Drosophila Muscle Research Articles

Dominant myosin storage myopathy mutations disrupt striated muscles in Drosophila and the myosin tail-tail interactome of human cardiac thick filaments.

Myosin storage myopathy (MSM) is a rare skeletal muscle disorder caused by mutations in the slow muscle/β-cardiac myosin heavy chain (MHC) gene. MSM missense mutations frequently disrupt the tail's stabilizing heptad repeat motif. Disease hallmarks include subsarcolemmal hyaline-like β-MHC aggregates, muscle weakness and, occasionally, cardiomyopathy. We generated transgenic, heterozygous Drosophila to examine the dominant physiological and structural effects of the L1793P, R1845W, and E1883K MHC MSM mutations on diverse muscles. The MHC variants reduced lifespan and flight and jump abilities. Moreover, confocal and electron microscopy revealed that they provoked indirect flight muscle breaks and myofibrillar disarray/degeneration with filamentous inclusions. Incorporation of GFP-myosin enabled in situ determination of thick filament lengths, which were significantly reduced in all mutants. Semi-automated heartbeat analysis uncovered aberrant cardiac function, which worsened with age. Thus, our fly models phenocopied traits observed among MSM patients. We additionally mapped the mutations onto a recently-determined, 6Å resolution, cryo-EM structure of the human cardiac thick filament. The R1845W mutation replaces a basic arginine with a polar-neutral, bulkier tryptophan, while E1883K reverses charge at critical filament loci. Both would be expected to disrupt the core and the outer shell of the backbone structure. Replacing L1793 with a proline, a potent breaker of alpha-helices, could disturb the coiled-coil of the myosin rod and alter the tail-tail interactome. Hence, all mutations likely destabilize and weaken the filament backbone. This may trigger disease in humans, while potentially analogous perturbations are likely to yield the observed thick filament and muscle disruption in our fly models.

Actin-driven nanotopography promotes stable integrin adhesion formation in developing tissue

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

EMC1 Is Required for the Sarcoplasmic Reticulum and Mitochondrial Functions in the Drosophila Muscle

EMC1 is part of the endoplasmic reticulum (ER) membrane protein complex, whose functions include the insertion of transmembrane proteins into the ER membrane, ER–mitochondria contact, and lipid exchange. Here, we show that the Drosophila melanogaster EMC1 gene is expressed in the somatic musculature and the protein localizes to the sarcoplasmic reticulum (SR) network. Muscle-specific EMC1 RNAi led to severe motility defects and partial late pupae/early adulthood lethality, phenotypes that are rescued by co-expression with an EMC1 transgene. Motility impairment in EMC1-depleted flies was associated with aberrations in muscle morphology in embryos, larvae, and adults, including tortuous and misaligned fibers with reduced size and weakness. They were also associated with an altered SR network, cytosolic calcium overload, and mitochondrial dysfunction and dysmorphology that impaired membrane potential and oxidative phosphorylation capacity. Genes coding for ER stress sensors, mitochondrial biogenesis/dynamics, and other EMC components showed altered expression and were mostly rescued by the EMC1 transgene expression. In conclusion, EMC1 is required for the SR network’s mitochondrial integrity and influences underlying programs involved in the regulation of muscle mass and shape. We believe our data can contribute to the biology of human diseases caused by EMC1 mutations.

