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Abstract: Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1 to 3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized nonevoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibitors of clathrin, Dnm, and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm, and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment., (Copyright © 2022 International Association for the Study of Pain.)

The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK 1 R), Gα q/i , and βarrestin-2. Whereas the FDA-approved NK 1 R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK 1 R+ve neurons in knockin mice expressing human NK 1 R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK 1 R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK 1 R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK 1 R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of diverse diseases.

Excitatory and inhibitory neurotransmission within the spinal dorsal horn is tightly controlled to regulate transmission of nociceptive signals to the brain. One aspect of this control is modulation of neuronal activity through cholinergic signaling. Nociceptive neurons in the dorsal horn express both nicotinic and muscarinic cholinergic receptors and activation of these receptors reduces pain in humans, while inhibition leads to nociceptive hypersensitivity. At a cellular level, acetylcholine (ACh) has diverse effects on excitability which is dependent on the receptor and neuronal subtypes involved. In the present study we sought to characterize the electrophysiological responses of specific subsets of lamina II interneurons from rat and marmoset spinal cord. Neurons were grouped by morphology and by action potential firing properties. Whole-cell voltage-clamp recordings from lamina II dorsal horn neurons of adult rats showed that bath applied acetylcholine increased, decreased or had no effect on spontaneous synaptic current activity in a cell-type specific manner. ACh modulated inhibitory synaptic activity in 80% of neurons, whereas excitatory synaptic activity was affected in less than 50% of neurons. In whole-cell current clamp recordings, brief somatic application of ACh induced cell-type specific responses in 79% of rat lamina II neurons, which included: depolarization and action potential firing, subthreshold membrane depolarization, biphasic responses characterized by transient depolarization followed by hyperpolarization and membrane hyperpolarization alone. Similar responses were seen in marmoset lamina II neurons and the properties of each neuron group were consistent across species. ACh-induced hyperpolarization was blocked by the muscarinic antagonist atropine and all forms of acetylcholine-induced depolarization were blocked by the nicotinic antagonist mecamylamine. The cholinergic system plays an important role in regulating nociception and this study contributes to our understanding of how circuit activity is controlled by ACh at a cellular level in primate and rodent spinal cord., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The adenosine A 1 receptor (A 1 R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain 1,2 . However, development of analgesic orthosteric A 1 R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects 3 . Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A 1 R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A 1 R co-bound to adenosine, MIPS521 and a G i2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Trigeminal neuropathic pain is a chronic pain condition caused by damage or inflammation of the trigeminal nerve or its branches, with both peripheral and central nervous system dysfunction contributing to the disorder. Trigeminal pain conditions present with diagnostic and therapeutic challenges to healthcare providers and often require multiple therapeutic approaches for pain reduction. This review will provide the overview of pathophysiology in peripheral and central nociceptive circuits that are involved in neuropathic pain conditions involving the trigeminal nerve and the current therapeutics that are used to treat these disorders. Recent advances in treatment of trigeminal pain, including novel therapeutics that target ion channels and receptors, gene therapy and monoclonal antibodies that have shown great promise in preclinical studies and clinical trials will also be described., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: Inhibitory neurotransmission plays an important role in controlling excitability within nociceptive circuits of the spinal cord dorsal horn. Loss of inhibitory signalling is thought to contribute to the development of pathological pain. Preclinical studies suggest that increasing inhibitory glycinergic signalling is a good therapeutic strategy for treating pain. One approach to increase synaptic glycine is to inhibit the activity of the glycine transporter 2 (GlyT2) on inhibitory nerve terminals. These transporters are involved in regulating glycine concentrations and recycling glycine into presynaptic terminals. Inhibiting activity of GlyT2 increases synaptic glycine, which decreases excitability in nociceptive circuits and provides analgesia in neuropathic and inflammatory pain models., Experimental Approach: We investigated the effects of reversible and irreversible GlyT2 inhibitors on inhibitory glycinergic and NMDA receptor-mediated excitatory neurotransmission in the rat dorsal horn. The effect of these drugs on synaptic signalling was determined using patch-clamp electrophysiology techniques to measure glycine- and NMDA-mediated postsynaptic currents in spinal cord slices in vitro., Key Results: We compared activity of four compounds that increase glycinergic tone with a corresponding increase in evoked glycinergic postsynaptic currents. These compounds did not deplete synaptic glycine release over time. Interestingly, none of these compounds increased glycine-mediated excitatory signalling through NMDA receptors. The results suggest that these compounds preferentially inhibit GlyT2 over G1yT1 with no potentiation of the glycine receptor and without inducing spillover from inhibitory to excitatory synapses., Conclusions and Implications: GlyT2 inhibitors increase inhibitory neurotransmission in the dorsal horn and have potential as pain therapeutics., Linked Articles: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc., (© 2018 The British Pharmacological Society.)

