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The large conductance, calcium- and voltage-activated potassium channel, known as the BK channel, is one of the central proteins that mediate alcohol intoxication and tolerance across species. Although ethanol targets BK channels through direct interaction, how ethanol-mediated BK channel activation causes behavioral intoxication is poorly understood. In. C. elegans, loss of function in SLO-1, the BK channel ortholog, confers profound ethanol resistance in movement and egg-laying behaviors. Here, we show that depletion of SLO-1 channels clustered at the active zones with no change in the overall channel expression level results in locomotory resistance to the intoxicating effect of ethanol, equivalent to that of slo-1 loss-of-function mutants. Likewise, depletion of clustered SLO-1 channels in the sarcolemma and neurons leads to ethanol-resistant egg-laying behavior. By contrast, reduction in the overall SLO-1 channel level by over 70% causes only moderate ethanol resistance in movement, and minimal, if any, resistance in egg laying. Our findings strongly suggest that behavioral ethanol sensitivity is conferred by local, but not global, depression of excitability via clustered BK channels. Given that clustered BK channels are functionally coupled to, and localize near, calcium channels, ethanol may mediate its behavioral effects by targeting BK channels and their coupled calcium channels.

Duchenne muscular dystrophy, a fatal degenerative muscle disease, is caused by mutations in the dystrophin gene. Loss of dystrophin in the muscle cell membrane causes muscle fiber necrosis. Previously, loss-of-function mutations in dys-1, the Caenorhabditis elegans dystrophin ortholog, were shown to cause a contractile defect and mild fiber degeneration in striated body wall muscle. Here, we show that loss of dystrophin function in C. elegans results in a shorter lifespan and stochastic, age-dependent muscle-cell death. Reduction of dystrophin function also accelerated age-dependent protein aggregation in muscle cells, suggesting a defect in proteostasis. Both muscle cell death and protein aggregation showed wide variability among the muscle cells. These observations suggest that muscle cell death in dys-1 mutants is greatly influenced by cellular environments. Thus, the manipulation of the cellular environment may provide an opportunity to thwart the cell death initiated by the loss of dystrophin. We found that reduced insulin-like growth factor (IGF) signaling, which rejuvenates the cellular environment to protect cells from a variety of age-dependent pathologies, prevented muscle cell death in the dys-1 mutants in a daf-16-dependent manner. Our study suggests that manipulation of the IGF signaling pathways in muscle cells could be a potent intervention for muscular dystrophy.

Voltage- and calcium-dependent BK channels regulate calcium-dependent cellular events such as neurotransmitter release by limiting calcium influx. Their plasma membrane abundance is an important factor in determining BK current and thus regulation of calcium-dependent events. In C. elegans, we show that ERG-28, an endoplasmic reticulum (ER) membrane protein, promotes the trafficking of SLO-1 BK channels from the ER to the plasma membrane by shielding them from premature degradation. In the absence of ERG-28, SLO-1 channels undergo aspartic protease DDI-1-dependent degradation, resulting in markedly reduced expression at presynaptic terminals. Loss of erg-28 suppressed phenotypic defects of slo-1 gain-of-function mutants in locomotion, neurotransmitter release, and calcium-mediated asymmetric differentiation of the AWC olfactory neuron pair, and conferred significant ethanol-resistant locomotory behavior, resembling slo-1 loss-of-function mutants, albeit to a lesser extent. Our study thus indicates that the control of BK channel trafficking is a critical regulatory mechanism for synaptic transmission and neural function. DOI: http://dx.doi.org/10.7554/eLife.24733.001

Background Large conductance, calcium-activated BK channels regulate many important physiological processes, including smooth muscle excitation, hormone release and synaptic transmission. The biological roles of these channels hinge on their unique ability to respond synergistically to both voltage and cytosolic calcium elevations. Because calcium influx is meticulously regulated both spatially and temporally, the localization of BK channels near calcium channels is critical for their proper function. However, the mechanism underlying BK channel localization near calcium channels is not fully understood. Results We show here that in C. elegans the localization of SLO-1/BK channels to presynaptic terminals, where UNC-2/CaV2 calcium channels regulate neurotransmitter release, is controlled by the hierarchical organization of CTN-1/α-catulin and DYB-1/dystrobrevin, two proteins that interact with cortical cytoskeletal proteins. CTN-1 organizes a macromolecular SLO-1 channel complex at presynaptic terminals by direct physical interaction. DYB-1 contributes to the maintenance or stabilization of the complex at presynaptic terminals by interacting with CTN-1. We also show that SLO-1 channels are functionally coupled with UNC-2 calcium channels, and that normal localization of SLO-1 to presynaptic terminals requires UNC-2. In the absence of UNC-2, SLO-1 clusters lose the localization specificity, thus accumulating inside and outside of presynaptic terminals. Moreover, CTN-1 is also similarly localized in unc-2 mutants, consistent with the direct interaction between CTN-1 and SLO-1. However, localization of UNC-2 at the presynaptic terminals is not dependent on either CTN-1 or SLO-1. Taken together, our data strongly suggest that the absence of UNC-2 indirectly influences SLO-1 localization via the reorganization of cytoskeletal proteins. Conclusion CTN-1 and DYB-1, which interact with cortical cytoskeletal proteins, are required for the presynaptic punctate localization of SLO-1 in a hierarchical manner. In addition, UNC-2 calcium channels indirectly control the fidelity of SLO-1 puncta localization at presynaptic terminals. We suggest that the absence of UNC-2 leads to the reorganization of the cytoskeletal structure that includes CTN-1, which in turn influences SLO-1 puncta localization. Electronic supplementary material The online version of this article (doi:10.1186/s12868-015-0166-2) contains supplementary material, which is available to authorized users.

Evolutionarily conserved signaling pathways and transcription factors that sense physiological and environmental states regulate aging processes. Lifespan extension was achieved by the modulation of these pathways in yeast, worms, flies and mice. Remarkably, slowing aging processes resulted in the amelioration of age-dependent degenerative diseases (reviewed by Kenyon, Nature, 2010, 464: 504). Our recent study (Oh, PNAS, 2013, 110: 19024) shows that lifespan-extending signaling pathways may provide opportunities for therapeutic interventions of muscle degenerative diseases. Muscular dystrophies are a heterogeneous group of muscle degenerative diseases. The most prevalent form of muscular dystrophies is Duchenne muscular dystrophy (DMD), which is caused by mutations in the dystrophin gene. Dystrophin connects intracellular structural components to the extracellular matrix, and its loss causes cellular damage and consequent myofiber necrosis. While intense efforts have been made to restore dystrophin expression through various methods, including gene transfer and exon skipping, without much success, the mechanism by which the loss of dystrophin function causes muscle cell death has not received much attention. Caenorhabditis elegans (C. elegans) is an invertebrate genetic model organism. Dystrophin and its associated proteins and major signaling and cell death pathways are highly conserved from worms to humans. In our recent study, we have established a C. elegans model of dystrophinopathy. Mutations in the dystrophin gene (dys-1) result in muscle cell death in an age-dependent manner. dys-1 mutant animals lost locomotory function earlier than wild-type animals, and their lifespan was shorter. dys-1 mutants also exhibited morphological and structural defects. A muscle structure that attaches thin filaments to the muscle membrane was lost in dys-1 mutants. Muscle nuclei and mitochondria were disintegrated in dys-1 mutants. This array of phenotypic defects is not apparent in young adults; however, as the mutant animals get older, this array becomes obvious and exacerbated. In addition to such age-dependent severity, the affected muscle cells were random in their positions in the body and in their developmental lineage, even though all of the muscle cells lacked dystrophin. Similarly, in humans, the onset and progression of muscle degeneration and the muscle groups affected are highly variable, even among siblings with the same mutation in the dystrophin gene. Hence, we concluded that although muscle cell death is caused by the loss of dystrophin, age-dependent, intrinsically variable cellular environments influence the onset, severity and progression of the development of muscle cell death. We hypothesize that these cellular environments are the modulators of susceptibility and will be excellent therapeutic targets to thwart muscle cell death. We reasoned that cellular stress responses would be the modulators of susceptibility because stress proteins and cellular calcium levels are elevated in the muscles of human DMD patients and mammalian models. The loss of dystrophin in C. elegans increases muscle excitability and contractility in an activity-dependent manner. The over-excitation of motoneurons leads to the disruption of calcium and protein homeostasis in C. elegans muscle cells. Therefore, we hypothesized that one of the cellular consequences of the loss of dystrophin could be the disruption of proteostasis. We tested this hypothesis using a polyglutamine protein aggregation model, where polyQ35 proteins in muscles form aggregates in an age-dependent manner and the onset and severity of polyQ35 aggregation serve as a proxy for protein homeostasis (Garcia SM et al. Genes Dev, 2007, 21:3006). Indeed, we found that polyQ35 forms aggregates earlier in dys-1 mutants than in the wild type animals, indicating that the loss of dystrophin leads to the disruption of protein homeostasis. Compromised protein-folding capacity can lead to the misfolding and dysfunction of structurally unstable proteins that are tolerated under normal conditions. This phenomenon may contribute to the pathology of dystrophinopathy. Our conclusion that muscle cell death in dys-1 mutants is determined by cellular environments that are variable and age-dependent led us to reason that a sustained, rejuvenating cellular environment may provide protection from muscle cell death. Reductions in IGF signaling enhance stress resistance, delay inherent protein aggregation, and extend lifespan. Indeed, we found that reducing IGF signaling prevented muscle cell death in the dys-1 mutant in a DAF-16 (homolog of FOXO) dependent manner. Consistent with our finding, the chronic activation of AMPK (AMP-activated protein kinase), which is a FOXO activator, was shown to increase sarcolemmal integrity in mdx mice (the mouse model of DMD) (Ljubicic V. et al. Human Mol Genetics, 2011, 20:3478). Rapamycin treatment, which inhibits the mTORC1 (the mammalian target of rapamycin complex 1), a protein complex down-regulated by FOXO under stress conditions, was shown to reduce muscle necrosis and fibrosis in the mouse model of Duchenne muscular dystrophy (Eghtesad S, Mol Medicine, 2011, 17:917). Many important questions remain to be answered. What is the trigger of cell death? How does the collapse of proteostasis contribute to the cell death pathway in muscle cells? Which component of the IGF signaling pathways is critical for the prevention of cell death? Are there other signaling pathways that can thwart cell death? The answers to these questions will be the foundation for discovering new therapeutic interventions and biomarkers for Duchenne muscular dystrophy.