Assembly of plant Shaker-like K(out) channels requires two distinct sites of the channel alpha-subunit

SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K(+) channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K(out) channels. Deletion mutants and chimeric proteins generated from SKOR and the K(in) channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K(T) domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K(out) alpha-subunits did not assemble with K(in) alpha-subunits because of the absence of interaction between their assembly sites.     

Beticolins, nonpeptidic, polycyclic molecules produced by the phytopathogenic fungus Cercospora beticola, as a new family of ion channel-forming toxins

Beticolins are toxins produced by Cercospora beticola, a phytopathogenic fungus responsible for the leaf spot disease of sugar beet. They form a family of 20 nonpeptidic compounds (named B0 to B19) that share the same polycyclic skeleton but differ by isomeric configuration (ortho- or para-) and by a variable residue R (bridging two carbons in one of the six cycles). It has been previously shown that B0 assembles itself into a multimeric structure and forms ion channels into planar lipid bilayers (C. Goudet, A.-A. Very, M.-L. Milat, M. Ildefonse, J.-B. Thibaud, H. Sentenac, and J.-P. Blein, Plant J. 14:359-364, 1998). In the present work, we investigate pore formation by three ortho-beticolins, B0, B2, and B4, and their related (i.e., same R) para-isomers, B13, B1, and B3, respectively, using planar lipid bilayers. All beticolins were able to form ion channels with multiple conductance states, although the type of cyclization (ortho- or para-) and residue (R) result in variations of channel conductance and ionic permeability, respectively. Channel formation by beticolins is likely to be involved in the biological activity of these toxins.     

Organophosphorus insecticides synergize pyrethroids in the resistant strain of cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) from West Africa

Helicoverpa armigera (Hübner) populations from West Africa recently developed resistance to pyrethroid insecticides through enhanced metabolism by mixed-function oxidases. The combination index method was used to study the synergism of pyrethroids by organophosphorus insecticides. Several mixtures of insecticides currently registered to control cotton pest complex in West Africa were tested, including: cypermethrin/ethion, cypermethrin/profenofos, deltamethrin/ triazophos, deltamethrin/chlorpyriphos, cyfluthrin/chlorpyriphos, and betacyfluthrin/chlorpyriphos. In the resistant strain, the organophosphorus insecticides significantly increased the toxicity of pyrethroids suppressing the resistance effect, either by additive or synergistic effects. Significant synergism was shown for the following mixtures: cypermethrin/ethion, deltamethrin/triazophos, and deltamethrin/chlorpyriphos. The use of synergism from these insecticide mixtures should prove to be an additional tool in the overall resistance management strategy because the pyrethroid resistance in H. armigera from West Africa is not yet stable, decreasing between cotton seasons and increasing with treatments. In absence of selection, the susceptibility of H. armigera to insecticides should be restored.     

H Cotransports in Corn Roots as Related to the Surface pH Shift Induced by Active H Excretion

The surface pH shift induced by active H⁺ excretion in corn (Zea mays L.) roots was estimated using acetic acid influx as a pH probe (H Sentenac, C Grignon 1987 Plant Physiol 84: 1367-1372). At constant bulk pH, buffering the medium strongly reduced the magnitude of the surface pH shift. This was used to study the effect of surface pH shift on H⁺ cotransports. In the absence of buffers, the surface pH shift increased with the bulk pH. Buffers decreased ³²Pi influx and this effect was stronger at pH 7.2 than at pH 5.8, and stronger in the absence than in the presence of an inhibitor of the proton pump (vanadate). Buffers exerted a similar depressive and pH-dependent effect on net NO₃⁻ uptake. They hyperpolarized the cell membrane, and stimulated ⁸⁶Rb⁺ influx, K⁺:H⁺ net exchange, and malate accumulation. These results are consistent with the hypothesis that H⁺ accumulation at the cell surface is effective in driving H⁺ reentry. We concluded that the surface pH shift due to proton pump activity is involved in the energetic coupling of H⁺ cotransports.     

Evidence for a Multi-ion Pore Behavior in the Plant Potassium Channel KAT1

KAT1 is a cloned voltage-gated K⁺ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of `anomalous' rectification of the i <sub>f</sub> pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb<sup>+</sup> solution than in pure K<sup>+</sup> solution, and negligible in pure Na<sup>+</sup> solution. Experiments in different K<sup>+</sup>/Na<sup>+</sup> or K<sup>+</sup>/Rb<sup>+</sup> mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K<sup>+</sup>/Rb<sup>+</sup> mole fraction (i.e., the ratio [K<sup>+</sup>]/([K<sup>+</sup>]+[Rb<sup>+</sup>])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called `foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K⁺/Rb⁺ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in P_Rb/P_K. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical `green' member of the Shaker superfamily of K⁺ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies. Received: 9 February 1998/Revised: 28 July 1998     

