-based STX bioassay.     

Evidence for differences in the metabolism of saxitoxin and C1+2 toxins in the freshwater cyanobacterium Cylindrospermopsis raciborskii T3

The activity of paralytic shellfish poisoning (PSP) toxins biosynthetic enzymes was assayed in the cyanobacterium Cylindrospermopsis raciborskii T3 after inhibiting protein synthesis with chloramphenicol (CAM). The production of C1+2 and saxitoxin (STX) was sensitive to CAM with STX levels decreasing by 70% after 24-h exposure to the antibiotic. PSP toxin production was strongly promoted by arginine supplementation, with a maximum 476% increase in intracellular STX concentrations after 24-h exposure to 10 mM of the amino acid. However, arginine had no stimulating effect on PSP toxin levels if supplemented in combination with CAM at 10 microg l(-1). Addition of agmatine and proline to C. raciborskii T3 cultures in the presence of 10 microg l(-1) CAM increased C1+2 toxins levels, while having a negative or no effect on STX accumulation. In vitro, PSP toxin levels increased naturally in cyanobacterial extracts, with CAM and arginine having no influence on either C1+2 or STX synthesis. The evidence presented in this study suggests a possible difference between the metabolism of STX and the C1+2 toxins and indicated a high turnover rate of STX biosynthetic enzymes in C. raciborskii T3.     

Detection of saxitoxin-producing cyanobacteria and Anabaena circinalis in environmental water blooms by quantitative PCR

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by marine "red tide" dinoflagellates and several species of freshwater filamentous cyanobacteria, including Anabaena circinalis, Aphanizomenon spp., Lyngbya wollei, and Cylindrospermopsis raciborskii. A specific quantitative PCR (qPCR) method based on SYBR green chemistry was developed to quantify saxitoxin-producing Anabaena circinalis cyanobacteria, which are major bloom-forming freshwater cyanobacteria. The aim of this study was to infer the potential toxigenicity of samples by determining the copy number of a unique and unusual polyketide synthase (PKS) sequence (sxtA) in the STX biosynthesis gene cluster identified in cyanobacteria. Our qPCR approach was applied to water samples collected from different Australian lakes, dams, and rivers. The STX concentration and cyanobacterial cell density of these blooms were also determined by high-pressure liquid chromatography (HPLC) and microscopic cell counting, respectively. STX concentrations correlated positively with STX gene copy numbers, indicating that the latter can be used as a measure of potential toxigenicity in Anabaena circinalis and possibly other cyanobacterial blooms. The qPCR method targeting STX genes can also be employed for both monitoring and ecophysiological studies of toxic Anabaena circinalis blooms and potentially several other STX-producing cyanobacteria.     

Antagonism by Local Anesthetics of Sodium Channel Activators in the Presence of Scorpion Toxins: Two Mechanisms for Competitive Inhibition

1. The interaction of veratridine (VTD), a Na+ channel activator, scorpion alpha-toxin (LQ), an open state Na+ channel stabilizer, and the local anesthetic, lidocaine (LID), a channel inhibitor, at the neuronal sodium channel was assessed by measuring VTD-dependent slow depolarizations of frog sciatic nerve using the sucrose-gap method. 2. The slow depolarizing action of veratridine was potentiated more than 10-fold by the peptide LQ toxin, whereas its competitive inhibition by lidocaine was unchanged by LQ. 3. We conclude that the antagonism between VTD and a LID molecule during slow depolarization is allosteric, involving a trapping of the Na+ channel by LID in the inactivated state that has a very low affinity for VTD. 4. The binding of VTD to the open state of the channel, which is stabilized by LQ, may be inhibited by orthosteric competition at overlapping sites since both LID and VTD bind avidly and rapidly to open channels.     

Uncoupling cell shrinkage from apoptosis reveals that Na+ influx is required for volume loss during programmed cell death

