Drosophila ryanodine receptors mediate general anesthesia by halothane

Abstract

Background: Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and the authors hypothesized that it has a central role in anesthesia.

Methods: Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca imaging and patch clamp electrophysiology.

Results: Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in the mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca flux that is altered in dRyr mutants and correlates with strong hyperpolarization.

Conclusions: In Drosophila, neurally expressed dRyr mediates a substantial proportion of the anesthetic effects of halothane in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. The authors' results provide support for Ryr as an important mediator of immobilization by volatile anesthetics.

Figure 1

dRyr mutants display preferential resistance to halothane. (A) Genetic map of dRyr region. The insertion sites of dRyr^GS21220 (blue triangle) and dRyr^k04913 (green triangle) are diagrammed above the genomic structure. The dashed line below the dRyr gene indicates the genomic segment deleted in dRyr^Δ25. The genomic extent of CH321-24D03, which was used to generate the duplication strain dRyr^24D03, is represented by the grey bar at the bottom. (B) dRyr mutants show resistance to the volatile anesthetic halothane in the reactive climbing assay. Canton-S flies (control, black triangles) respond to halothane in a concentration-dependent fashion (solid line is a fit of the data in the logit model). The concentration-response curves of dRyr^GS21220/+ (blue squares) and dRyr^k04913/+ (green circles) shift to the right relative to the control, indicating increased resistance. dRyr^k04913/dRyr^GS21220 (red diamonds) shows an even stronger halothane resistance. (C) dRyr protein expression is decreased in dRyr mutants. (C1) Quantification of dRyr, normalized to Na,K-ATPase. Values are mean ± SEM, with statistical significance indicated by an asterisk (*; P = 0.0005). (n = 8). (C2) A representative western blot. (D) dRyr mutants affect halothane anesthesia in preference to the effects of other anesthetics. Data are plotted as the shift in EC₅₀, the concentration at which half of the flies are down, compared to the wildtype. dRyr mutants, dRyr^GS21220/+ (blue); dRyr^k04913/+ (green); and dRyr^k04913/dRyr^GS21220 (red) have a strong effect on halothane sensitivity, a weak effect on the sensitivity to sevoflurane, enflurane, and isoflurane. Detailed comparisons are described in Results. Values are mean ± SEM.

Figure 2

Sensitivity to halothane follows dRyr copy number. (A) Reduced dRyr copy number is associated with resistance to halothane. Heterozygous dRyr deletion mutant dRyr^Δ25/+ (medium grey), carrying one copy of dRyr, is resistant to halothane compared to the control, Canton-S, which carries two copies. dRyr^Δ25/dRyr^GS21220 transheterozygotes (light grey) are resistant compared to the wildtype and dRyr^GS21220/+ (dark gray). (B) The dRyr duplication strain dRyr^24D03/+ (black), carrying three copies of dRyr, is more sensitive to halothane than its control (VK33 without insertion, carrying two copies of dRyr). Introducing dRyr^24D03 into insertion mutant dRyr^GS21220 (dRyr^GS21220/+;dRyr^24D03/+, dark grey) rescues the resistant phenotype of dRyr^GS21220/+ (gray) to normal levels. Values are mean ± SEM (C) In the absence of anesthetic dRyr^Δ25/+ (grey), dRyr^GS21220/+ (dark grey), and dRyr^24D03(black) display normal locomotor activity, while Ryr^Δ25/dRyr^GS21220 (light grey) is significantly less active than control flies. Values are mean ± SEM (n = 3-5), and asterisk denotes statistical significance (P = 0.0006, one-way ANOVA and Bonferroni-Dunn post-hoc tests). (D) dRyr copy number affects dRyr protein expression. (D1) Quantification of dRyr protein levels. Asterisks denote statistically significant difference from the wildtype (P < 0.0001 and P < 0.0001, one-way ANOVA and Bonferroni-Dunn post-hoc tests). Values are mean ± s.e.m. (n = 8) (D2) A representative western blot, showing trend of increasing dRyr expression with the number of dRyr copies.

