Using planar lipid-bilayers to study plant ion channels

Ion channels are found in most plant membranes. They catalyse the rapid passive uniport of particular ions with varying selectivity. Planar lipid-bilayer (PLB) techniques have been developed to study the electrical activities of single ion channels in well-defined lipid and aqueous environments. They greatly facilitate both the biophysical and biochemical characterisation of ion channels and complement both conventional impaling electrode and membrane-patch voltage-clamping (patch-clamping) electrophysiological techniques applied in vivo. Bilayers can be formed across the end of patch-clamp pipettes or across apertures in specifically designed chambers. Ion channels in native membranes and purified, genetically altered or synthetic ion channels, proteins and peptides can all be studied in PLBs. The main applications of PLBs are (1) to study ion channels in membranes inaccessible to patch-clamp electrodes, (2) to provide a functional assay system during channel-protein purification and (3) to investigate the relationship between the molecular structure of ion channels and their conductance properties. In the present article we describe the techniques available for reconstitution and analysis of ion channels in PLBs and discuss how the PLB technique has been, and may be, useful to the study of plant ion channels.     

Temperature and chemical shocks induce chilling tolerance in germinating Cucumis sativus (cv. Poinsett 76) seeds

Roots of 24-h-old germinated cucumber ( Cucumis sativus cv. Poinsett 76) seeds were subjected to thermal and chemical stresses, equilibrated at 25°C for 2 h and chilled at 2.5°C for 96 h. The germinated seeds were then held at 25°C for 72 h after they were chilled and the elongation of the primary root was used as a measure of chilling tolerance. Control roots elongated from an initial length of 0.2 cm to a final length of 6.3 cm at the end of 72 h. while chilled roots elongated to a final length of only 0.4 to 0.6 cm. Exposure to 0.4 M ethanol for 4 h or to 40°C for 1 h induced substantial chilling tolerance and the roots had a final length of 4.1 and 3.1 cm. respectively. Exposure to 7.5°C for 3 h conferred less chilling tolerance (elongation to 1.4 cm). while exposure to other chemicals (i.e. aqueous solutions of Ca(NO₃)₂, mannitol. methanol and NaCl) produced less, though still significant increases in chilling tolerance. A more severe chilling treatment of 144 h at 2.5°C was required to consistently induce elevated rates of ion leakage. Only the heat and the ethanol shock treatments significantly reduced chilling-induced ion leakage. Inclusion of the protein synthesis inhibitor cycloheximide negated the protective effects of these shock treatments. It appears that de novo protein synthesis is required for induction of chilling tolerance by a variety of chemical and thermal shock treatments.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Simultaneous extraction of soil phytoavailable cadmium,copper, and lead by chelating resin membrane

Cadmium, copper, and lead were extracted from suspensions of contaminated soils using metal chelating exchange resin membranes. Nine soils with widely varying properties and Cd, Cu and Pb levels were tested. Soil suspensions made up with 4 g in 40 mL deionized water were equilibrated with 5 cm² Ca-saturated Chelex exchange resin membrane which was retained inside a polypropylene bag and shaken at 150 rpm for 24 hrs. Resin membrane extractable Cd, Cu and Pb of the soils were correlated with Cd, Cu, and Pb uptake by young wheat seedlings grown in these soils and compared with soil Cd, Cu, and Pb extracted by 0.1 M HCl, 0.01 M CaCl₂, and 0.005 M Diethylenetriamine pentaacetic acid (DTPA). The amounts of Cd, Cu and Pb extracted by the Ca-saturated Chelex membrane from all tested soils correlated well with those absorbed by young wheat seedlings. The Ca-saturated Chelex membrane extractable Cd, Cu and Pb of the soil had the strongest correlation with plant uptake Cd, Cu and Pb among the extraction methods we tested. It was demonstrated that the Ca-saturated Chelex membrane extraction is an appropriate method in simultaneously estimating Cd, Cu and Pb phytoavailability of soil and is applicable to a wide range of soils.     

Do salt bridges stabilize proteins? A continuum electrostatic analysis

The electrostatic contribution to the free energy of folding was calculated for 21 salt bridges in 9 protein X-ray crystal structures using a continuum electrostatic approach with the DELPHI computer-program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5 kcal/mol. This is fundamentally different from stability measurements using pKa shifts, which effectively measure the strength of a salt bridge relative to 1 or more charged hydrogen bonds. The calculated effect was due to a large, unfavorable desolvation contribution that was not fully compensated by favorable interactions within the salt bridge and between salt-bridge partners and other polar and charged groups in the folded protein. Some of the salt bridges were studied in further detail to determine the effect of the choice of values for atomic radii, internal protein dielectric constant, and ionic strength used in the calculations. Increased ionic strength resulted in little or no change in calculated stability for 3 of 4 salt bridges over a range of 0.1-0.9 M. The results suggest that mutation of salt bridges, particularly those that are buried, to "hydrophobic bridges" (that pack at least as well as wild type) can result in proteins with increased stability. Due to the large penalty for burying uncompensated ionizable groups, salt bridges could help to limit the number of low free energy conformations of a molecule or complex and thus play a role in determining specificity (i.e., the uniqueness of a protein fold or protein-ligand binding geometry).     

Optimization of the electrostatic interactions in proteins of different functional and folding type

The 3-dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge-charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge-charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed αβ folding type are electrostatically better optimized than pure α-helical or β-strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge-charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.     

Effect of penicillin on GABA-gated chloride ion influx

To investigate the mechanism of penicillin-induced convulsions, we have studied the effects of penicillin G (PC-G) on GABA-gated chloride ion influx in brain microsac preparations of mice. In the presence of 10^–4 M GABA, PC-G inhibited GABA-gated chloride ion influx in a dose-dependent manner. The dose-response curve for GABA in the presence of 10^–3 M PC-G was shifted rightward and there was a decrease in maximum response. The inhibitory effects of PC-G were not reversed by RO 15-1788, an antagonist of benzodiazepine (BZ) receptors, but were reversed by washing the microsac membranes. Therefore, PC-G probably exerts its proconvulsant effect by inhibiting GABA-gated chloride ion influx. However, it appears not to act through the BZ receptor of the GABA/BZ receptor complex.     

Effects of sub-minimal inhibitory concentrations of EDTA on growth of Escherichia coli and the release of lipopolysaccharide

Abstract Release of lipopolysaccharide from E. coli was studied in the presence of sub-minimal inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). In untreated cells no release was detected with 50 mM Mg²⁺ in the medium, but a steady release of over 50% of the synthesized lipopolysaccharide was observed with 0.1 mM Mg²⁺. EDTA at MIC/8 led to a 2- to 3-fold higher release, presumably by an adjustment of the concentration of unchelated Mg²⁺ to a value still sustaining normal growth but giving rise to a highly unstable outer membrane. No structural difference was observed between cell-bound and released lipopolysaccharide.     

短杆菌肽通道内离子K~＋，Na~＋，Li~＋与水的相关性

基于右手螺旋短杆菌肽Ａ离子通道模型,利用分子动力学计算机模拟方法研究了通道内离子Ｋ＋,Ｎａ＋,Ｌｉ＋与水分子的相关性．