Dysregulated FOXO1 activity drives skeletal muscle intrinsic dysfunction in amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder, with accumulating evidence indicating metabolic disruptions in the skeletal muscle preceding disease symptoms, rather than them manifesting as a secondary consequence of motor neuron (MN) degeneration. Hence, energy homeostasis is deeply implicated in the complex physiopathology of ALS and skeletal muscle has emerged as a key therapeutic target. Here, we describe intrinsic abnormalities in ALS skeletal muscle, both in patient-derived muscle cells and in muscle cell lines with genetic knockdown of genes related to familial ALS, such as TARDBP (TDP-43) and FUS. We found a functional impairment of myogenesis that parallels defects of glucose oxidation in ALS muscle cells. We identified FOXO1 transcription factor as a key mediator of these metabolic and functional features in ALS muscle, via gene expression profiling and biochemical surveys in TDP-43 and FUS-silenced muscle progenitors. Strikingly, inhibition of FOXO1 mitigated the impaired myogenesis in both the genetically modified and the primary ALS myoblasts. In addition, specific in vivo conditional knockdown of TDP-43 or FUS orthologs (TBPH or caz) in Drosophila muscle precursor cells resulted in decreased innervation and profound dysfunction of motor nerve terminals and neuromuscular synapses, accompanied by motor abnormalities and reduced lifespan. Remarkably, these phenotypes were partially corrected by foxo inhibition, bolstering the potential pharmacological management of muscle intrinsic abnormalities associated with ALS. The findings demonstrate an intrinsic muscle dysfunction in ALS, which can be modulated by targeting FOXO factors, paving the way for novel therapeutic approaches that focus on the skeletal muscle as complementary target tissue.

The effects of doxapram and its potential interactions with K2P channels in experimental model preparations.

The channels commonly responsible for maintaining cell resting membrane potentials are referred to as K2P (two-P-domain K+ subunit) channels. These K+ ion channels generally remain open but can be modulated by their local environment. These channels are classified based on pharmacology, pH sensitivity, mechanical stretch, and ionic permeability. Little is known about the physiological nature of these K2P channels in invertebrates. Acidic conditions depolarize neurons and muscle fibers, which may be caused by K2P channels given that one subtype can be blocked by acidic conditions. Doxapram is used clinically as a respiratory aid known to block acid-sensitive K2P channels; thus, the effects of doxapram on the muscle fibers and synaptic transmission in larval Drosophila and crawfish were monitored. A dose-dependent response was observed via depolarization of the larval Drosophila muscle and an increase in evoked synaptic transmission, but doxapram blocked the production of action potentials in the crawfish motor neuron and had a minor effect on the resting membrane potential of the crawfish muscle. This indicates that the nerve and muscle tissues in larval Drosophila and crawfish likely express different K2P channel subtypes. Since these organisms serve as physiological models for neurobiology and physiology, it would be of interest to further investigate what types of K2P channel are expressed in these tissues. (212 words).

Activin is a neural inducer of a male-specific muscle in Drosophila

Drosophila melanogaster has a pair of male-specific muscles called the muscle of Lawrence (MOL) in abdominal segment 5 (A5) of adult flies. The MOL is produced only when its innervating motoneuron expresses FruitlessM (FruM) neural masculinizing proteins. We show that MOL induction is hampered by: (1) silencing electrical activities in the motoneuron, (2) blocking vesicular release from the motoneuron, and (3) knocking down Activin ß (Actß) in the motoneuron or knocking down Actß signaling pathway components in the myoblasts. Our timelapse live imaging of the developing neuromuscular system reveals that, upon contact with the presumptive MOL, the motoneuronal axon retracts concomitant with the progression of MOL degeneration resulting from neural silencing. We conclude that MOL formation depends on the bidirectional trophic interactions between pre- and postsynaptic cells, with motoneuron-derived Actß playing an inducing role in MOL formation.

Fluvastatin-induced myofibrillar damage is associated with elevated ROS, and impaired fatty acid oxidation, and is preceded by mitochondrial morphological changes