Figure 3

Point mutations in dRyr change halothane sensitivity of Drosophila. (A) Schematic illustration shows predicted motifs in the primary sequence of Drosophila Ryr, including MIR domains, SPRY domains, FKBP12- binding domain, Ca²⁺-binding domain, calmodulin-binding domain, six trans-membrane segments (TM), and the Ca²⁺ pore. Sites of amino acid alterations (arrows) in the five dRyr alleles described in the main text. Red arrows indicate nonsense mutations and black arrows indicate missense mutations. (B) Alignment of dRyr (top line; accession NP_476994.1) and the human cardiac Ryr, hRyr2 (second line; accession NP_001026.2), in the region spanning the missense alleles dRyr^R4305C and dRyr^E4340K. Sites of amino acid substitutions are indicated by boxes. In the third line, identities between the sequences are indicated by the amino acid symbol, conservative substitutions by plus signs, and non-conservative substitutions by blank spaces. (C) Point mutations in dRyr alter halothane sensitivity. Nonsense mutations dRyr^Y4452X/+ and dRyr^Q3878X/+ (red) are resistant to halothane, whereas missense mutations dRyr^R4305C/+, dRyr^E4340K/+, and dRyr^P2773L/+ (black) display hypersensitivity to halothane. All mutant values are significantly different from their matched wild-type controls (P = 0.0043 for dRyr^R4305C/+ and P < 0.0001 for all others, one-way ANOVA and Bonferroni-Dunn post-hoc tests). Values are mean ± SEM.

Figure 4

dRyr expressed in the nervous system is required for normal halothane sensitivity. (A) dRyr activity in the nervous system, but not in muscle, is required for normal halothane sensitivity in Drosophila. Flies expressing RNAi against dRyr (UAS-dRyr^RNAi) under the control of the muscle-specific driver MHC-GAL4 (MHC->dRyr^RNAi) are not different from controls (MHC-GAL4 alone) in the reactive climbing assay for halothane (P = 1, two-way ANOVA and Bonferroni-Dunn post-hoc tests). In contrast, elav->dRyr^RNAi and Appl->dRyr^RNAi flies, in which dRyr expression in neurons is specifically inhibited, display strong resistance to halothane compared to controls (elav-GAL4 and Appl-GAL4, respectively). Expressing UAS-dRyr^RNAi using the glial-specific driver repo-GAL4 (repo->dRyr^RNAi) significantly increases resistance to halothane (P = 0.0373 two-way ANOVA and Bonferroni-Dunn post-hoc tests). Broad expression in neurons and glia, driven by nrv2-GAL4 (nrv2->dRyr^RNAi), does not cause significant resistance (P = 0.0862. two-way ANOVA and Bonferroni-Dunn post-hoc tests). Values are mean ± SEM. (B) dRyr expressed in the nervous system is sufficient for halothane sensitivity. Restoring dRyr expression in the nervous system using nrv2-GAL4 to drive UAS-dRyr rescues the resistant phenotype in mutant dRyr^k04913/dRyr^GS21220. (C) Restoring dRyr expression in the nervous system in rescue flies (dRyr^k04913/dRyr^GS21220; nrv2->dRyr) is confirmed by western blot of membrane extracts from fly heads. (C1) The bar graph shows that dRyr levels increase significantly (Asterisk) over the negative control (P < 0.0001, Student's t-test; n = 8). Values are mean ± s.e.m. (C2) Representative western blot. As shown in the bar graph, dRyr protein is present in the mutants, but not visible in this exposure.

