Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots

The aim of the present work was to characterize Na(+) currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca(2+) by digesting tissue in elevated Ca(2+). Experiments in whole-cell and outside-out modes were carried out. We found that Na(+) currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium(+) and verapamil, had no time-dependent activation (permanently opened or completely activated within 1-2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K(+) (1.49) > NH(4)(+) (1.24) > Rb(+) (1.15) approximately equal to Cs(+) (1.10) approximately equal to Na(+) (1.00) > Li(+) (0.73) > tetraethylammonium(+) (0.47). Arabidopsis root NSCCs were blocked by H(+) (pK approximately equal to 6.0), Ca(2+) (K(1/2) approximately equal to 0.1 mM), Ba(2+), Zn(2+), La(3+), Gd(3+), quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca(2+)-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na(+) influx to the cell and probably having other important roles in physiological processes of plants.     

Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots

The polypeptide complement of enriched soybean (Glycine max [L.] Merr. cult. wells) root plasma membrane fractions was studied by two-dimensional gel electrophoresis. Good resolution was obtained when polypeptides were solubilized in sodium dodecyl sulfate and when butylated hydroxytoluene was included in the vesicle isolation and solubilization media. The pattern obtained on the two-dimensional slab gel for root plasma membrane was characteristic for that membrane. The polypeptide complements from mitochondrial membranes and from enriched fractions of three other endomembrane components were solubilized and electrophoresed for comparison. Each membrane preparation was identifiable on the basis of its characteristic electrophoretogram. Electrophoresis of protein solubilized from plasma membrane fractions isolated from meristematic and mature root tissue revealed both qualitative and quantitative differences in the respective protein complements.     

Phytochrome and calcium ions are involved in light-induced membrane depolarization in Nitella

Isolated internodes of Nitella (N. opaca, N. flexilis) and Nitellopsis spec. were punctured with single microelectrodes and their membrane potentials were recorded continuously during various light treatments. In red light the initial response was always a depolarization. This depolarization began with a lag-time of 0.4-3.5s and reached a steady state within 1–2 min of continuous illumination. Repolarization began within several seconds after turning off the light. The magnitude of the red-light-induced depolarization increased with the Ca²⁺-concentration of the medium. The largest depolarizations were recorded in 5 m mol l^-1 Ca²⁺. Ca²⁺ could not be replaced in this function by Na⁺, Mg²⁺, La³⁺ or mannitol. Far-red light alone had no effect on the resting membrane potential. Far-red light applied immediately after red light accelerated the repolarization of the membrane potential. Far-red light applied simultaneously with red light reduced the amount of depolarization and increased the rate of repolarization. The results indicate that phytochrome and Ca²⁺ are involved in the light-induced depolarization of the membrane. They are consistent with the hypothesis that phytochrome may act by triggering a Ca²⁺-influx at the plasma membrane.Abbreviations APW artificial pond water - Pfr far-red absorbing form of phytochrome - DCMU 3-(3,4-Dichlorphenyl)-1,1-dimethylurea     

Regulation of plasma membrane calcium fluxes by mitochondria

The role of mitochondria in cell signaling is becoming increasingly apparent, to an extent that the signaling role of mitochondria appears to have stolen the spotlight from their primary function as energy producers. In this chapter, we will review the ionic basis of calcium handling by mitochondria and discuss the mechanisms that these organelles use to regulate the activity of plasma membrane calcium channels and transporters.     

Chlorpromazine induces membrane potential depolarization and ATP loss,and inhibits microsomal ATPase in soybean (Glycine Max L.) roots

In intact soybean roots, chlorpromazine causes a depolarization of the membrane potential at low concentrations (as low as 30 μM, half-maximally at about 150 μM), and induces a marked decrease in ATP levels at higher concentrations (half-maximal at about 0.5 mM) over longer periods of time. In root microsomal suspensions, chlorpromazine inhibits an apparently specific ATPase activity component (half-maximally at about 0.3 mM). Chlorpromazine inhibits $\text{N,N′-}\text{dicyclohexylcarbodiimide}$-, diethylstilbesterol- and azide-inhibited ATPase activities. On linear sucrose gradients, chlorpromazine inhibition of ATPase activity occurs in two peaks, at 1.12 g/ml and 1.14–1.17 g/ml, which may represent a tonoplast and plasma membrane ATPase, respectively. Neither peak corresponds to the F₁ ATPase. It is unclear whether ATPase inhibition or ATP loss is the cause of the membrane potential depolarization. Clearly chlorpromazine has multiple effects which are probably unrelated to its calmodulin-inhibition activity.     