G protein-coupled receptors (GPCRs) are considered to function primarily at the plasma membrane, where they interact with extracellular ligands and couple to G proteins that transmit intracellular signals. Consequently, therapeutic drugs are designed to target GPCRs at the plasma membrane. Activated GPCRs undergo clathrin-dependent endocytosis. Whether GPCRs in endosomes control pathophysiological processes in vivo and are therapeutic targets remains uncertain. We investigated the contribution of endosomal signaling of the calcitonin receptor-like receptor (CLR) to pain transmission. Calcitonin gene-related peptide (CGRP) stimulated CLR endocytosis and activated protein kinase C (PKC) in the cytosol and extracellular signal regulated kinase (ERK) in the cytosol and nucleus. Inhibitors of clathrin and dynamin prevented CLR endocytosis and activation of cytosolic PKC and nuclear ERK, which derive from endosomal CLR. A cholestanol-conjugated antagonist, CGRP 8-37 , accumulated in CLR-containing endosomes and selectively inhibited CLR signaling in endosomes. CGRP caused sustained excitation of neurons in slices of rat spinal cord. Inhibitors of dynamin, ERK, and PKC suppressed persistent neuronal excitation. CGRP 8-37 -cholestanol, but not unconjugated CGRP 8-37 , prevented sustained neuronal excitation. When injected intrathecally to mice, CGRP 8-37 -cholestanol inhibited nociceptive responses to intraplantar injection of capsaicin, formalin, or complete Freund's adjuvant more effectively than unconjugated CGRP 8-37 Our results show that CLR signals from endosomes to control pain transmission and identify CLR in endosomes as a therapeutic target for pain. Thus, GPCRs function not only at the plasma membrane but also in endosomes to control complex processes in vivo. Endosomal GPCRs are a drug target that deserve further attention., Competing Interests: Conflict of interest statement: Research in the N.W.B. laboratory is funded in part by Takeda Pharmaceuticals.

Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK 1 R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK 1 R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and β-arrestin blocked SP-induced NK 1 R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK 1 R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK 1 R antagonists. These results reveal a critical role for endosomal signaling of the NK 1 R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists., (Copyright © 2017, American Association for the Advancement of Science.)

Inhibitory glycinergic neurotransmission in the spinal cord dorsal horn plays an important role in regulating nociceptive signalling by inhibiting neuronal excitation. Blocking glycinergic transmission in the dorsal horn causes normally innocuous stimuli to become painful (allodynia) and increases sensitivity to noxious stimuli (hyperalgesia). Loss of inhibitory signalling is thought to contribute to the development of pathological pain. Management of neuropathic pain with current therapeutics is challenging and there is a great need for more effective treatments. Preclinical studies using drugs that increase glycinergic signalling by potentiating glycine receptor activity or inhibiting transporter activity suggest that targeting this system is a good therapeutic strategy. The spatially restricted expression of glycine receptors and transporters is an advantage for targeting specific pathologies such as pain. However, until recently there have been few pharmacological modulators identified and most of which do not specifically target glycinergic signalling. This mini-review provides an overview of recent advances in the development of therapeutics and novel approaches that aim to increase glycinergic neurotransmission for the treatment of persistent pain., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Human genetic studies have implicated the voltage-gated sodium channel Na V 1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits Na V 1.7 (IC 50 0.9 nM) with at least 40-1000-fold selectivity over all other Na V subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na V 1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective Na V 1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na V 1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na V 1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

The development of neuropathic pain involves persistent changes in signalling within pain pathways. Reduced inhibitory signalling in the spinal cord following nerve-injury has been used to explain sensory signs of neuropathic pain but specific circuits that lose inhibitory input have not been identified. This study shows a specific population of spinal cord interneurons, radial neurons, lose glycinergic inhibitory input in a rat partial sciatic nerve ligation (PNL) model of neuropathic pain. Radial neurons are excitatory neurons located in lamina II of the dorsal horn, and are readily identified by their morphology. The amplitude of electrically-evoked glycinergic inhibitory post-synaptic currents (eIPSCs) was greatly reduced in radial neurons following nerve-injury associated with increased paired-pulse ratio. There was also a reduction in frequency of spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSC) in radial neurons without significantly affecting mIPSC amplitude. A subtype selective receptor antagonist and western blots established reversion to expression of the immature glycine receptor subunit GlyRα2 in radial neurons after PNL, consistent with slowed decay times of IPSCs. This study has important implications as it identifies a glycinergic synaptic connection in a specific population of dorsal horn neurons where loss of inhibitory signalling may contribute to signs of neuropathic pain.