Cell shrinkage, or the loss of cell volume, is a ubiquitous characteristic of programmed cell death that is observed in all examples of apoptosis, independent of the death stimulus. This decrease in cell volume occurs in synchrony with other classical features of apoptosis. The molecular basis for cell shrinkage during apoptosis involves fluxes of intracellular ions including K+, Na+, and Cl-. Here we show for the first time that these ion fluxes, but not cell shrinkage, are necessary for apoptosis. Using sodium-substituted medium during anti-Fas treatment of Jurkat cells, we observed cellular swelling, a property normally associated with necrosis, in contrast to the typical cell shrinkage. Surprisingly, these swollen cells displayed all of the other classical features of apoptosis, including chromatin condensation, externalization of phosphatidylserine, caspase activity, poly(ADP)-ribose polymerase cleavage, and internucleosomal DNA degradation. These swollen cells had a marked decrease in intracellular potassium, and subsequent inhibition of this potassium loss completely blocked apoptosis. Reintroduction of sodium ions in cell cultures reversed this cellular swelling, resulting in a dramatic loss of cell volume and the characteristic apoptotic morphology. Additionally, inhibition of sodium influx using a sodium channel blocker saxitoxin completely prevented the onset of anti-Fas-induced apoptosis in Jurkat cells. These findings suggest that sodium influx can control not only changes in cell size but also the activation of apoptosis, whereas potassium ion loss controls the progression of the cell death process. Therefore cell shrinkage can be separated from other features of apoptosis.     

Periodate treatment reduces the tetrodotoxin-sensitivity of voltage-gated Na+ channels

(1) Voltage-clamped nerve fibres of the frog Rana esculenta were treated with periodate in the extracellular solution. (2) Periodate treatment irreversibly reduced the effect of tetrodotoxin (TTX) on the Na+ currents. (3) The effect of saxitoxin (STX) was also reduced but less than that of TTX. (4) The presence of STX during the application of periodate to the nerve fibre almost completely prevented the effect of the chemical reagent on the TTX sensitivity of the Na+ channels. (5) The reduction of the TTX effect is not due to the reaction of small amounts of periodate with the diol group of this toxin, because the effect was seen after prolonged washing with reagent-free Ringer solution with or without high amounts of ribose. (6) Carboxyl groups present in the Na+ channel seem to be quite important for the binding of TTX and STX. Periodate modifies several amino acid side chains, however, it does not attack carboxyl groups in a peptide chain. Thus, these results suggest that periodate modifies a further group critically involved in the binding of TTX and STX.     

Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Evidence for a sulfhydryl group in the Zn2+/saxitoxin binding site.

Mammalian heart Na+ channels exhibit approximately 100-fold higher affinity for block by external Zn2+ than other Na+ channel subtypes. With batrachotoxin-modified Na+ channels from dog or calf heart, micromolar concentrations of external Zn2+ result in a flickering block to a substate level with a conductance of approximately 12% of the open channel at -50 mV. We examined the hypothesis that, in this blocking mode, Zn2+ binds to a subsite of the saxitoxin (STX) binding site of heart Na+ channels by single-channel analysis of the interaction between Zn2+ and STX and also by chemical modification experiments on single heart Na+ channels incorporated into planar lipid bilayers in the presence of batrachotoxin. We found that external Zn2+ relieved block by STX in a strictly competitive fashion. Kinetic analysis of this phenomenon was consistent with a scheme involving direct binding competition between Zn2+ and STX at a single site with intrinsic equilibrium dissociation constants of 30 nM for STX and 30 microM for Zn2+. Because high-affinity Zn2(+)-binding sites often include sulfhydryl groups as coordinating ligands of this metal ion, we tested the effect of a sulfhydryl-specific alkylating reagent, iodoacetamide (IAA), on Zn2+ and STX block. For six calf heart Na+ channels, we observed that exposure to 5 mM IAA completely abolished Zn2+ block and concomitantly modified STX binding with at least 20-fold reduction in affinity. These results lead us to propose a model in which Zn2+ binds to a subsite within or near the STX binding site of heart Na+ channels. This site is also presumed to contain one or more cysteine sulfhydryl groups.     

Reconstitution of the voltage-sensitive sodium channel of rat brain from solubilized components

The voltage-sensitive sodium channel of rat brain synaptosomes was solubilized with sodium cholate. The solubilized sodium channel migrated on a sucrose density gradient with an apparent S20,w of approximately 12 S, retained [3H]saxitoxin ([3H]STX) binding activity that was labile at 36 degrees C but no longer bound 125I-labeled scorpion toxin (125I-ScTX). Following reconstitution into phosphatidylcholine vesicles, the channel regained 125I-ScTX binding and thermal stability of [3H]STX binding. Approximately 50% of the [3H]STX binding activity and 58% of 125I-ScTX binding activity were recovered after reconstitution. The reconstituted sodium channel bound STX and ScTX with KD values of 5 and 10 nM, respectively. Under depolarized conditions, veratridine enhanced the binding of 125I-ScTX with a K0.5 of 20 microM. These KD and K0.5 values are similar to those of the native synaptosome sodium channel. 125I-ScTX binding to the reconstituted sodium channel, as with the native channel, was voltage dependent. The KD for 125I-ScTX increased with depolarization. This voltage dependence was used to demonstrate that the reconstituted channel transports Na+. Activation of sodium channels by veratridine under conditions expected to cause hyperpolarization of the reconstituted vesicles increased 125I-ScTX binding 3-fold. This increased binding was blocked by STX with K0.5 = 5 nM. These data indicate that reconstituted sodium channels can transport Na+ and hyperpolarize the reconstituted vesicles. Thus, incorporation of solubilized synaptosomal sodium channels into phosphatidylcholine vesicles results in recovery of toxin binding and action at each of the three neurotoxin receptor sites and restoration of Na+ transport by the reconstituted channels.     

