C. Michael Crowder

The Sodium-Activated Potassium Channel Is Encoded by a Member of the Slo Gene Family

Na(+)-activated potassium channels (K(Na)) have been identified in cardiomyocytes and neurons where they may provide protection against ischemia. We now report that K(Na) is encoded by the rSlo2 gene (also called Slack), the mammalian ortholog of slo-2 in C. elegans. rSlo2, heterologously expressed, shares many properties of native K(Na) including activation by intracellular Na(+), high conductance, and prominent subconductance states. In addition to activation by Na(+), we report that rSLO-2 channels are cooperatively activated by intracellular Cl(-), similar to C. elegans SLO-2 channels. Since intracellular Na(+) and Cl(-) both rise in oxygen-deprived cells, coactivation may more effectively trigger the activity of rSLO-2 channels in ischemia. In C. elegans, mutational and physiological analysis revealed that the SLO-2 current is a major component of the delayed rectifier. We demonstrate in C. elegans that slo-2 mutants are hypersensitive to hypoxia, suggesting a conserved role for the slo-2 gene subfamily.

Survival from Hypoxia in C. elegans by Inactivation of Aminoacyl-tRNA Synthetases

Hypoxia is important in a wide range of biological processes, such as animal hibernation and cell survival, and is particularly relevant in many diseases. The sensitivity of cells and organisms to hypoxic injury varies widely, but the molecular basis for this variation is incompletely understood. Using forward genetic screens in Caenorhabditis elegans, we isolated a hypoxia-resistant reduction-of-function mutant of rrt-1 that encodes an arginyl–transfer RNA (tRNA) synthetase, an enzyme essential for protein translation. Knockdown of rrt-1, and of most other genes encoding aminoacyl-tRNA synthetases, rescued animals from hypoxia-induced death, and the level of hypoxia resistance was inversely correlated with translation rate. The unfolded protein response was induced by hypoxia and was required for the hypoxia resistance of the reduction-of-function mutant of rrt-1. Thus, translational suppression produces hypoxia resistance, in part by reducing unfolded protein toxicity.

Autophagy protects against hypoxic injury in C. elegans

Macroautophagy has been implicated in a variety of pathological processes. Hypoxic/ischemic cellular injury is one such process in which autophagy has emerged as an important regulator. In general, autophagy is induced after an hypoxic/ischemic insult; however, whether the induction of autophagy promotes cell death or recovery is controversial and appears to be context dependent. We have developed C. elegans as a genetically tractable model for the study of hypoxic cell injury. Both necrosis and apoptosis are mechanisms of cell death following hypoxia in C. elegans. However, the role of autophagy in hypoxic injury in C. elegans has not been examined. Here, we found that RNAi knockdown of the C. elegans homologs of beclin 1/Atg6 (bec-1) and LC3/Atg8 (lgg-1, lgg-2), and mutation of Atg1 (unc-51) decreased animal survival after a severe hypoxic insult. Acute inhibition of autophagy by the type III phosphatidylinositol 3-kinase inhibitors, 3-methyladenine and Wortmannin, also sensitized animals to hypoxic death. Hypoxia-induced neuronal and myocyte injury as well as necrotic cellular morphology were increased by RNAi knockdown of BEC-1. Hypoxia increased the expression of a marker of autophagosomes in a bec-1-dependent manner. Finally, we found that the hypoxia hypersensitive phenotype of bec-1(RNAi) animals could be blocked by loss-of-function mutations in either the apoptosis or necrosis pathway. These results argue that inhibition of autophagy sensitizes C. elegans and its cells to hypoxic injury and that this sensitization is blocked or circumvented when either of the two major cell death mechanisms is inhibited.