In the spinal cord and periphery, adenosine inhibits neuronal activity through activation of the adenosine A1 receptor (A1R), resulting in antinociception and highlighting the potential of therapeutically targeting the receptor in the treatment of neuropathic pain. This study investigated the changes in adenosine tone and A1R signaling, together with the actions of a novel A1R positive allosteric modulator (PAM), VCP171 [(2-amino-4-(3-(trifluoromethyl)phenyl)thiophen-3-yl)(phenyl)methanone], on excitatory and inhibitory neurotransmission at spinal cord superficial dorsal horn synapses in a rat partial nerve-injury model of neuropathic pain. In the absence of A1R agonists, superfusion of the A1R antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 μM), produced a significantly greater increase in electrically evoked α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic current (eEPSC) amplitude in both lamina I and II neurons from nerve-injured animals than in controls, suggesting that endogenous adenosine tone is increased in the dorsal horn. Inhibitory GABAergic and glycinergic synaptic currents were also significantly increased by DPCPX in controls but there was no difference after nerve injury. The A1R agonist, N6-cyclopentyladenosine, produced greater inhibition of eEPSC amplitude in lamina II but not lamina I of the spinal cord dorsal horn in nerve-injured versus control animals, suggesting a functional increase in A1R sensitivity in lamina II neurons after nerve injury. The A1R PAM, VCP171, produced a greater inhibition of eEPSC amplitude of nerve-injury versus control animals in both lamina I and lamina II neurons. Enhanced adenosine tone and A1R sensitivity at excitatory synapses in the dorsal horn after nerve injury suggest that new generation PAMs of the A1R can be effective treatments for neuropathic pain., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

Opioids, local anesthetics, anticonvulsant drugs, antidepressants, and non-steroidal anti-inflammatory drugs (NSAIDs) are used to provide pain relief but they do not provide adequate pain relief in a large proportion of chronic pain patients and are often associated with unacceptable side effects. Inhibitory glycinergic neurotransmission is impaired in chronic pain states, and this provides a novel target for drug development. Inhibitors of the glycine transporter 2 (GlyT2) enhance inhibitory neurotransmission and show particular promise for the treatment of neuropathic pain. N-arachidonyl-glycine (NAGly) is an endogenous lipid that inhibits glycine transport by GlyT2 and also shows potential as an analgesic, which may be further exploited in drug development. In this review we discuss the role of glycine neurotransmission in chronic pain and future prospects for the use of glycine transport inhibitors in the treatment of pain., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Miniature neurotransmission is the transsynaptic process where single synaptic vesicles spontaneously released from presynaptic neurons induce miniature postsynaptic potentials. Since their discovery over 60 years ago, miniature events have been found at every chemical synapse studied. However, the in vivo necessity for these small-amplitude events has remained enigmatic. Here, we show that miniature neurotransmission is required for the normal structural maturation of Drosophila glutamatergic synapses in a developmental role that is not shared by evoked neurotransmission. Conversely, we find that increasing miniature events is sufficient to induce synaptic terminal growth. We show that miniature neurotransmission acts locally at terminals to regulate synapse maturation via a Trio guanine nucleotide exchange factor (GEF) and Rac1 GTPase molecular signaling pathway. Our results establish that miniature neurotransmission, a universal but often-overlooked feature of synapses, has unique and essential functions in vivo., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Spinal muscular atrophy (SMA) is a lethal human disease characterized by motor neuron dysfunction and muscle deterioration due to depletion of the ubiquitous survival motor neuron (SMN) protein. Drosophila SMN mutants have reduced muscle size and defective locomotion, motor rhythm, and motor neuron neurotransmission. Unexpectedly, restoration of SMN in either muscles or motor neurons did not alter these phenotypes. Instead, SMN must be expressed in proprioceptive neurons and interneurons in the motor circuit to nonautonomously correct defects in motor neurons and muscles. SMN depletion disrupts the motor system subsequent to circuit development and can be mimicked by the inhibition of motor network function. Furthermore, increasing motor circuit excitability by genetic or pharmacological inhibition of K(+) channels can correct SMN-dependent phenotypes. These results establish sensory-motor circuit dysfunction as the origin of motor system deficits in this SMA model and suggest that enhancement of motor neural network activity could ameliorate the disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pre-mRNA alternative splicing is an important mechanism for the generation of synaptic protein diversity, but few factors governing this process have been identified. From a screen for Drosophila mutants with aberrant synaptic development, we identified beag, a mutant with fewer synaptic boutons and decreased neurotransmitter release. Beag encodes a spliceosomal protein similar to splicing factors in humans and Caenorhabditis elegans. We find that both beag mutants and mutants of an interacting gene dsmu1 have changes in the synaptic levels of specific splice isoforms of Fasciclin II (FasII), the Drosophila ortholog of neural cell adhesion molecule. We show that restoration of one splice isoform of FasII can rescue synaptic morphology in beag mutants while expression of other isoforms cannot. We further demonstrate that this FasII isoform has unique functions in synaptic development independent of transsynaptic adhesion. beag and dsmu1 mutants demonstrate an essential role for these previously uncharacterized splicing factors in the regulation of synapse development and function.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The heart generates and propagates action potentials through synchronized activation of ion channels allowing inward Na(+) and Ca(2+) 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 microM), lolitrem B (1 microM), and iberiotoxin (0.23 microM), 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.

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.

"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 beta4 BK channel accessory subunit (Kcnmb4(-/-)), only low-level ataxia was observed. Our study therefore reveals a new role for the accessory BK beta4 subunit in motor control. The beta4 subunit could be considered as a potential target for treatment of ataxic conditions in animals and in humans.