Julie E. Dalziel

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.

A role for BK channels in heart rate regulation in rodents.

The heart generates and propagates action potentials through synchronized activation of ion channels allowing inward Na+ and Ca2+ and outward K+ currents. There are a number of K+ channel types expressed in the heart that play key roles in regulating the cardiac cycle. Large conductance calcium-activated potassium (BK) ion channels are not thought to be directly involved in heart function. Here we present evidence that heart rate can be significantly reduced by inhibiting the activity of BK channels. Agents that specifically inhibit BK channel activity, including paxilline and lolitrem B, slowed heart rate in conscious wild-type mice by 30% and 42%, respectively. Heart rate of BK channel knock-out mice (Kcnma1−/−) was not affected by these BK channel inhibitors, suggesting that the changes to heart rate were specifically mediated through BK channels. The possibility that these effects were mediated through BK channels peripheral to the heart was ruled out with experiments using isolated, perfused rat hearts, which showed a significant reduction in heart rate when treated with the BK channel inhibitors paxilline (1 µM), lolitrem B (1 µM), and iberiotoxin (0.23 µM), of 34%, 60%, and 42%, respectively. Furthermore, paxilline was shown to decrease heart rate in a dose-dependent manner. These results implicate BK channels located in the heart to be directly involved in the regulation of heart rate.

BK channels regulate sinoatrial node firing rate and cardiac pacing in vivo.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels play prominent roles in shaping muscle and neuronal excitability. In the cardiovascular system, BK channels promote vascular relaxation and protect against ischemic injury. Recently, inhibition of BK channels has been shown to lower heart rate in intact rodents and isolated hearts, suggesting a novel role in heart function. However, the underlying mechanism is unclear. In the present study, we recorded ECGs from mice injected with paxilline (PAX), a membrane-permeable BK channel antagonist, and examined changes in cardiac conduction. ECGs revealed a 19 ± 4% PAX-induced reduction in heart rate in wild-type but not BK channel knockout (Kcnma1(-/-)) mice. The heart rate decrease was associated with slowed cardiac pacing due to elongation of the sinus interval. Action potential firing recorded from isolated sinoatrial node cells (SANCs) was reduced by 55 ± 15% and 28 ± 9% by application of PAX (3 μM) and iberiotoxin (230 nM), respectively. Furthermore, baseline firing rates from Kcnma1(-/-) SANCs were 33% lower than wild-type SANCs. The slowed firing upon BK current inhibition or genetic deletion was due to lengthening of the diastolic depolarization phase of the SANC action potential. Finally, BK channel immunoreactivity and PAX-sensitive currents were identified in SANCs with HCN4 expression and pacemaker current, respectively, and BK channels cloned from SANCs recapitulated similar activation as the PAX-sensitive current. Together, these data localize BK channels to SANCs and demonstrate that loss of BK current decreases SANC automaticity, consistent with slowed sinus pacing after PAX injection in vivo. Furthermore, these findings suggest BK channels are potential therapeutic targets for disorders of heart rate.

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.

Molecular divergence of two orthologous scorpion toxins affecting potassium channels

Alpha-KTxs are a diverse group of scorpion short-chain peptide toxins that affect animal potassium channels. We report the biochemical purification, gene cloning, and functional characterization of a new α-KTx named MeuTx3B, from venom of the scorpion Mesobuthus eupeus. MeuTx3B is an orthologue of BmTx3B/Martentoxin (α-KTx16 subfamily) from Mesobuthus martensii that differs by three amino acid substitutions. We found that despite their orthologous relationship, MeuTx3B and BmTx3B exhibit different post-transcriptional processing patterns due to nucleotide mutations in their untranslated regions (UTRs). Our results show that MeuTx3B also differs functionally from BmTx3B in that it lacks inhibitory activity on large conductance calcium-activated potassium channels (BK), implicating the amino acids of difference in conferring the inhibitory activity of BmTx3B. Furthermore, we show that MeuTx3B (2μM) partially inhibits human voltage-gated potassium channel Kv1.3. By using codon-substitution models, we detected signals of positive selection that could drive adaptive evolution of MeuTx3B and related toxins in the functional region associated with pharmacological diversification of toxins in the α-KTx 1 and 16 subfamilies.

Combined effects of fungal alkaloids on intestinal motility in an in vitro rat model.

Diarrhea is caused by factors that alter absorption and secretion of water and ions across the intestinal epithelium and disrupt motility. Parasitic infection, stress, poor nutrition, and exposure to plant or fungal toxins predispose livestock to noninfectious diarrhea. This is more prevalent in sheep that graze pastures infected with wild-type endophytic fungus, suggesting the involvement of fungal alkaloids. These increase smooth muscle contraction: ergovaline/ergotamine (ergot alkaloid) activates serotonin (5-HT) receptors, and lolitrem B (indole diterpene) inhibits large-conductance Ca2+-activated K+ (BK) channels. Because of their separate mechanisms of action the objective of this study was to investigate whether they act synergistically to increase smooth muscle contraction. Effects of ergotamine (1 µM) and lolitrem B (0.1 µM) on the tension and frequency of spontaneous contractions were investigated in a longitudinal preparation of isolated distal colon. The compounds were dissolved in 0.1% dimethyl sulfoxide (DMSO) and applied separately or together for 1 h. Ergotamine increased contractile tension compared to the pretreatment control (P<0.01) and produced a short-lived increase in frequency (P<0.001). Lolitrem B increased contractile tension (P<0.05) but had no effect on frequency. When applied together, the contractile tension was greater than the sum of the compounds applied separately (P<0.05). The frequency of contractions was increased (P<0.05) but was not significantly different from that for ergotamine alone. The increased contractile tension when both compounds were applied together indicates that ergotamine and lolitrem B acted synergistically to increase smooth muscle contraction, suggesting that they would alter motility in vivo.

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.

Tracking gastrointestinal transit of solids in aged rats as pharmacological models of chronic dysmotility

Dysmotility in the gastrointestinal (GI) tract often leads to impaired transit of luminal contents leading to symptoms of diarrhea or constipation. The aim of this research was to develop a technique using high resolution X‐ray imaging to study pharmacologically induced aged rat models of chronic GI dysmotility that mimic accelerated transit (diarrhea) or constipation. The 5‐hydroxytryptamine type 4 (5‐HT4) receptor agonist prucalopride was used to accelerate transit, and the opioid agonist loperamide was used to delay transit.

Porous Materials to Support Bilayer Lipid Membranes for Ion Channel Biosensors

To identify materials suitable as membrane supports for ion channel biosensors, six filter materials of varying hydrophobicity, tortuosity, and thickness were examined for their ability to support bilayer lipid membranes as determined by electrical impedance spectroscopy. Bilayers supported by hydrophobic materials (PTFE, polycarbonate, nylon, and silanised silver) had optimal resistance (14–19 GΩ) and capacitance (0.8–1.6 μF) values whereas those with low hydrophobicity did not form BLMs (PVDF) or were short-lived (unsilanised silver). The ability of ion channels to function in BLMs was assessed using a method recently reported to improve the efficiency of proteoliposome incorporation into PTFE-supported bilayers. Voltage-gated sodium channel activation by veratridine and inhibition by saxitoxin showed activity for PTFE, nylon, and silanised silver, but not polycarbonate. Bilayers on thicker, more tortuous, and hydrophobic materials produced higher current levels. Bilayers that self-assembled on PTFE filters were the longest lived and produced the most channel activity using this method.