"Ryegrass staggers" is a neurological condition of unknown mechanism that impairs motor function in livestock. It is caused by infection of perennial ryegrass pastures by an endophytic fungus that produces neurotoxins, predominantly the indolediterpenoid compound lolitrem B. Animals grazing on such pastures develop uncontrollable tremors and become uncoordinated in their movement. Lolitrem B and the structurally related tremor inducer paxilline both act as potent large conductance calcium-activated potassium (BK) channel inhibitors. Using patch clamping, we show that their different apparent affinities correlate with their toxicity in vivo. To investigate whether the motor function deficits produced by lolitrem B and paxilline are due to inhibition of BK ion channels, their ability to induce tremor and ataxia in mice deficient in this ion channel (Kcnma1 Ϫ/Ϫ ) was examined. Our results show that mice lacking Kcnma1 are unaffected by these neurotoxins. Furthermore, doses of these substances known to be lethal to wild-type mice had no effect on Kcnma1 Ϫ/Ϫ mice. These studies reveal the BK channel as the molecular target for the major components of the motor impairments induced by ryegrass neurotoxins. Unexpectedly, when the response to lolitrem B was examined in mice lacking the 4 BK channel accessory subunit (Kcnmb4 Ϫ/Ϫ ), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK 4 subunit in motor control. The 4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.
The membrane potentials of aged, excised barley (Hordeum vulgare L.) root cells were rapidly depolarized by the addition of salicylic acid (o-hydroxybenzoic acid) The phenomenon of allelopathy, in which one species of plant may dramatically inhibit the development of others by producing and releasing specific compounds into the environment, is well documented (18). In a few cases, the allelopathic compounds and their mode of transmission have been elucidated (11,12). In a large number of reported cases, however, the modes of action of the allelopathic compounds (allelochemicals) are not understood. This paper reports further investigations into the mode of action of an important group of allelochemicals, phenolic acids.Earlier papers by Glass (3)(4)(5) have established that one ubiquitous group of phenolics, namely benzoic and cinnamic acids, are potent inhibitors of active K and Pi absorption by excised barley roots. The effect is readily reversible and the inhibitory capacity of the benzoic acids is strongly correlated with their lipid solubilities. From these results it was suggested that phenolics act directly on the cell membrane, modifying its permeability and thereby increasing the rate of efflux of ions. To test this hypothesis the effect of phenolics on cell membrane electrical potentials has been studied. According to the Goldman (7) where E is the electropotential difference (PD) between o, the outer solution and i, the inner solution; R, the gas constant (joules degree mole); T, the absolute temperature; F, the Faraday (coulombs equivalent-'); P subscript is the permeability coefficient of a univalent cation, j+, or a univalent anion, j-; C,+°, the concentration of j+ outside, and Cj,+, the concentration of j+ inside. Predictions of E based only upon K+ and Clcharacteristics, derived from the above equation, may under certain conditions be remarkably consistent with measured values for root cell membrane potentials (14). Clearly any alterations of the permeability properties of the cell membrane with regard to K+ or Cl-will be reflected by changes in E. If the cell membrane became extremely permeable, the imbalance of ion concentration across the cell membrane, which is necessary for E, would disappear and E would fall to near zero. MATERIALS AND METHODSSeedlings of Hordeum vulgare L. cv. Karlsberg were grown for 3 or 4 days in aerated 5 X 10-' M CaSO, solution following a 1-day germination in aerated distilled water. At the end of this period, the last 1 to 1.5 cm of roots were excised from the root tip and allowed to age in aerated 5 X 10-M CaSO4 solution. It was found that the aging process produced a very significant increase in the cell membrane potential of the order reported by Pitman et al. (14). Membrane potentials of cells in the root tips were measured with microcapillary electrodes using the apparatus described by Dunlop and Bowling (2). During determinations of membrane potentials the root tips were held in a 5 ml Plexiglas chamber which, under control conditions, contained a ...
The influx of K(+) into excised roots of barley (Hordeum vulgare L.) and ryegrass (Lolium multiflorum L.) previously grown with or without K(+) was measured in K(+) solutions ranging in concentration from 0.01 to 50 mM. In both species the K(+) influx was lower in the roots with high K(+) content. The extent of reduction by high internal [K(+)] decreased with external concentration above 1 mM. These results support the contention that at high external concentrations passive diffusion makes significant contributions to observed fluxes.
The effects of phosphorus status on the kinetics of phosphate absorption by 3-day-old Arabidopsis thaliana (L.) Heynh. seedlings were studied. Seeds were germinated for 3 days on agarose gel containing different amounts of phosphate and the absorption of 32P-labelled phosphate by these seedlings was measured at a range of phosphate concentrations. The absorption rates for seeds germinated in the absence of phosphate were up to four times faster than for seedlings germinated on gels containing 1, 5 or 10 mM phosphate. The kinetic constants Km and Vmax were determined by fitting the data to models with one and two mechanisms operating in parallel. A model with two mechanisms was needed to provide a good fit to the data for concentrations ranging from 0.5 µM to 5 mM. For the high affinity mechanism, Km,1 (mean value 12.3 µM ) was not affected as phosphorus status increased but Vmax,1 decreased. For the low affinity mechanism the values for both Km,2 (5697–16547 µM) and Vmax,2 increased with increasing phosphorus status. Arsenate inhibited phosphate absorption but only moderately. Varying the pH over the range 4.8 to 7.3 did not affect the rate of absorption. It is concluded that a kinetic approach may not have sufficient resolution to discriminate between different mechanisms unless there are large quantitative differences in the constants Kmor Vmax.
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