Phospholipid composition of a plasma membrane-enriched fraction from developing soybean roots

Phospholipid polar head group and fatty acid composition were determined for plasma membrane enriched fractions from developing soybean root (Glycine max [L.] Merr. cult. Wells II). Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots at pH 7.8 and in the presence of 5 millimolar ethylenediaminetetraacetate, 5 millimolar ethyleneglycol-bis (β-aminoethyl ether)N,N tetraacetic acid, and 10 millimolar NaF. Lipid extracts analyzed for phospholipid composition revealed two major phospholipid components: phosphatidylcholine and phosphatidylethanolmine. Minor phospholipid components identified were phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and diphosphatidylglycerol. Lipid degradation by endogenous phospholipase D during membrane isolation at pH 6.5 and in the absence of chelating agents and NaF resulted in the recovery of large amounts of phosphatidic acid. Phosphatidylcholine was the principal substrate for phospholipase D.     

Cation selectivity of the plasma membrane of tobacco protoplasts in the electroporated state

Cation selectivity of the cellular membrane of tobacco culture cells (cell line ‘bright yellow-2’) exposed to pulsed electric fields in the millisecond range was investigated. The whole cell configuration of the patch clamp technique was established on protoplasts prepared from these cells. Ion selectivity of the electroporated membrane was investigated by measuring the reversal potential of currents passing through field-induced pores. To this end the membrane was hyper- or depolarized for 10 ms (prepulse); subsequently the voltage was driven to opposite polarity at a constant rate (+ 40 or ? 40 mV/ms, respectively). The experiment was started by polarizing the membrane to moderately negative or positive voltages (prepulse potential ± 150 mV) that would not induce pore formation. Subsequently, an extended voltage range was scanned in the porated state of the membrane (prepulse potential ± 600 mV). IV curves in the porated and the non-porated state (obtained at the same prepulse polarity) were superimposed to determine the voltage at which both curves intersected (‘Intersection potential’). Using a modified version of the Goldmann–Hodgkin–Katz equation relative permeabilities to Ca^2 + and various monovalent alkali and organic cations were calculated. Pores were found to be fairly cation selective, with a selectivity sequence determined to be Ca^2 + > Li⁺ > Rb⁺ ≈ K⁺ ≈ Na⁺ > TEA⁺ ≈ TBA⁺ > Cl^?. Relative permeability to monovalent cations was inversely related to the ionic diameter. By fitting a formalism suggested by Dwyer at al. (J. Gen. Physiol. 75 (1980), 469–492) the effective average diameter of field induced pores was estimated to be about 1.8 nm. Implications of these results for biotechnology and electroporation theory are discussed.     

Cloning of putative subunits of the soybean plasma membrane NADPH oxidase involved in the oxidative burst by antibody expression screening

Summary Plant cells respond to a variety of external signals with the production of reactive-oxygen species. The enzyme system generating these reactive-oxygen species is believed to be an NADPH oxidase located in the plasma membrane and sharing similarities with the NADPH oxidase from mammalian macrophages. Antibodies directed against individual subunits (p22^phox, p47^phox, p67^phox) of the human NADPH oxidase cross-react with soybean proteins of a similar size and subcellular location. An extensive expression screening of a soybean cDNA-library with the anti-human NADPH oxidase antibodies gave a single class of cDNA-clones for each antibody. However, the sequence analysis of these clones clearly demonstrates that the different antibodies recognise proteins which are unrelated to the expected oxidase subunits. The anti-p22^phox antibody recognised a microsomal protein with no significant homology to any known protein in the database. One anti-p47^phox antibody cross-reacted with the UDP-glucose dehydrogenase and another antibody bound to the chaperon peptidyl prolyl-cis-trans isomerase, both soluble cytosolic proteins. The anti-p67^phox antibody detected the soluble enzyme acetohydroxy acid reductoisomerase. Chromatography of soybean protein extracts on an ion-exchange column (MonoQ, FPLC) gave a perfect comigration of the enzyme activity with the antibody signal, thus confirming these unexpected results by independent biochemical experiments.Abbreviations AARI acetohydroxy acid reductoisomerase - DPI diphenylene iodonium - GST glutathione-S-transferase - phox NADPH oxidase of phagocytes - ROS reactive-oxygen species     

Comparisons of plasma membrane polypeptides from soybean and alfalfa

Polypeptides were solubilized with sodium dodecyl sulfate from plasma membrane vesicles of eight varieties of soybean roots [Glycine max (L.) Merr.] and of cultured alfalfa cells (Medicago sativa L.). The solubilized polypeptides were analysed by 2D-polyacrylamide gel electrophoresis. Apparent isoelectric point and MW values were obtained for 80 soybean plasma membrane polypeptides and 44 alfalfa plasma membrane polypeptides. From these data composite distribution patterns were constructed, which are representative of the soybean or alfalfa 2D-gels, respectively. The results showed that the general polypeptide staining patterns were similar for all the soybean varieties, but some minor differences were evident. The alfalfa electrophoretograms differed markedly from the soybean electrophoretograms in specific details, though some general pattern similarities were noted. The data are discussed in terms of a physiological role for the integral plasma membrane polypeptides and in terms of the potential for distinguishing among soybean varieties and between species at the plasma membrane polypeptide level.     