Figure 5

Halothane induces Ca²⁺ release from internal stores in Sf9 cells stably transfected with dRyr (Sf9+dRyr). Flow cytometry was used to measure [Ca²⁺]_i in Sf9 cells loaded with Fluo5-AM. (A) Representative histograms from flow cytometry analysis of Sf9+dRyr cells treated with halothane. Plots show the number of cells at each level of fluorescence (arbitrary units, A.U.), normalized to the modal value (% of Max). (A1) Treatment with 1.43 mM halothane results in a high proportion of cells with high fluorescence intensity. (A2, A3) Intermedate concentrations of halothane shift the proportions of cells with high vs. low fluorescence. (A4) In the absence of halothane, Sf9+dRyr cells show low levels of fluorescence. Each histogram represents 10,000 cells. (B) Depletion of intracellular Ca²⁺ with thapsigargin blocks the halothane-induced [Ca²⁺]_i increase in Sf9+dRyr cells. (B1) Pre-treatment with thapsigargin, which blocks the sarco/endoplasmic reticulum Ca²⁺ ATPase and results in store depletion, blocks the effect of halothane, compared to cells treated with thapsigargin alone (B2). (B3) Treatment of cells with a high concentration of halothane (2.5 mM), the majority show elevated fluorescence compared to untreated cells (B4). Note that a subpopulation of cells in this experiment did not respond to halothane. (C) Untransfected Sf9 cells do not respond to halothane. Fluorescence does not increase in Sf9 cells treated with 0.86 mM halothane (grey line), compared to untreated controls (black line). (D) Concentration-response curves for anesthetic-induced Ca²⁺ flux. Each data point represents the proportion of cells with elevated [Ca²⁺]_i in a sample of 10,000 cells. The proportion of Sf9+dRyr cells with elevated [Ca²⁺]_i increases as a function of anesthetic concentration, with halothane (filled circles) being many-fold more potent than sevoflurane (filled squares) or isoflurane (open triangles) in stimulating Ca²⁺ release via dRyr. Untransfected Sf9 cells do not respond to halothane (open diamonds), even at high concentrations (>2mM).

Figure 6

Halothane increases [Ca²⁺]_i and hyperpolarizes motoneuron RP2. (A) Halothane treatment increases [Ca²⁺]_i in larval motoneuron RP2. Changes in [Ca²⁺]_i in RP2 cell bodies are visualized using the genetically encoded Ca²⁺ indicator UAS-GCaMP3 driven by ShakB-GAL4. As shown in this false color image of four cells in two abdominal segments, [Ca²⁺]_i increases with the addition of halothane (2.5 mM). Scale bar: 10 μm. (B) Representative traces of calcium transients observed in the somata of RP2 neurons with halothane treatment. The entire soma was defined as the region of interest, and fluorescence intensity was converted into a % change (ΔF/F) as described in Experimental Procedures. (B1) All cells produced an immediate response, with a sharp rise, that occurred within a short period of halothane entering the chamber. [Ca²⁺]_i began to decay before halothane was removed. (B2) At higher concentrations, a pronounced undershoot, in which [Ca²⁺]_i dropped below baseline, followed halothane removal (arrow). (B3) A minority of cells produced a delayed response at the time of halothane removal, consisting of a plateau with superimposed spiky transients (arrowhead). (C) The concentration-response relationship for the peak amplitude of the halothane-induced calcium transient response in RP2 neurons. RP2 responded to halothane in a concentration-dependent manner, with EC₅₀ = 0.61 mM. Values are mean ± SEM (n = 3-8 preparations per data point). (D) dRyr mutations affect RP2's response to halothane. RP2 neurons in larvae heterozygous for dRyr^E4340K, which causes hypersensitivity to halothane in adult flies, respond more strongly to 0.5 mM halothane than controls. In contrast, halothane appears to induce smaller responses in RP2 neurons heterozygous for the deletion mutant dRyr^Δ25and the nonsense mutation dRyr^Q3878X. Error bars represent s.e.m., and asterisk denotes statistical significance by one-way ANOVA and Bonferroni-Dunn post-hoc tests (P = 0.0484). (E) dRyr mutations reduce sensitivity to caffeine. RP2 motoneurons from larvae heterozygous for dRyr^Q3878X and dRyr^E4340K respond more weakly to 5 mM caffeine than controls (P = 0.0375 and P = 0.0453, one-way ANOVA and Bonferroni-Dunn post-hoc tests). (F) Halothane hyperpolarizes wild-type RP2 neurons. In whole-cell current clamp recordings, halothane application produces a strong hyperpolarization that is proportional to halothane concentration. Recovery is delayed at 1 mM compared to 0.5 mM halothane. Membrane potentials are recorded using whole-cell current clamp recording. Values are mean ± SEM (n = 5 - 7).