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.     

Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C. raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.     

Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.     

Na+ channel and Na+-K+ ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb.

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.     

Calcium calmodulin in altered NaCl transport by heat-labile enterotoxin of Escherichia coli

The mucosal-to-serosal and serosal-to-mucosal fluxes of Na+ and Cl- were carried out in control and experimental groups treated with different doses of heat-labile enterotoxin in the presence or absence of Ca2+-ionophore, Ca2+ channel blocker and calmodulin inhibitor. There was net secretion of Na+ and Cl- in 16 and 32 units of heat-labile enterotoxin treated groups in comparison to net absorption in control group, however, in animals treated with 8 units of heat-labile enterotoxin, no change in Na+ and Cl- fluxes was found when compared to control. Ca2+- ionophore increased net secretion of Na+ and Cl- in 16 and 32 units of heat-labile enterotoxin treated groups and also caused secretion in control group instead of net absorption. Ca2+ channel blocker and calmodulin inhibitor partially reversed the effect of heat-labile enterotoxin. The effect of Ca2+-ionophore was more pronounced in the control group while that of Ca2+ channel blocker and calmodulin inhibitor was more pronounced in 16 and 32 units of heat-labile enterotoxin treated groups. The findings suggest the involvement of Ca2+ and calmodulin in the action of heat-labile enterotoxin of Escherichia coli in mice.     

A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel.

Biophysical evidence has placed the binding site for the naturally occurring marine toxins tetrodotoxin (TTX) and saxitoxin (STX) in the external mouth of the Na+ channel ion permeation pathway. We developed a molecular model of the binding pocket for TTX and STX, composed of antiparallel beta-hairpins formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel. For TTX the guanidinium moiety formed salt bridges with three carboxyls, while two toxin hydroxyls (C9-OH and C10-OH) interacted with a fourth carboxyl on repeats I and II. This alignment also resulted in a hydrophobic interaction with an aromatic ring of phenylalanine or tyrosine residues for the brainII and skeletal Na+ channel isoforms, but not with the cysteine found in the cardiac isoform. In comparison to TTX, there was an additional interaction site for STX through its second guanidinium group with a carboxyl on repeat IV. This model satisfactorily reproduced the effects of mutations in the S5-S6 regions and the differences in affinity by various toxin analogs. However, this model differed in important ways from previously published models for the outer vestibule and the selectivity region of the Na+ channel pore. Removal of the toxins from the pocket formed by the four beta-hairpins revealed a structure resembling a funnel that terminated in a narrowed region suitable as a candidate for the selectivity filter of the channel. This region contained two carboxyls (Asp384 and Glu942) that substituted for molecules of water from the hydrated Na+ ion. Simulation of mutations in this region that have produced Ca2+ permeation of the Na+ channel created a site with three carboxyls (Asp384, Glu942, and Glu1714) in proximity.     