Oxygen-sensitivity of potassium fluxes across plasma membrane of cerebellar granule cells

This study focuses on the oxygen-dependence of active and passive K⁺ fluxes across membranes of cerebellar granule cells of neonatal rats. Maximal Na⁺,K⁺-ATPase activity along with minimal passive K⁺ influx was observed within oxygen concentration range characteristic for neonatal rat cerebellum. Prolonged exposure to hypoxia as well as hyperoxia resulted in suppression of the Na⁺,K⁺-ATPase and activation of the passive K⁺ flux. Toxic effects of hypoxia could be partially prevented by inhibition of NO production with L-NAME. This was accomplished by suppression of Na⁺,K⁺-ATPase with subsequent reduction in ATP consumption concurrently with the reduction in passive K⁺ flux. Activation of the Na⁺,K⁺-ATPase by NO at physiological pO₂ could be abolished by inhibition of NO synthase by L-NAME or soluble guanylyl cyclase with ODQ. However, treatment of cells with activator of PKG Rp-8-CTP did not mimic normoxic activation of the active K⁺ influx. Oxygen-induced responses under normoxic conditions were differentially mediated by α1 isoform of the Na⁺,K⁺-ATPase catalytic subunit, whereas α2/3 isoform was predominantly active under conditions of severe hypoxia. We conclude that both hypoxia and hyperoxia trigger a gradual dissipation of transmembrane K⁺ gradient and loss of excitability of cerebellar neurons. The latter may be partially reversed by suppression of NO production under hypoxic conditions     

ADAMs参与哺乳动物精-卵结合与融合

ADAMs家族是含多结构域的跨膜蛋白。睾丸特异的ADAMs,在精子发生与附睾精子转运过程中,经过蛋白水解成为成熟精子的分子形式,与精．卵质膜结合和融合有关。对于精-卵质膜相互作用,ADAMs去整合素域具有关键氨基酸残基和特殊模体。模拟ADAM2和ADAM3去整合素域的短肽能用于鉴别特异性卵子识别蛋白。精子ADAMs去整合素域与卵子膜蛋白整合素β1、α4／α9、α6和CD9相互作用,介导了精卵质膜的结合与融合。     

Electrical properties of soybean plasma membrane measured in heterotrophic suspension callus

Previous work on heterotrophic suspension-cultured cells has failed to detect the electrogenic processes normally associated with the plasma membranes of non-animal cells. This study reports measurements on heterotrophic cells from soybean (Glycine max L.) suspension cultures, which are shown to be amenable to impalement with microelectrodes. The plasma membrane clearly exhibits fundamental characteristics which are common to many other plant cell types: (i) a resting membrane potential significantly more negative than-100mV (measured value:121±4mV); (ii) obvious electrogenic activity, as evidenced by the marked depolarization of the membrane (87±6mV) by cyanide, and by the fact the membrane potential was frequently more negative than the equilibrium potential for K⁺; (iii) a finite permeability to K⁺ ions; (iv) electrophoretic transport of glucose. The development of a recording medium consisting primarily of 1:5 diluted growth medium was critical for successful impalement of these cells. It is proposed that the novel identification of electrogenic processes in heterotrophic suspension-cultured cells results from the deployment of electrodes with relatively dilute filling solutions, thus avoiding substantial changes in intracellular ion concentrations.The overwhelming majority of cells in soybean suspension cultures exist in small clusters, and the possibility of intercellular coupling potentially precludes assessment of membrane specific resistance and current density. Furthermore, as with most higher-plant cells, the vacuole occupies a large fraction of the intracellular volume. However, a model in which the measuring electrode is cytosolically located and the cells are electrically well-coupled is the only one which satisfactorily generates values for membrane specific resistance in a manner which is not strongly dependent on the number of cells in the cluster: other models in which the electrode tip is located in the vacuole and-or the impaled cell is electrically isolated from the others do not seem to apply. The measured values of membrane specific resistance are in the range 5.4 to 8.4 ·m², which is in excellent agreement with comparable measurements on other plant and fungal cells. The results are discussed with respect to mechanisms of transmembrane signalling in soybean, as well as to general electrophysiological studies on higher-plant cells in suspension culture and in tissues.Abbreviations and symbols R_m membrane resistance - r_p plasma-membrane resistivity - SRB Soybean Recording Buffer - V_m membrane potential     

Binding site for chitin oligosaccharides in the soybean plasma membrane.

