Departments of 1 Anesthesiology and 2 Pharmacology, Weill Medical College of Cornell University, New York, NY, USA
* Corresponding author: Department of Anesthesiology, Box 50, Weill Cornell Medical College, 525 E. 68th Street, New York, NY 10021, USA. E-mail: hchemmi{at}med.cornell.edu
Accepted for publication April 12, 2005.
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Abstract |
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Methods. We analysed the effects of halothane and isoflurane on the binding of purified recombinant rat synaptotagmin 1 C2A, C2B and C2AB domains to radiolabelled phospholipid liposomes.
Results. Halothane and isoflurane had no significant effects on the maximal binding or Ca2+ dependence of binding of synaptotagmin 1 C2 domains to mixed phospholipid vesicles composed of either phosphatidylserine/phosphatidylcholine or phosphatidylinositol/phosphatidylcholine.
Conclusions. Inhibition of synaptic vesicle exocytosis by volatile anaesthetics does not appear to involve an effect on the critical Ca2+/phospholipid binding properties of synaptotagmin 1, a Ca2+ sensor involved in regulating evoked Ca2+-dependent neurotransmitter release.
Keywords: anaesthetics, volatile ; pharmacology, neurotransmission effects ; theories of anaesthetic action, molecular
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Introduction |
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Recent progress in defining the molecular and cellular details of neurotransmitter release and its regulation makes it possible to analyse anaesthetic effects on specific targets and steps involved in the synaptic vesicle exocyotosis/endocytosis cycle.67 Action potential-evoked neurotransmitter release is highly regulated and Ca2+-dependent. Docked vesicles fuse when triggered by Ca2+ influx through associated voltage-gated Ca2+ channels in a process catalysed by highly conserved presynaptic proteins which interact to form the four helical core soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.8 Screening existing mutants of the nematode Caenorhabditis elegans for alterations in anaesthetic sensitivity identified mutations in all three protein components of the core vesicle fusion complex.9 This evidence implicated the SNARE protein machinery involved in mediating synaptic vesicle exocytosis as a target for volatile anaesthetic effects on neurotransmitter release.
Synaptotagmins are synaptic vesicle-associated proteins that interact with SNARE complexes and are proposed to act as Ca2+ sensors for transmitter release (Fig. 1).6 Hippocampal neurons cultured from homozygous synaptotagmin 1-deficient mutant mice show selective loss of fast Ca2+-dependent neurotransmitter release while spontaneous and Ca2+-independent release are unimpaired.10 Synaptotagmin 1 contains a single transmembrane domain and two cytoplasmic C2 domains that bind Ca2+ and anionic phospholipids;11 the affinity of Ca2+ binding is greatly increased by the simultaneous binding of phospholipids.12 A C2A domain mutation that decreases Ca2+-dependent phospholipid binding by synaptotagmin 1 also impairs synaptic vesicle release, indicating the functional importance of this interaction.12 13 The critical role of synaptotagmin 1 in regulating synaptic exocytosis and its interactions with SNARE proteins that have been implicated by genetic screening as potential targets for volatile anaesthetics prompted us to examine the effects of an anaesthetic ether (isoflurane) and alkane (halothane) on Ca2+/phospholipid binding to isolated C2 domains of synaptotagmin 1.
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Materials and methods |
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Synaptotagmin 1 constructs
cDNA clones encompassing the cytoplasmic domain of rat synaptotagmin 1 were kindly provided by Dr R. H. Scheller.14 All constructs were fused to glutathione S-transferase (GST) on the 5'-end in the expression vector pGEX-KG using the restriction sites EcoRI and NcoI. The synaptotagmin 1 constructs used were: C2A, a 507 base-pair insert encoding amino acid residues 96265 encompassing the C2A domain; C2B, a 519 base-pair insert encoding amino acid residues 249421 encompassing the C2B domain; and C2AB, a 975 base-pair insert encoding amino acid residues 96421 encompassing both the C2A and C2B domains, as confirmed by DNA sequencing.
Preparation of GST fusion proteins
cDNA clones were introduced in BL21-competent E. coli cells (Stratagene, Cedar Creek, TX, USA), and the transformed bacteria were grown in 2x YT broth (Q-BIOgene, Carlsbad, CA, USA) plus 50 µg ml1 ampicillin with vigorous agitation at 37°C to an OD600 of 0.60.8, and then induced with 200 µM isopropyl ß-D-thiogalactopyranoside for 5 h. Cells were harvested by centrifugation, resuspended in PBS (10 mM K phosphate, 150 mM NaCl, pH 7.4) plus protease inhibitors (1 µg ml1 aprotinin, 10 µM leupeptin and 1 µg ml1 pepstatin A), and lysed by sonication for 60 s three times on ice. The lysate was centrifuged at 12 000 g at 4°C for 20 min, and the supernatant was subjected to batch purification using glutathioneSepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and elution with glutathione according to the manufacturer's instructions. Protein purity was >90% as assessed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Protein concentrations were determined using a Coomassie blue binding assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard.