Previously, we showed that fluvastatin treatment induces myofibrillar damage and mitochondrial phenotypes in the skeletal muscles of Drosophila. However, the sequential occurrence of mitochondrial phenotypes and myofibril damage remains elusive. To address this, we treated flies with fluvastatin for two and five days and examined their thorax flight muscles using confocal microscopy. In the two-day fluvastatin group, compared to the control, thorax flight muscles exhibited mitochondrial morphological changes, including fragmentation, rounding up and reduced content, while myofibrils remained organized in parallel. In the five-day fluvastatin treatment, not only did mitochondrial morphological changes become more pronounced, but myofibrils became severely disorganized with significantly increased thickness and spacing, along with myofilament abnormalities, suggesting myofibril damage. These findings suggest that fluvastatin-induced mitochondrial changes precede myofibril damage. Moreover, in the five-day fluvastatin group, the mitochondria demonstrated elevated H2O2 and impaired fatty acid oxidation compared to the control group, indicating potential mitochondrial dysfunction. Surprisingly, knocking down Hmgcr (Drosophila homolog of HMGCR) showed normal mitochondrial respiration in all parameters compared to controls or five-day fluvastatin treatment, which suggests that fluvastatin-induced mitochondrial dysfunction might be independent of Hmgcr inhibition. These results provide insights into the sequential occurrence of mitochondria and myofibril damage in statin-induced myopathy for future studies.

Myosin storage myopathy mutations disrupt striated muscles in Drosophila and the myosin tail-tail interactome of human cardiac thick filaments

Myosin storage myopathy mutations disrupt striated muscles in Drosophila and the myosin tail-tail interactome of human cardiac thick filaments

PIGB maintains nuclear lamina organization in skeletal muscle of Drosophila.

The nuclear lamina (NL) plays various roles and participates in nuclear integrity, chromatin organization, and transcriptional regulation. Lamin proteins, the main components of the NL, form a homogeneous meshwork structure under the nuclear envelope. Lamins are essential, but it is unknown whether their homogeneous distribution is important for nuclear function. Here, we found that PIGB, an enzyme involved in glycosylphosphatidylinositol (GPI) synthesis, is responsible for the homogeneous lamin meshwork in Drosophila. Loss of PIGB resulted in heterogeneous distributions of B-type lamin and lamin-binding proteins in larval muscles. These phenotypes were rescued by expression of PIGB lacking GPI synthesis activity. The PIGB mutant exhibited changes in lamina-associated domains that are large heterochromatic genomic regions in the NL, reduction of nuclear stiffness, and deformation of muscle fibers. These results suggest that PIGB maintains the homogeneous meshwork of the NL, which may be essential for chromatin distribution and nuclear mechanical properties.

Identification of CryAB as a target of NUAK kinase activity in Drosophila muscle tissue.

Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. Here, we characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. We further show that a phospho-mimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis.

The RNF220 domain nuclear factor Teyrha-Meyrha (Tey) regulates the migration and differentiation of specific visceral and somatic muscles in Drosophila.

Development of the visceral musculature of the Drosophila midgut encompasses a closely coordinated sequence of migration events of cells from the trunk and caudal visceral mesoderm that underlies the formation of the stereotypic orthogonal pattern of circular and longitudinal midgut muscles. Our study focuses on the last step of migration and morphogenesis of longitudinal visceral muscle precursors and shows that these multinucleated precursors utilize dynamic filopodial extensions to migrate in dorsal and ventral directions over the forming midgut tube. The establishment of maximal dorsoventral distances from one another, and anteroposterior alignments, lead to the equidistant coverage of the midgut with longitudinal muscle fibers. We identify Teyrha-Meyhra (Tey), a tissue-specific nuclear factor related to the RNF220 domain protein family, as a crucial regulator of this process of muscle migration and morphogenesis that is further required for proper differentiation of longitudinal visceral muscles. In addition, Tey is expressed in a single somatic muscle founder cell in each hemisegment, regulates the migration of this founder cell, and is required for proper pathfinding of its developing myotube to specific myotendinous attachment sites.

