James Dunlop

The Molecular Mechanism of “Ryegrass Staggers,” a Neurological Disorder of K+ Channels

“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 indole-diterpenoid 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-/-) 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-/- 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 β4 BK channel accessory subunit (Kcnmb4-/-), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK β4 subunit in motor control. The β4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.

Phosphate absorption by Arabidopsis thaliana: interactions between phosphorus status and inhibition by arsenate.

The effects of phosphorus status and arsenate on the absorption of phosphate by roots of intact sterile seedlings of Arabidopsis thaliana were studied by analysing the rate of depletion of phosphate from solutions initially containing 10 M KH2PO4. Depletion of phosphate from the experimental solutions was measured both chemically and by labelling with 32P. There was a substantial efflux of phosphate coincident with a rapid influx of phosphate, with efflux increasing with increasing phosphorus status. The highest rates of absorption were obtained for the plants initially grown with a high level of phosphorus but then deprived of phosphate for 5 d prior to the experiments, with the next highest rates obtained for the most phosphorus-deficient plants. Kinetic analysis suggests that changes in both the affinity and capacity of the absorption mechanism contribute to differences in the rate of phosphate influx between plants of different phosphorus status. Arsenate as 20 M KH2AsO4 inhibited phosphate influx in a manner such that all plants, regardless of their phosphorus status, had the same phosphate influx rate. This was reflected in identical values for the Michaelis constant, Km, and maximum velocity as used in Michaelis–Menten kinetics, Vmax. Arsenate had its greatest effect on phosphate movement to the shoot. The simultaneous elimination of differences in phosphate influx between plants of different phosphorus status suggest that phosphate movement to the shoot may be important in the regulation of influx by phosphorus status.

Bilayer lipid membranes supported on Teflon filters: a functional environment for ion channels.

Many ion channel proteins have binding sites for toxins and pharmaceutical drugs and therefore have much promise as the sensing entity in high throughput technologies and biosensor devices. Measurement of ionic conductance changes through ion channels requires a robust biological membrane with sufficient longevity for practical applications. The conventional planar BLM is 100-300 μm in diameter and typically contains fewer than a dozen channels whereas pharmaceutical screening methods in cells use current recordings for many ion channels. We present a new, simple method for the fabrication of a disposable porous-supported bilayer lipid membrane (BLM) ion channel biosensor using hydrated Teflon (polytetrafluoroethylene, PTFE) filter material (pore size 5 μm, filter diameter=1 mm). The lipid layer was monitored for its thickness and mechanical stability by electrical impedance spectroscopy. The results showed membrane capacitances of 1.8±0.2 nF and membrane resistances of 25.9±4.1 GΩ, indicating the formation of lipid bilayers. The current level increased upon addition of the pore-forming peptide gramicidin. Following addition of liposomes containing voltage-gated sodium channels, small macroscopic sodium currents (1-80 pA) could be recorded. By preloading the porous Teflon with sodium channel proteoliposomes, prior to BLM formation, currents of 1-10 nA could be recorded in the presence of the activator veratridine that increased with time, and were inhibited by tetrodotoxin. A lack of rectification suggests that the channels incorporated in both orientations. This work demonstrates that PTFE filters can support BLMs that provide an environment in which ion channels can maintain their functional activity relevant for applications in drug discovery, toxin detection, and odour sensing.

Structural determinants of lolitrems for inhibition of BK large conductance Ca2+-activated K+ channels

Lolitrem B is an indole-diterpenoid neurotoxin which is the main causative agent of ryegrass staggers, an animal disease associated with tremors and incoordination. It is also a potent inhibitor of large conductance calcium-activated potassium (BK) channel activity (IC(50)=4 nM). Furthermore, we have recently shown that the motor function deficits induced by lolitrem B are specifically mediated by BK channels, making the toxin a valuable tool for investigating the molecular function and physiological roles of these channels. To determine what structural features of BK channel agents are required for high potency, the effect of lolitrem B and seven structurally-related lolitrems on BK channel activity has been measured. Concentration-responses and conductance-voltage (G-V) relationships were determined for each compound and related to the different structure types. This study has identified seven new BK channel inhibitors and has allowed the identification of two key structural features required for high potency BK channel activity by lolitrems.

Inheritance of phosphorus response in white clover (Trifolium repens L.)

Genotypes of white clover that exhibited divergent responses to P were identified in a glasshouse pot trial. Six high P-responding genotypes were selected from previously identified high P-responding cultivars and 5 low P-responding genotypes were selected from previously identified low P-responding cultivars. These were crossed in a full diallel design without selfing and reciprocals were kept separate. The P-response of progeny lines was compared with parents. High P-response was dominant over low P-response with progeny from crosses between high and low P-response genotypes being similar to the high P-response parent. Reciprocal effects were not significant. The general combining abilities of high P-response genotypes were generally greater than that of the low P-response genotypes, although there were significant specific combining abilities. Narrow sense heritabilities for P response were moderate, 0.46 based on the linear coefficient and 0.33 based on the quadratic coefficient of the fitted response curves.