Xenon Acts by Inhibition of Non–N -methyl-d-aspartate Receptor–mediated Glutamatergic Neurotransmission in Caenorhabditis elegans

Background:Electrophysiologic experiments in rodents have found that nitrous oxide and xenon inhibit N-methyl-d-aspartate (NMDA)–type glutamate receptors. These findings led to the hypothesis that xenon and nitrous oxide along with ketamine form a class of anesthetics with the identical mechanism, NMDA receptor antagonism. Here, the authors ask in Caenorhabditis elegans whether xenon, like nitrous oxide, acts by a NMDA receptor–mediated mechanism. Methods:Xenon:oxygen mixtures were delivered into sealed chambers until the desired concentration was achieved. The effects of xenon on various behaviors were measured on wild-type and mutant C. elegans strains. Results:With an EC50 of 15–20 vol% depending on behavioral endpoint, xenon altered C. elegans locomotion in a manner indistinguishable from that of mutants in glutamatergic transmission. Xenon reduced the frequency and duration of backward locomotion without altering its speed or other behaviors tested. Mutation of glr-1, encoding a non-NMDA glutamate receptor subunit, abolished the behavioral effects of xenon; however, mutation of nmr-1, which encodes the pore-forming subunit of an NMDA glutamate receptor previously shown to be required for nitrous oxide action, did not significantly alter xenon response. Transformation of the glr-1 mutant with the wild-type glr-1 gene partially restored xenon sensitivity, confirming that glr-1 was necessary for the full action of xenon. Conclusions:Xenon acts in C. elegans to alter locomotion through a mechanism requiring the non-NMDA glutamate receptor encoded by glr-1. Unlike for the action of nitrous oxide in C. elegans, the NMDA receptor encoded by nmr-1 is not essential for sensitivity to xenon.

Hypoxic Preconditioning Requires the Apoptosis Protein CED-4 in C. elegans

Hypoxic preconditioning (HP) is a rapid and reversible proadaptive response to mild hypoxic exposure with such a response protecting cells from subsequent hypoxic or ischemic insult. HP mechanisms are of great interest because of their therapeutic potential and insight into metabolic adaptation and cell death. HP has been widely demonstrated in the vertebrate subphylum but not in invertebrates. Here, we report that the nematode Caenorhabditis elegans has a potent HP mechanism that protects the organism as well as its neurons and myocytes from hypoxic injury. The time course of C. elegans HP was consistent with vertebrate-delayed HP, appearing 16 hr after preconditioning and lasting at least 36 hr. The apoptosis pathway has been proposed as either a trigger or target of HP. Testing of mutations in the canonical C. elegans apoptosis pathway showed that in general, genes in this pathway are not required for HP. However, loss-of-function mutations in ced-4, which encodes an Apaf-1 homolog, completely blocked HP. RNAi silencing of ced-4 in adult animals immediately preceding preconditioning blocked HP, indicating that CED-4 is required in adults during or after preconditioning. CED-4/Apaf-1 is essential for HP in C. elegans and acts through a mechanism independent of the classical apoptosis pathway.

Ethanol targets: a BK channel cocktail in C. elegans

A genetic screen for resistance to ethanol intoxication in Caenorhabditis elegans isolated mutants of the gene slo-1. slo-1 encodes the pore-forming subunit of a large-conductance Ca(2+)-activated K(+) channel previously shown to limit excitatory neurotransmitter release in C. elegans. Electrophysiological data recorded in vivo are consistent with a model in which ethanol potentiation of SLO-1 produces intoxication in C. elegans by reducing excitatory neurotransmitter release.

Volatile Anesthetics Bind Rat Synaptic Snare Proteins

Background: Volatile general anesthetics (VAs) have a number of synaptic actions, one of which is to inhibit excitatory neurotransmitter release; however, no presynaptic VA binding proteins have been identified. Genetic data in Caenorhabditis elegans have led to the hypothesis that a protein that interacts with the presynaptic protein syntaxin 1A is a VA target. Motivated by this hypothesis, the authors measured the ability of syntaxin 1A and proteins that interact with syntaxin to bind to halothane and isoflurane. Methods: Recombinant rat syntaxin 1A, SNAP-25B, VAMP2, and the ternary SNARE complex that they form were tested. Binding of VAs to these proteins was detected by 19F-nuclear magnetic resonance relaxation measurements. Structural alterations in the proteins were examined by circular dichroism and ability to form complexes. Results: Volatile anesthetics did not bind to VAMP2. At concentrations in the clinical range, VAs did bind to SNAP-25B; however, binding was detected only in preparations containing SNAP-25B homomultimers. VAs also bound at clinical concentrations to both syntaxin and the SNARE complex. Addition of an N-terminal His6 tag to syntaxin abolished its ability to bind VAs despite normal secondary structure and ability to form SNARE complexes; thrombin cleavage of the tag restored VA binding. Thus, the VA binding site(s) has structural requirements and is not simply any α-helical bundle. VAs at supraclinical concentrations produced an increase in helicity of the SNARE complex; otherwise, VA binding produced no gross alteration in the stability or secondary structure of the SNARE complex. Conclusion: SNARE proteins are potential synaptic targets of volatile anesthetics.