Atl (atlastin) regulates mTor signaling and autophagy in Drosophila muscle through alteration of the lysosomal network

ABSTRACT The hereditary spastic paraplegias (HSPs) represent a family of genetic disorders comprising at least 72 different genes with the common pathology of progressive locomotor deficits and spasticity. ATL1/SPG3A (atlastin GTPase 1) encodes an ER fusion protein that controls ER morphology, which implicates ER structure as a causal factor in HSP. Here we use Drosophila to study effects of decreased atl (atlastin) on properties of the larval body wall muscle. We found that muscle atl loss causes accumulation of aggregates containing polyubiquitin (polyUB), mostly bound to the autophagy receptor ref(2)P/SQSTM1/p62. Muscle atl loss also decreased volume and complexity of the endolysosomal network and decreased lysosome number. To determine effects of these lysosomal deficits on progression through the basal autophagy pathway, we expressed Atg8a tagged with both GFP and mCherry in a wild-type and atl mutant background. We found numerous structures containing mCherry but not GFP fluorescence in wild type, indicating that Atg8a was found mostly in mature autolysosomes. In contrast, muscles lacking atl exhibited significant amounts of GFP signal, indicating failure of autophagosome maturation with acidic lysosomes. Many of these GFP-positive puncta contained the late-endosome marker Rab7 but not Lamp1, indicating that some autophagy cargo was accumulating within amphisomes. We also found that this autophagy block was accompanied by an inability to activate the mTor kinase. Our results provide mechanistic insights into the role of atl in maintaining proper function of the autophagy pathway and suggests that certain pathologies in patients with mutations in ATL1/SPG3A might result from altered MTOR signaling. Abbreviations atl atlastin; ALR autophagic lysosome reformation; ER endoplasmic reticulum; GFP green fluorescent protein; HSP hereditary spastic paraplegia; Lamp1 lysosomal associated membrane protein 1 PolyUB polyubiquitin; RFP red fluorescent protein; spin spinster; mTor mechanistic Target of rapamycin; VCP valosin containing protein

A Novel Interaction between MFN2/Marf and MARK4/PAR-1 Is Implicated in Synaptic Defects and Mitochondrial Dysfunction.

As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2 (MFN2)/mitochondrial assembly regulatory factor (Marf), plays a role in mitochondrial fusion, mutations of which are implicated in age-related human diseases, including several neurodegenerative disorders. However, the regulation of MFN2/Marf-mediated mitochondrial fusion, as well as the pathologic mechanism of neurodegeneration, is not clearly understood. Here, we identified a novel interaction between MFN2/Marf and microtubule affinity-regulating kinase 4 (MARK4)/PAR-1. In the Drosophila larval neuromuscular junction, muscle-specific overexpression of MFN2/Marf decreased the number of synaptic boutons, and the loss of MARK4/PAR-1 alleviated the synaptic defects of MFN2/Marf overexpression. Downregulation of MARK4/PAR-1 rescued the mitochondrial hyperfusion phenotype caused by MFN2/Marf overexpression in the Drosophila muscles as well as in the cultured cells. In addition, knockdown of MARK4/PAR-1 rescued the respiratory dysfunction of mitochondria induced by MFN2/Marf overexpression in mammalian cells. Together, our results indicate that the interaction between MFN2/Marf and MARK4/PAR-1 is fine-tuned to maintain synaptic integrity and mitochondrial homeostasis, and its dysregulation may be implicated in neurologic pathogenesis.

Bypassing mitochondrial defects rescues Huntington's phenotypes in Drosophila

Huntington's disease (HD) is a fatal neurodegenerative disease with limited treatment options. Human and animal studies have suggested that metabolic and mitochondrial dysfunctions contribute to HD pathogenesis. Here, we use high-resolution respirometry to uncover defective mitochondrial oxidative phosphorylation and electron transfer capacity when a mutant huntingtin fragment is targeted to neurons or muscles in Drosophila and find that enhancing mitochondrial function can ameliorate these defects. In particular, we find that co-expression of parkin, an E3 ubiquitin ligase critical for mitochondrial dynamics and homeostasis, produces significant enhancement of mitochondrial respiration when expressed either in neurons or muscles, resulting in significant rescue of neurodegeneration, viability and longevity in HD model flies. Targeting mutant HTT to muscles results in larger mitochondria and higher mitochondrial mass, while co-expression of parkin increases mitochondrial fission and decreases mass. Furthermore, directly addressing HD-mediated defects in the fly's mitochondrial electron transport system, by rerouting electrons to either bypass mitochondrial complex I or complexes III-IV, significantly increases mitochondrial respiration and results in a striking rescue of all phenotypes arising from neuronal mutant huntingtin expression. These observations suggest that bypassing impaired mitochondrial respiratory complexes in HD may have therapeutic potential for the treatment of this devastating disorder.