Saxitoxin is a gating modifier of HERG K+ channels

Potassium (K+) channels mediate numerous electrical events in excitable cells, including cellular membrane potential repolarization. The hERG K+ channel plays an important role in myocardial repolarization, and inhibition of these K+ channels is associated with long QT syndromes that can cause fatal cardiac arrhythmias. In this study, we identify saxitoxin (STX) as a hERG channel modifier and investigate the mechanism using heterologous expression of the recombinant channel in HEK293 cells. In the presence of STX, channels opened slower during strong depolarizations, and they closed much faster upon repolarization, suggesting that toxin-bound channels can still open but are modified, and that STX does not simply block the ion conduction pore. STX decreased hERG K+ currents by stabilizing closed channel states visualized as shifts in the voltage dependence of channel opening to more depolarized membrane potentials. The concentration dependence for steady-state modification as well as the kinetics of onset and recovery indicate that multiple STX molecules bind to the channel. Rapid application of STX revealed an apparent "agonist-like" effect in which K+ currents were transiently increased. The mechanism of this effect was found to be an effect on the channel voltage-inactivation relationship. Because the kinetics of inactivation are rapid relative to activation for this channel, the increase in K+ current appeared quickly and could be subverted by a decrease in K+ currents due to the shift in the voltage-activation relationship at some membrane potentials. The results are consistent with a simple model in which STX binds to the hERG K+ channel at multiple sites and alters the energetics of channel gating by shifting both the voltage-inactivation and voltage-activation processes. The results suggest a novel extracellular mechanism for pharmacological manipulation of this channel through allosteric coupling to channel gating.     

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.     

Non-selective voltage-activated cation channel in the human red blood cell membrane

Using the patch-clamp technique, a non-selective voltage-activated Na+ and K+ channel in the human red blood cell membrane was found. The channel operates only at positive membrane potentials from about +30 mV (inside positive) onwards. For sodium and potassium ions, similar conductances of about 21 pS were determined. Together with the recently described K+(Na+)/H+ exchanger, this channel is responsible for the increase of residual K+ and Na+ fluxes across the human red blood cell membrane when the cells are suspended in low ionic strength medium.     

Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx.     

Progress and perspective on cyanobacterial glycogen metabolism engineering

As important oxygenic photoautotrophs, cyanobacteria are also generally considered as one of the most promising microbial chassis for photosynthetic biomanufacturing. Diverse synthetic biology and metabolic engineering approaches have been developed to enable the efficient harnessing of carbon and energy flow toward the synthesis of desired metabolites in cyanobacterial cell factories. Glycogen metabolism works as the most important natural carbon sink mechanism and reserve carbon source, storing a large portion of carbon and energy from the Calvin-Benson-Bassham (CBB) cycle, and thus is traditionally recognized as a promising engineering target to optimize the efficacy of cyanobacterial cell factories. Multiple strategies and approaches have been designed and adopted to engineer glycogen metabolism in cyanobacteria, leading to the successful regulation of glycogen synthesis and storage contents in cyanobacteria cells. However, disturbed glycogen metabolism results in weakened cellular physiological functionalities, thereby diminishing the robustness of metabolism. In addition, the effects of glycogen removal as a metabolic engineering strategy to enhance photosynthetic biosynthesis are still controversial. This review focuses on the efforts and effects of glycogen metabolism engineering on the physiology and metabolism of cyanobacterial chassis strains and cell factories. The perspectives and prospects provided herein are expected to inspire novel strategies and tools to achieve ideal control over carbon and energy flow for biomanufacturing.     

Futile Na+ cycling at the root plasma membrane in rice (Oryza sativa L.): kinetics, energetics, and relationship to salinity tolerance

Globally, over one-third of irrigated land is affected by salinity, including much of the land under lowland rice cultivation in the tropics, seriously compromising yields of this most important of crop species. However, there remains an insufficient understanding of the cellular basis of salt tolerance in rice. Here, three methods of 24Na+ tracer analysis were used to investigate primary Na+ transport at the root plasma membrane in a salt-tolerant rice cultivar (Pokkali) and a salt-sensitive cultivar (IR29). Futile cycling of Na+ at the plasma membrane of intact roots occurred at both low and elevated levels of steady-state Na+ supply ([Na+]ext=1 mM and 25 mM) in both cultivars. At 25 mM [Na+]ext, a toxic condition for IR29, unidirectional influx and efflux of Na+ in this cultivar, but not in Pokkali, became very high [>100 micromol g (root FW)(-1) h(-1)], demonstrating an inability to restrict sodium fluxes. Current models of sodium transport energetics across the plasma membrane in root cells predict that, if the sodium efflux were mediated by Na+/H+ antiport, this toxic scenario would impose a substantial respiratory cost in IR29. This cost is calculated here, and compared with root respiration, which, however, comprised only approximately 50% of what would be required to sustain efflux by the antiporter. This suggests that either the conventional 'leak-pump' model of Na+ transport or the energetic model of proton-linked Na+ transport may require some revision. In addition, the lack of suppression of Na+ influx by both K+ and Ca2+, and by the application of the channel inhibitors Cs+, TEA+, and Ba2+, questions the participation of potassium channels and non-selective cation channels in the observed Na+ fluxes.