Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

α2-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

A proper transmembrane Ca2+ gradient is essential for the stimulation of adenylate cyclase activity by stimulatory GTP-binding protein

Stimulatory GTP-binding Protein (Gs) and adenylate cyclase prepared from bovine brain cortices were co-reconstituted into asolectin vesicles with or without 1000-fold transmembrane Ca²⁺ gradient. The results showed that both basal activity and Gs-stimulated activity of adenylate cyclase were highest in proteoliposomes with a transmembrane Ca²⁺ gradient similar to physiological condition (1 M Ca²⁺ outside and 1 mM Ca²⁺ inside) and lowest when the transmembrane Ca²⁺ gradient was in the inverse direction. Such a difference could be diminished following dissipation of the transmembrane Ca²⁺ gradient by A23187. Comparable conformational changes of Gs in proteoliposomes were also observed when Gs was labeled with the fluorescence probe, acrylodan. These results may indicate that a proper transmembrane Ca²⁺ gradient is essential not only for higher adenylate cyclase activity but also for its stimulation by Gs.     

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).     

Structure and functions of arrestins.

Transmembrane signal transductions in a variety of cell types that mediate signals as diverse as those carried by neurotransmitters, hormones, and sensory signals share basic biochemical mechanisms that include: (1) an extracellular perturbation (neurotransmitter, hormone, odor, light); (2) specific receptors; (3) coupling proteins, such as G proteins; and (4) effector enzymes or ion channels. Parallel to these amplification reactions, receptors are precisely inactivated by mechanisms that involve protein kinases and regulatory proteins called arrestins. The structure and functions of arrestins are the focus of this review.