Role of apoplast acidification by the H+ pump

We have studied the mechanism of the response to iron deficiency in rape (Brassica napus L.), taking into account our previous results: net H⁺ extrusion maintains a pH shift between the root apoplast and the solution, and the magnitude of the pH shift decreases as the buffering power in the solution increases. The ferric stress increased the ability of roots to reduce Fe[III]EDTA. Buffering the bulk solution (without change in pH) inhibited Fe[III]EDTA reduction. At constant bulk pH, the inhibition (ratio of the Fe[III]EDTA-reduction rates measured in the presence and in the absence of buffer) increased with the rate of H⁺ extrusion (modulated by the length of a pretreatment in 0.2 mM CaSO₄). These results support the hypothesis that the apoplastic pH shift caused by H⁺ excretion stimulated Fe[III] reduction. The shape of the curves describing the pH-dependency of Fe[III]EDTA reduction in the presence and in the absence of a buffer fitted this hypothesis. When compared to the titration curves of Fe[III]citrate and of Fe[III]EDTA, the curves describing the dependency of the reduction rate of these chelates on pH indicated that the stimulation of Fe[III] reduction by the apoplastic pH shift due to H⁺ excretion could result from changes in electrostatic interactions between the chelates and the fixed chargers of the cell wall and-or plasmalemma. Blocking H⁺ excretion by vanadate resulted in complete inhibiton of Fe[III] reduction, even in an acidic medium in which there was neither a pH shift nor an inhibitory effect of a buffer. This indicates that the apoplastic pH shift resulting from H⁺ pumping is not the only mechanism which is involved in the coupling of Fe[III] reduction to H⁺ transport. Our results shed light on the way by which the strong buffering effect of HCO ₃ ^- in some soils may be involved in iron deficiency encountered by some of the plants which grow in them.     

Molecular determinants of the Arabidopsis AKT1 K+ channel ionic selectivity investigated by expression in yeast of randomly mutated channels

The Arabidopsis thaliana K⁺ channel AKT1 was expressed in a yeast strain defective for K⁺ uptake at low K⁺ concentrations (<3 m M ). Besides restoring K⁺ transport in this strain, AKT1 expression increased its tolerance to salt (NaCl or LiCl), whatever the external K⁺ concentration used (50 μ M , 5 m M , or 50 m M ). We took advantage of the latter phenomenon for screening a library of channels randomly mutated in the region that shares homologies with the pore forming domain (the so-called P domain) of animal K⁺ channels (Shaker family). Cassette mutagenesis was performed using a degenerate oligonucleotide that was designed to ensure, theoretically, a single mutation per P cassette. The mean number of amino acid exchanges per cassette turned out to be 1.4. Mutant channels that conferred on the transformed cells a reduction in salt tolerance (increased Na⁺ content, decreased K⁺ content, and lower growth rate, as compared to control cells expressing the wild-type channel) were selected. By co-expressing them with the wild-type AKT1 cDNA, it was shown that the mutated polypeptides were expressed, stable and correctly targeted to the cell membrane where they formed channels with altered properties. Analysis of the mutation distribution in these channels suggests that the AKT1 P domain has a structure similar to that of animal Shaker channels (a strongly constrained central region lining the tunnel that includes the highly conserved consensus motif TXXTXGYGD, and flanking regions forming the outer mouth of the pore), with an additional selectivity filter located upstream from the tunnel and formed by residues present in the N-terminal flanking region.     

Role of oxidative stress in cardiac dysfunction of PPARalpha-/- mice

This study was designed to determine the effects of PPARalpha lack on cardiac mechanical performance and to identify potential intracellular mechanisms linking PPARalpha pathway deficiency to cardiac contractile dysfunction. Echocardiography, ex vivo papillary muscle assays, and in vitro motility assays were used to assess global, intrinsic ventricular muscle performance and myosin mechanical properties, respectively, in PPARalpha(-/-) and age-matched wild-type mice. Three-nitrotyrosine formation and 4-hydroxy-2-nonenal protein-adducts, both markers of oxidative damage, were analyzed by Western blot analysis and immunolabeling. Radical scavenging capacity was analyzed by measuring protein levels and/or activities of the main antioxidant enzymes, including catalase, glutathione peroxidase, and manganese and copper-zinc superoxide dismutases. Echocardiographic left ventricular fractional shortening in PPARalpha(-/-) was 16% lower than that in wild-type. Ex vivo left ventricular papillary muscle exhibited reduced shortening velocity and isometric tension (three- and twofold, respectively). In vitro myosin-based velocity was approximately 20% slower in PPARalpha(-/-), indicating that myosin itself was involved in the contractile dysfunction. Staining of 3-nitrotyrosine was more pronounced in PPARalpha(-/-), and myosin heavy chain was the main nitrated protein. Formation of 3-nitrotyrosine myosin heavy chain was twofold higher in PPARalpha(-/-) and 4-hydroxy-2-nonenal protein-adducts were threefold higher. The expression and activity of manganese superoxide dismutase were respectively 33% and 50% lower in PPARalpha(-/-), with no changes in copper-zinc superoxide dismutase, catalase, or glutathione peroxidase. These findings demonstrate that PPARalpha pathway deficiency impairs cardiac function and also identify oxidative damage to myosin as a link between PPARalpha deficiency and contractile dysfunction.     

Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF‐α

Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14⁺ blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human‐induced pluripotent stem cell‐derived RPE cells, we show that activated Mos markedly inhibit RPEOTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF‐α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF‐α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age‐related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD.