Davis, Scott J et al. (2011) International Worm Meeting "Uncovering the molecular basis for ethanol activation of the BK channel using random mutagenesis."

Pierce-Shimomura, Jon, Davis, Scott J, Milman, Kelly, Song, Sam

[International Worm Meeting, 2011]

Previous research has demonstrated a strong genetic component in the development of alcohol abuse. Specifically, people with a high resistance to intoxication at the time of their first drink are at risk. This suggests protein targets of alcohol or their downstream effectors that mediate behavioral intoxication are critical for the potential of an individual to abuse alcohol. A highly conserved target protein that is emerging as a key mediator for behavioral intoxication and tolerance is the big conductance potassium (BK) channel(1). The BK channel is activated by low levels of alcohol (~20 mM) across many species including C. elegans, rodent and human, which is equivalent to the legal level of intoxication. In mice, this channel contributes to both behavioral intoxication and tolerance. A gain-of function mutation in the BK channel results in hypersensitivity to alcohol in humans 2. In addition, a genetic screen identified a null mutation in the slo-1 gene, which encodes the BK channel, as critical for resistance to behavioral intoxication in C. elegans3. In order to elucidate the interaction of ethanol with the BK channel at the molecular level, we are using a genetic screen to uncover novel non-null mutations throughout the SLO-1 channel that result in resistance to behavioral intoxication. In addition, we are using random mutagenesis to uncover novel non-null mutations in the C-terminus of the SLO-1 channel, a region previously implicated in the response to ethanol in mice4. Non-null candidates will be distinguished from null mutants by differences in locomotory pattern and subsequent sequence analysis. So far, one of the 20 mutants has been identified as a candidate non-null mutant. After a non-null mutant is identified, we will perform in vivo patch-clamp recordings to assess how the mutation alters basal BK channel function and the response to alcohol. This study may provide knowledge on how ethanol acts on the BK channel at the molecular level to induce intoxication across species. 1. Treistman and Martin. BK Channels: mediators and models for alcohol tolerance, Trends Neurosci. 2009. 32(12)629-37; 2. Du et al., Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder Nat Genet. 2005. 733-8; 3. Davies et al., A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell. 2003. 115(6) 655-66; 4. Liu et al., Ethanol modulates BKCa channels by acting as an adjuvant of calcium. Mol Pharmacol. 2008. 74(3) 628-40.

Davis S J et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Uncovering the Molecular Basis for Ethanol Activation of the BK Channel Using a Genetic Screen"

Davis S J, Song S S, Pierce-Shimomura J T

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Ethanol acts on dozens of ion channel types across many species. However, the molecular basis for how ethanol affects these ion channels is poorly understood, as are their individual contributions to altered behavior. Previously, a genetic screen in C. elegans defined the voltage and calcium-activated BK potassium channel as the major target of ethanol to produce behavioral intoxication (Davies et al., 2003). Moreover, the BK channel was activated by typical human intoxication conditions of 20 mM ethanol with in vivo patch-clamp recording. The BK channel has recently been implicated in sensitivity to intoxication in humans, and tolerance in both mice and Drosophila. The genetic tractability of C. elegans allows for rapid screening of mutations in the BK channel gene. In vitro studies have already identified specific regions and residues of the BK channel that are critical for BK channel activation by ethanol. We hypothesize that amino acid residues in the BK channel exist that when mutated render the channel insensitive to activation by ethanol in vivo, without severely compromising channel function. Such mutations might reveal potential binding sites for ethanol or parts of the channel that undergo ethanol-induced conformational changes. To test this hypothesis, we are performing a large-scale screen to obtain novel mutations in the BK channel that confer behavioral resistance to ethanol in vivo. Currently, this ongoing screen has uncovered 20 candidate BK channel mutants that demonstrate resistance to intoxication. In order to predict whether new mutants are null or non-null we are quantitatively comparing the pattern of movement made by these mutants compared to those made by wild type and a known null mutant. Non-null candidate mutants will undergo genetic sequencing and further analyzed with in vivo patch-clamp recordings to reveal how mutations change the basal properties and/or ethanol sensitivity of the BK channel. Uncovering residues critical to behavioral intoxication will elucidate the molecular interaction between ethanol and the BK channel that can be tested further in mammalian species.

Davis KC et al. (2016) J Physiol "A mechanism for sickness sleep: lessons from invertebrates."