An Evolutionarily Conserved Presynaptic Protein Is Required for Isoflurane Sensitivity in Caenorhabditis elegans

Background:Volatile general anesthetics inhibit neurotransmitter release by an unknown mechanism. A mutation in the presynaptic soluble NSF attachment protein receptor (SNARE) protein syntaxin 1A was previously shown to antagonize the anesthetic isoflurane in Caenorhabditis elegans. The mechanism underlying this antagonism may identify presynaptic anesthetic targets relevant to human anesthesia. Methods:Sensitivity to isoflurane concentrations in the human clinical range was measured in locomotion assays on adult C. elegans. Sensitivity to the acetylcholinesterase inhibitor aldicarb was used as an assay for the global level of C. elegans neurotransmitter release. Comparisons of isoflurane sensitivity (measured by the EC50) were made by simultaneous curve fitting and F test as described by Waud. Results:Expression of a truncated syntaxin fragment (residues 1–106) antagonized isoflurane sensitivity in C. elegans. This portion of syntaxin interacts with the presynaptic protein UNC-13, suggesting the hypothesis that truncated syntaxin binds to UNC-13 and antagonizes an inhibitory effect of isoflurane on UNC-13 function. Consistent with this hypothesis, overexpression of UNC-13 suppressed the isoflurane resistance of the truncated syntaxins, and unc-13 loss-of-function mutants were highly isoflurane resistant. Normal anesthetic sensitivity was restored by full-length UNC-13, by a shortened form of UNC-13 lacking a C2 domain, but not by a membrane-targeted UNC-13 that might bypass isoflurane inhibition of membrane translocation of UNC-13. Isoflurane was found to inhibit synaptic localization of UNC-13. Conclusions:These data show that UNC-13, an evolutionarily conserved protein that promotes neurotransmitter release, is necessary for isoflurane sensitivity in C. elegans and suggest that its vertebrate homologs may be a component of the general anesthetic mechanism.

Ceramides--Friend or Foe in Hypoxia?

Molecular details of how nematode cells respond to a low-oxygen environment suggest a possible mechanism by which mammalian cells survive hypoxic conditions.

Volatile Anesthetic Preconditioning Present in the Invertebrate Caenorhabditis elegans

Background:Volatile anesthetics (VAs) have been found to induce a delayed protective response called preconditioning to subsequent hypoxic/ischemic injury. VA preconditioning has been primarily studied in canine and rodent heart. A more genetically tractable model of VA preconditioning would be extremely useful. Here, the authors report the development of the nematode Caenorhabditis elegans as a model of VA preconditioning. Methods:Wild-type and mutant C. elegans were exposed to isoflurane, halothane, or air under otherwise identical conditions. After varying recovery periods, the animals were challenged with hypoxic, azide, or hyperthermic incubations. After recovery from these incubations, mortality was scored. Results:Isoflurane- and halothane-preconditioned animals had significantly reduced mortality to all three types of injuries compared with air controls. Concentrations as low as 1 vol% isoflurane (0.64 mm) and halothane (0.71 mm) induced significant protection. The onset and duration of protection after anesthetic were 6 and 9 h, respectively. A mutation that blocks inhibition of neurotransmitter release by isoflurane did not attenuate the preconditioning effect. A loss-of-function mutation of the Apaf-1 homolog CED-4 blocked the preconditioning effect of isoflurane, but mutation of the downstream caspase CED-3 did not. Conclusions:Volatile anesthetic preconditioning extends beyond the vertebrate subphylum. This markedly broadens the scope of VA preconditioning and suggests that its mechanisms are widespread across species and is a fundamental and evolutionarily conserved cellular response. C. elegans offers a means to dissect genetically the mechanism for VA preconditioning as illustrated by the novel finding of the requirement for the Apaf-1 homolog CED-4.