Phospholipid binding assays
Liposome preparation
3H-labelled liposomes were prepared by the method of Davletov and Südhof15 with modifications. In 15-ml Corex glass tubes, 1.25 mg of phosphatidylcholine (PC) plus 0.5 mg of phosphatidylserine (PS) or phosphatidylinositol (PI) was combined with 5 µCi of L--1,2-dipalmitoyl-3-phosphatidyl[N-methyl-3H]choline (specific activity 76 Ci mmol1) as a tracer. The lipids were mixed, dried under a stream of argon, resuspended in 5 ml of Buffer A (50 mM HEPES, pH 7.2, 0.1 M NaCl) by vigorous vortexing for 3 min, and then sonicated for 30 s three times on ice using a probe sonicator at 20 kHz. The liposome suspension was centrifuged at 15 000 g at 4°C for 20 min to remove aggregates and stored at 4°C for use within 1 week.
Binding assays
Binding assays were performed in a reaction volume of 100 µl using 0.5-ml tubes (Sarstedt, Newton, NC, USA). Reactions contained GSTsynaptotagmin 1 fusion proteins immobilized on glutathioneSepharose beads (3 µg protein/10 µl beads), 10 µl of calcium solution in Buffer A, 50 µl (17.5 µg lipid) of liposome preparation, and 10 µl of stock anaesthetic solution in Buffer A, brought up to 100 µl with Buffer A. Sealed tubes were incubated at room temperature (25°C) for 15 min with brief vortexing every 23 min, before chilling on ice. The tubes were centrifuged at 6000 g for 3 min at 4°C, and the supernatant was carefully aspirated without disturbing the beads. The beads were washed three times with 300 µl of ice-cold buffer containing the same components as in the reaction buffer except the liposomes, and centrifuged at the same speed. The beads were transferred to scintillation vials for quantification of liposome binding by liquid scintillation counting (Beckman LS6000IC).
Anaesthetic preparation
Saturated anaesthetic stock solutions were prepared daily by stirring excess halothane or isoflurane with Buffer A at room temperature overnight. Concentrations of the saturated stock solutions and dilutions prepared in Buffer A were determined by gas chromatography after heptane extraction as described.16
Data analysis
Concentrationeffect data were fitted by least-squares analysis to estimate Emax, EC50 and Hill slope with standard errors (Prism v. 3.02; GraphPad Software, San Diego, CA, USA).
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Results |
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Discussion |
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We analysed the effects of volatile anaesthetics on Ca2+/phospholipid binding to synaptotagmin 1, a critical presynaptic component of the SNARE apparatus involved in mediating Ca2+-evoked exocytosis. Previous studies have implicated voltage-gated Na+ channels,17 19 21 22 Ca2+ channels18 and/or exocytotic SNARE proteins9 as targets for the presynaptic effects of volatile anaesthetics. Neurochemical evidence indicates that inhibition of transmitter release from isolated rodent cortical nerve terminals19 or of exocytosis in cultured hippocampal neurons22 occurs primarily at a target upstream of Ca2+ entry via the voltage-gated Ca2+ channels coupled to exocytosis. Electrophysiological evidence2325 supports depression of presynaptic action potential amplitude through Na+ channel block as a plausible mechanism for this effect, though actions on other presynaptic ion channels have not been ruled out. However, isoflurane also has a significant inhibitory action on exocytosis at a site downstream of Ca2+ entry,22 perhaps involving coupling of Ca2+ entry to exocytosis by synaptotagmin 1. The identification of C. elegans mutants with altered sensitivity to volatile anaesthetics that harbour mutations in the homologues of SNARE proteins suggests an effect of volatile anaesthetics on the regulation of vesicle fusion.9 We find that the volatile anaesthetics halothane and isoflurane do not affect the interactions of the critical C2 domains of the synaptic vesicle protein synaptotagmin 1 with phospholipids and Ca2+ in vitro. Although anaesthetic effects on other targets downstream from Ca2+ entry into the nerve terminal may contribute to the presynaptic effects of volatile anaesthetics, effects on the lipid and Ca2+ binding properties of the Ca2+ sensor synaptotagmin 1 do not appear to be involved.
Inhibition of excitatory glutamatergic transmission appears to be due to presynaptic inhibition of glutamate release: halothane inhibits NMDA (N-methyl-D-aspartate) and non-NMDA receptor-mediated excitatory postsynaptic currents in rat hippocampal slices at clinical concentrations that do not affect postsynaptic responses to exogenously applied agonists.3 In contrast, anaesthetic facilitation of GABAergic inhibitory transmission appears to primarily involve direct potentiation of postsynaptic and possibly extrasynaptic GABAA receptors.1 26 Isoflurane inhibits synaptic vesicle exocytosis evoked by action potential stimulation in cultured hippocampal neurons22 and glutamate release chemically evoked by 4-aminopyridine from isolated cortical nerve terminals2 19 with greater potency than that evoked by elevated KCl. This differential sensitivity suggests that inhibition of glutamate release results primarily from Na+ channel antagonism2 17 19 2427 with depression of presynaptic action potential amplitude23 25 28 and/or activation of anaesthetic activated two-pore domain K+ channels,29 although blockade of Ca2+ channels18 30 and effects on the synaptic vesicle fusion apparatus may also contribute to reduced transmitter release. The functional heterogeneity of presynaptic terminals suggests that the relative contributions of specific molecular mechanisms underlying presynaptic anaesthetic actions may vary between nerve terminal types, as seen in the differential sensitivity of glutamatergic and GABAergic terminals to volatile anaesthetics.19 Our results indicate that a critical function of the presynaptic Ca2+ sensor protein synaptotagmin 1 is not affected by volatile anaesthetics, though other components of the SNARE apparatus may be anaesthetic-sensitive targets downstream of Ca2+ entry.
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Acknowledgments |
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References |
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