Davis KC, Raizen DM

[J Physiol, 2016]

During health, animal sleep is regulated by an internal clock and by the duration of prior wakefulness. During sickness, sleep is regulated by cytokines released from either peripheral cells or from cells within the nervous system. These cytokines regulate central nervous system neurons to induce sleep. Recent research in the invertebrates Caenorhabditis elegans and Drosophila melanogaster has led to new insights into the mechanism of sleep during sickness. Sickness is triggered by exposure to environments such as infection, heat, or ultraviolet light irradiation, all of which cause cellular stress. Epidermal growth factor is released from stressed cells and signals to activate central neuroendocrine cell(s). These neuron(s) release neuropeptides including those containing an amidated arginine(R)-phenylalanine(F) motif at their C-termini (RFamide peptides). Importantly, mechanisms regulating sickness sleep are partially distinct from those regulating healthy sleep. We will here review key findings, which have elucidated the central neuroendocrine mechanism of sleep during sickness. Adaptive mechanisms employed in the control of sickness sleep may play a role in correcting cellular homeostasis after various insults. We speculate that these mechanisms may play a maladaptive role in human pathological conditions such as in the fatigue and anorexia associated with autoimmune diseases, with major depression, and with unexplained chronic fatigue. This article is protected by copyright. All rights reserved.

Davies AG et al. (2015) J Vis Exp "An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans."

Raabe RC, Bettinger JC, Blackwell GG, Davies AG

[J Vis Exp, 2015]

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C. elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C. elegans.

Davies SK et al. (2015) J Proteome Res "Metabolic Youth in Middle Age: Predicting Aging in Caenorhabditis elegans Using Metabolomics."

Bundy JG, Leroi AM, Davies SK

[J Proteome Res, 2015]

Many mutations and allelic variants are known that influence the rate at which animals age, but when in life do such variants diverge from normal patterns of aging? Is this divergence visible in their physiologies? To investigate these questions, we have used (1)H NMR spectroscopy to study how the metabolome of the nematode Caenorhabditis elegans changes as it grows older. We identify a series of metabolic changes that, collectively, predict the age of wild-type worms. We then show that long-lived mutant daf-2(m41) worms are metabolically youthful compared to wild-type worms, but that this relative youth only appears in middle age. Finally, we show that metabolic age predicts the timing and magnitude of differences in age-specific mortality between these strains. Thus, the future mortality of these two genotypes can be predicted long before most of the worms die.

Davis BO et al. (1986) J Chem Ecol "Laser microbeam studies of role of amphid receptors in chemosensory behavior of nematode Caenorhabditis elegans."

Dusenbery DB, Goode M, Davis BO

[J Chem Ecol, 1986]

Amphid sensilla, historically considered the primary chemosensory structures of nematodes, were found to be necessary for the detection of only one of the six chemical stimuli that were tested. Only the attraction to cAMP was eliminated by damaging the two lateral lips, which bear the amphid sensilla. The inner labial sensilla, one of which occurs on each of the six lips, are probably the primary receptor structures for the other chemical stimuli. Damaging all six lips, which should destroy all anterior chemosensory input, not only eliminated the attraction to sodium and chloride ions, but reversed the nematodes' response to them. Nematodes with all six lips destroyed showed reversal behavior when exposed to these attractants. Nematodes with damage to all six lips appeared to recover much of their normal chemosensory function within 24 hr after treatment.

Davis SJ et al. (2014) J Neurosci "Conserved single residue in the BK potassium channel required for activation by alcohol and intoxication in C. elegans."

Davis SJ, Scott LL, Pierce-Shimomura JT, Hu K

[J Neurosci, 2014]