Affinity cross-linking of the plasma membrane fraction to an (125)I-labeled chitin oligosaccharide led to the identification and characterization of an 85-kD, chitin binding protein in plasma membrane-enriched fractions from both suspension-cultured soybean cells and root tissue. Inhibition analysis indicated a binding preference for larger (i.e. degrees of polymerization = 8) N-acetylated chitin molecules with a 50% inhibition of initial activity value of approximately 50 nM. N-Acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 microM. It is noteworthy that the major lipo-chitin oligosaccharide Nod signal produced by Bradyrhizobium japonicum was also shown to be a competitive inhibitor of ligand binding. However, the binding site appeared to recognize the chitin portion of the Nod signal, and it is unlikely that this binding activity represents a specific Nod signal receptor. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity. Taken together, the presence of the chitin binding protein in the plasma membrane fraction and the specificity and induction of a biological response upon ligand binding suggest a role for the protein as an initial response mechanism for chitin perception in soybean (Glycine max).     

Anion channel activation and proton pumping inhibition involved in the plasma membrane depolarization induced by ABA in Arabidopsis thaliana suspension cells are both ROS dependent

In Arabidopsis thaliana suspension cells, ABA was previously shown to promote the activation of anion channels and the reduction of proton pumping that both contribute to the plasma membrane depolarization. These two ABA responses were shown to induce two successive [Ca(2+)](cyt) spikes. As reactive oxygen species (ROS) have emerged as components of ABA signaling pathways especially by promoting [Ca(2+)](cyt) variations, we studied whether ROS were involved in the regulation of anion channels and proton pumps activities. Here we demonstrated that ABA induced ROS production which triggered the second of the two [Ca(2+)](cyt) increases observed in response to ABA. Blocking ROS generation using diphenyleneiodonium (DPI) impaired the proton pumping reduction, the anion channel activation and the RD29A gene expression in response to ABA. Furthermore, H(2)O(2) was shown to activate anion channels and to inhibit plasma membrane proton pumping, as did ABA. However, ROS partially mimicked ABA's effects since H(2)O(2) treatment elicited anion channel activation but not the subsequent expression of the RD29A gene as did ABA. This suggests that expression of the RD29A gene in response to ABA results from the activation of multiple concomitant signaling pathways: blocking of one of them would impair gene expression whereas stimulating only one would not. We conclude that ROS are a central messenger of ABA in the signaling pathways leading to the plasma membrane depolarization induced by ABA.     

Cation channels in the plasma membrane of retinal rod outer segments activated by cGMP

Properties of cGMP-activated cation channels were investigated on isolated patches of the ROS plasma membrane using the "patch clamp" technique. The channels were shown to be characterized by ideal cation selectivity under physiological conditions and are nearly equally permeable for cations of alkaline metals. At the same time they are permeable for some bivalence cations (PNa approximately PCa). Other channel properties are described and their comparative analysis is given. It suggests that cGMP-activated cation channels represent a new type of cation channels.     

Pump-leak sodium fluxes in low salt corn roots

Summary The influx and efflux of sodium from 4-hr washed, low salt corn roots (Zea mays L.) has been studied for characterization of passive and active components. Initial Na⁺ content of the roots is very low, 2.25±0.4 mol/g fresh weight. Na⁺ influx in the presence of 0.2mm Ca²⁺ and 0.002 to 20mm K⁺ is passive (a leak) based upon Goldman-type models, being determined by Na⁺ and cell potential (). Na⁺ was not transported by the K⁺ carrier and influx was unaffected by 50 m dicyclohexylcarbodiimide (DCCD). Permeability of the cells to Na⁺ was of the same order asP _k.Efflux of Na⁺ was by an efficient and rapid active transport system (a pump), thus accounting for the failure of these roots to accumulate high levels of Na⁺. In short-term loading and efflux experiments, internal Na⁺ turnover had a half-time of about 5 min. Sodium efflux was unaffected by DCCD. Net H⁺ flux was zero in the presence of DCCD regardless of sodium efflux, indicating absence of Na⁺/H⁺ antiport. Efflux of Na⁺ was equally rapid into medium containing no Na⁺ and only 0.002mm K⁺. K⁺ influx accounted for less than 4% of Na⁺ efflux, prompting the hypothesis that the Na⁺ (or cation?) efflux pump is the second electrogenic system previously defined based upon electrophysiological measurements.