Lmpt regulates the function of Drosophila muscle by acting as a repressor of Wnt signaling

BackgroundLIM domain is considered to be important in mediating protein–protein interactions, and members of the LIM protein family can co-regulate tissue-specific gene expression by interacting with different transcription factors. However, its exact function in vivo remains unclear. Our study demonstrates that the LIM protein family member Lmpt may act as a cofactor that interacts with other transcription factors to regulate cellular functions. MethodsIn this study, we generated Lmpt knockdown Drosophila (Lmpt-KD) using the UAS-Gal4 system. We assessed the lifespan and motility of Lmpt-KD Drosophila and analyzed the expression of muscle-related and metabolism-related genes using qRT-PCR. Additionally, we utilized Western blot and Top-Flash luciferase reporter assay to evaluate the level of the Wnt signaling pathway. ResultsOur study revealed that knockdown of the Lmpt gene in Drosophila resulted in a shortened lifespan and reduced motility. We also observed a significant increase in oxidative free radicals in the fly gut. Furthermore, qRT-PCR analysis indicated that knockdown of Lmpt led to decreased expression of muscle-related and metabolism-related genes in Drosophila, suggesting that Lmpt plays a crucial role in maintaining muscle and metabolic functions. Finally, we found that reduction of Lmpt significantly upregulated the expression of Wnt signaling pathway proteins. ConclusionOur results demonstrate that Lmpt is essential for motility and survival in Drosophila and acts as a repressor in Wnt signaling.

Sorbitol reduction via AT-007 (govorestat) ameliorates synaptic dysfunction and neurodegeneration in models of sorbitol dehydrogenase deficiency.

Sorbitol dehydrogenase (SORD) deficiency has been identified as the most frequent autosomal recessive form of hereditary neuropathy. Loss of SORD causes high sorbitol levels in tissues due to the inability to convert sorbitol to fructose in the two-step polyol pathway, leading to degenerative neuropathy. The underlying mechanisms of sorbitol-induced degeneration have not been fully elucidated, and no current FDA-approved therapeutic options are available to reduce sorbitol levels in the nervous system. Here, in a Drosophila model of SORD deficiency, we showed synaptic degeneration in the brain, neurotransmission defect, locomotor impairment, and structural abnormalities in the neuromuscular junctions. In addition, we found reduced ATP production in the brain and reactive oxygen species accumulation in the central nervous system (CNS) and muscle, indicating mitochondrial dysfunction. Applied Therapeutics, Inc has developed a CNS-penetrant next-generation aldose reductase inhibitor (ARI), AT-007 (govorestat), which inhibits the conversion of glucose to sorbitol. AT-007 significantly reduced sorbitol levels in patient-derived fibroblasts, iPSC-derived motor neurons, and Drosophila brains. AT-007 feeding in Sord-deficient Drosophila mitigated synaptic degeneration and significantly improved synaptic transduction, locomotor activity, and mitochondrial function. Moreover, AT-007 treatment significantly reduced ROS accumulation in Drosophila CNS, muscle, and patient-derived fibroblasts. These findings uncover the molecular and cellular pathophysiology of SORD neuropathy and provide a potential treatment strategy for patients with SORD deficiency.

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells.