Alcohol directly modulates the BK potassium channel to alter behaviors in species ranging from invertebrates to humans. In the nematode Caenorhabditis elegans, mutations that eliminate the BK channel, SLO-1, convey dramatic resistance to intoxication by ethanol. We hypothesized that certain conserved amino acids are critical for ethanol modulation, but not for basal channel function. To identify such residues, we screened C. elegans strains with different missense mutations in the SLO-1 channel. A strain with the SLO-1 missense mutation T381I in the RCK1 domain was highly resistant to intoxication. This mutation did not interfere with other BK channel-dependent behaviors, suggesting that the mutant channel retained normal in vivo function. Knock-in of wild-type versions of the worm or human BK channel rescued intoxication and other BK channel-dependent behaviors in a slo-1-null mutant background. In contrast, knock-in of the worm T381I or equivalent human T352I mutant BK channel selectively rescued BK channel-dependent behaviors while conveying resistance to intoxication. Single-channel patch-clamp recordings confirmed that the human BK channel engineered with the T352I missense mutation was insensitive to activation by ethanol, but otherwise had normal conductance, potassium selectivity, and only subtle differences in voltage dependence. Together, our behavioral and electrophysiological results demonstrate that the T352I mutation selectively disrupts ethanol modulation of the BK channel. The T352I mutation may alter a binding site for ethanol and/or interfere with ethanol-induced conformational changes that are critical for behavioral responses to ethanol.

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

Monsalve, G.C. et al. (2009) International Worm Meeting "Timing the Molting Cycle."

Monsalve, G.C., Davie, J., Frand, A.R.

[International Worm Meeting, 2009]

Nematodes develop through periodic molts but molecular mechanisms that set the pace of the molting cycle are not well understood. Molting involves detachment of the old exoskeleton from the underlying hypodermis and synthesis of the new exoskeleton, an extracellular matrix composed largely of collagens. Epidermal stem cells divide in sync with the first three molts but terminally differentiate at the final molt. A period of behavioral quiescence (lethargus) accompanies remodeling of the exoskeleton. Animals subsequently use specialized movements to escape the old skeleton (ecdyse). Improper coordination of these cell biological and behavioral events can be fatal. We aim to identify genes and molecules that regulate the timing of the molting cycle. The best-characterized biological timers control circadian rhythms in the physiology and behavior of humans and other animals. In theory, worm counterparts of the core circadian clock proteins might also regulate the ultradian rhythm of the molting cycle. We are therefore investigating the role of clock gene homologs in the reiterative molecular and behavioral events characteristic of molting. As a complementary approach, we conducted a forward genetic screen for mutants that molt prematurely, using expression of the mlt-10p::gfp-pest reporter to indicate synthesis of a new cuticle. In a pilot screen of approximately 18,000 genomes, we indentified one candidate that expressed the mlt-10 reporter too early and then partly shed the L1 exoskeleton. This particular animal perished, possibly due to the exposure of an incomplete L2 skeleton to the environment. In ongoing work, a clonal screen will allow the recovery of lethal mutations from siblings of affected animals. To further investigate the physiologic regulation of the molting cycle, we have cloned and characterized new mutations that cause supernumerary molts after puberty. These alleles emerged from a genetic screen conducted by Alison Frand and Sascha Russel in the laboratory of Gary Ruvkun. By studying the regulation of the molting cycle, we expect to uncover novel but conserved mechanisms for the endocrine control of development and behavior.

Davis MB et al. (2017) Am J Physiol Cell Physiol "Biphasic adaptation to osmotic stress in the C. elegans germ line."

Davis MB, Wood MP, Schisa JA, Montalbano A

[Am J Physiol Cell Physiol, 2017]

Cells respond to environmental stress in multiple ways. In the germ line, heat shock and nutritive stress trigger the assembly of large ribonucleoprotein (RNP) granules via liquid-liquid phase separation (LLPS). The RNP granules are hypothesized to maintain the quality of oocytes during stress. The goal of this study was to investigate the cellular response to glucose in the germ line and determine if it is an osmotic stress response. We found that exposure to 500 mM glucose induces the assembly of RNP granules in the germ line within one hour. Interestingly, the RNP granules are maintained for up to three hours; however, they dissociate after longer periods of stress. The RNP granules include P body and stress granule proteins, suggesting shared functions. Based on several lines of evidence, the germ line response to glucose largely appears to be an osmotic stress response, thus identifying osmotic stress as a trigger of LLPS. Although RNP granules are not maintained beyond three hours of osmotic stress, the quality of oocytes does not appear to decrease after longer periods of stress, suggesting a secondary adaptation in the germ line. We used an indirect marker of glycerol and observed high levels after five and twenty hours of glucose exposure. Moreover, in gpdh-1;gpdh-2 germ lines glycerol levels are reduced concomitant with RNP granules being maintained for an extended period. We speculate that increased glycerol levels may function as a secondary osmoregulatory adaptive response in the germ line, following a primary response of RNP granule assembly.