Response to phosphorus of a world collection of white clover cultivars

White clover (Trifolium repens L.) is the predominant legume of temperate pastures. Nitrogen (N) fixed by white clover is essential for maintaining the production of grasses in agricultural systems where N fertilisers are not used. However, white clover is a poor competitor for soil phosphate (P). This is of particular significance when many soils in New Zealand and throughout the world are P deficient. The increasing cost of manufacture and application of P fertilisers has stimulated a breeding programme to develop a white clover with improved P nutrition.

Relationships between shoot and root characteristics of white clover cultivars differing in response to phosphorus

Abstract Root dry weight and the relationship between shoot and root characteristics for 119 white clover (Trifolium repens L.) cultivars and breeding lines were examined at five (40, 120, 200, 400, and 500 mg P/kg soil) phosphorus (P) levels. There were significant (P<0.01) differences among cultivars for root dry weight and root/shoot ratio. Instantaneous root/shoot ratio decreased with increasing P supply up to 200 mg P/kg, and then increased again from 400 to 500 mg P/kg soil. Allometric analysis indicated that root growth relative to shoot growth increased with increasing P supply. There was a significant (P<0.01) cultivar x P level interaction such that for the cultivars, the root/shoot ratio was either unaffected, increasing, or decreasing with increasing level of P supply. However, root/shoot ratio appeared to be unrelated to the functions used to describe the P response of the 119 cultivars studied. Shoot characters such as dry weight, leaf number, and stolon length were most closely correlated w...

Transporter genes to enhance nutrient uptake: Opportunities and challenges

Recently, there has been very rapid progress in understanding the transport of mineral nutrients across plant membranes. Genes for a number of primary ion pumps, cotransporters and ion channels have been cloned and the characteristics of their function are being investigated. While these advances have yet to produce cultivars that are better able to cope with nutrient deficient soils, they provide powerful tools to address some important gaps in our knowledge, particularly the regulation of transporter genes. While the current focus is on nutrient influx into roots, other processes are also significant in determining nutrient acquisition. This is illustrated by research on the relationship between the high affinity phosphate absorption mechanism and phosphate transport to the shoot, and with data on relating to the importance of phosphate efflux relative to influx. Molecular biology and plant physiology are providing information and technology that will be valuable in improving the suitability of crops for nutrient deficient environments. However, there is an urgent need for new ways to integrate this information so that the significance of individual processes to the performance of the whole system can be understood.

Mechanism of action of lolitrem B, a fungal endophyte derived toxin that inhibits BK large conductance Ca2+-activated K+ channels

The aim of this study was to compare the mode of action of the commonly used BK inhibitor paxilline with that of the more recently discovered lolitrem B. Similarities and differences in characteristics of inhibition between the two compounds were investigated. We have previously shown that lolitrem B does not affect the BK channel G-V, in contrast to the rightward shift produced by paxilline. These different effects on the voltage-dependence of activation suggest different modes of action for these two compounds. In this study we show that inhibition by both paxilline and lolitrem B is characterized by an open state preference for BK (hSlo) channels. Both compounds had a 3-fold higher apparent affinity under conditions likely to favour the open state, suggesting they have a similar BK conformational preference for binding. Furthermore, both compounds had a calcium concentration-dependence to their inhibitory effects. The G-V shift induced by paxilline was calcium concentration-dependent.

Physiological responses of wheat phosphorus-efficient and -inefficient genotypes in field and effects of mixing other nutrients on mobilization of insoluble phosphates in hydroponics

Abstract To elucidate the mechanism of mobilization of insoluble phosphates, a field trial and a split root experiment in a growth chamber were conducted. The results showed that a phosphorus (P)‐efficient wheat genotype, Yanzhong 144 (YZ), transpired 50% less than a P‐inefficient genotype, 80‐55. At the grain‐filling stage, the free water content in YZs rhizosphere was 2.4 times of that of 80‐55s. When either tricalcium phosphate [Ca3(PO4)2] or ferric phosphate (FePO4) were combined with the other nutrients in the same solution, total dry‐matter production was 140% greater for Ca3(PO4)2 and 60% greater for FePO4 than when these P sources were each supplied alone to one half of the root system while the other nutrients were supplied to the other half. The excess absorption of cations over anions by the roots facilitated mobilization of insoluble phosphates. The P‐efficient genotype was also water efficient, and the insoluble phosphates TCP and FP were phyto‐available in water culture.