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods areused to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment isvalidated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Role of muscle FOXO gene in exercise against the skeletal muscle and cardiac age-related defects and mortality caused by high-salt intake in Drosophila

FOXO has long been associated with aging, exercise, and tissue homeostasis, but it remains unclear what the role is of the muscle FOXO gene in E against high-salt intake(HSI)-induced age-related defects of the skeletal muscle, heart, and mortality. In this research, overexpression and RNAi of the FOXO gene in the skeletal and heart muscle of Drosophila were constructed by building Mhc-GAL4/FOXO-UAS-overexpression and Mhc-GAL4/FOXO-UAS-RNAi system. The skeletal muscle and heart function, the balance of oxidation and antioxidant, and mitochondrial homeostasis were measured. The results showed that exercise reversed the age-related decline in climbing ability and downregulation of muscle FOXO expression induced by HSI. Muscle-specific FOXO-RNAi (FOXO-RNAi) and -overexpression (FOXO-OE) promoted or slowed the age-related decline in climbing ability, heart function, and skeletal muscle and heart structure damage, which was accompanied by the inhibition or activation of FOXO/PGC-1α/SDH and FOXO/SOD pathway activity, and oxidative stress (ROS) increased or decreased in both skeletal muscle and heart. The protective effect of exercise on the skeletal muscle and heart was blocked by FOXO-RNAi in aged HSI flies. FOXO-OE prolonged its lifespan, but it did not resist the HSI-induced lifespan shortening. Exercise did not improve HSI-induced lifespan shortening in FOXO-RNAi flies. Therefore, current results confirmed that the muscle FOXO gene played a vital role in exercise against age-related defects of the skeletal muscle and heart induced by HSI because it determined the activity of muscle FOXO/SOD and FOXO/PGC-1α/SDH pathways. The muscle FOXO gene also played an important role in exercise against HSI-induced mortality in aging flies.

Longitudinal visceral muscles in Drosophila fully dedifferentiate and fragment prior to their reestablishment during metamorphosis.

Although the Drosophila longitudinal visceral muscles have been shown to undergo major morphological changes during the transition from larval to adult gut musculature, there have been conflicting views as to whether these muscles persist as such during metamorphosis or whether they are built anew (Klapper 2000; Aghajanian et al. 2016). Here we present our independent analysis using HLH54Fb-eGFP as a cell type specific marker, which strengthens the proposition by Aghajanian et al. (2016) that the syncytial larval longitudinal gut muscles completely dedifferentiate and fragment into mononucleated myoblasts during pupariation before they fuse again and redifferentiate to form the adult longitudinal gut muscles.

Hyperpolarization Induced by Lipopolysaccharides but Not by Chloroform Is Inhibited by Doxapram, an Inhibitor of Two-P-Domain K+ Channel (K2P)

Bacterial septicemia is commonly induced by Gram-negative bacteria. The immune response is triggered in part by the secretion of bacterial endotoxin lipopolysaccharide (LPS). LPS induces the subsequent release of inflammatory cytokines which can result in pathological conditions. There is no known blocker to the receptors of LPS. The Drosophila larval muscle is an amendable model to rapidly screen various compounds that affect membrane potential and synaptic transmission such as LPS. LPS induces a rapid hyperpolarization in the body wall muscles and depolarization of motor neurons. These actions are blocked by the compound doxapram (10 mM), which is known to inhibit a subtype of the two-P-domain K+ channel (K2P channels). However, the K2P channel blocker PK-THPP had no effect on the Drosophila larval muscle at 1 and 10 mM. These channels are activated by chloroform, which also induces a rapid hyperpolarization of these muscles, but the channels are not blocked by doxapram. Likewise, chloroform does not block the depolarization induced by doxapram. LPS blocks the postsynaptic glutamate receptors on Drosophila muscle. Pre-exposure to doxapram reduces the LPS block of these ionotropic glutamate receptors. Given that the larval Drosophila body wall muscles are depolarized by doxapram and hyperpolarized by chloroform, they offer a model to begin pharmacological profiling of the K2P subtype channels with the potential of identifying blockers for the receptors to mitigate the actions of the Gram-negative endotoxin LPS.