Volatile anaesthetic effects on phospholipid binding to synaptotagmin 1, a presynaptic Ca2+ sensor

D. Fu1, P. Vissavajjhala1 and H. C. Hemmings, Jr1,2,*

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.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Volatile anaesthetics have important effects on synaptic transmission in the CNS. Depression of excitatory transmission involves reduced transmitter release via unidentified presynaptic mechanisms. Synaptotagmin 1 is a synaptic vesicle-associated protein that regulates Ca2+-evoked transmitter release involving critical Ca2+/phospholipid interactions within its C2 domains.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite intense interest and scrutiny, the detailed mechanisms by which general anaesthetics alter neuronal function to produce amnesia, unconsciousness and immobility remain largely unknown. Ligand-gated ion channels are sensitive to clinical concentrations of a variety of volatile and intravenous general anaesthetics, and appear to underlie most postsynaptic anaesthetic effects.1 However, the synaptic effects of volatile anaesthetics are not fully explained by their postsynaptic actions on ligand-gated ion channels. Volatile anaesthetics also depress excitatory neurotransmission by poorly characterized presynaptic mechanisms.25

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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Fig 1 Role of synaptotagmin C2 domains in mediating Ca2+-dependent synaptic vesicle–membrane interactions. Interaction of Ca2+ with the tandem C2A and C2B domains of synaptotagmin 1 facilitates its interaction with negatively charged phospholipids in the plasma membrane. This interaction can trigger synchronous vesicle fusion mediated by further interactions with the t-SNARE proteins syntaxin and SNAP-25, components of the core vesicle fusion complex.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Reagents were purchased from Sigma (St Louis, MO, USA) or VWR (West Chester, PA, USA) unless stated otherwise. Phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 3H-labelled phosphatidylcholine was purchased from PerkinElmer (Boston, MA, USA). Inorganic analysis grade water (Fluka, now Sigma) was used for preparation of reaction buffers, calcium standards (Sigma) and anaesthetic stock solutions. Thymol-free halothane was from Halocarbon Products (River Edge, NJ, USA) and isoflurane was from Abbott Laboratories (North Chicago, IL, USA).

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 96–265 encompassing the C2A domain; C2B, a 519 base-pair insert encoding amino acid residues 249–421 encompassing the C2B domain; and C2AB, a 975 base-pair insert encoding amino acid residues 96–421 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 ml–1 ampicillin with vigorous agitation at 37°C to an OD600 of 0.6–0.8, 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 ml–1 aprotinin, 10 µM leupeptin and 1 µg ml–1 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 glutathione–Sepharose 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 sulphate–polyacrylamide 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-{alpha}-1,2-dipalmitoyl-3-phosphatidyl[N-methyl-3H]choline (specific activity 76 Ci mmol–1) 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 GST–synaptotagmin 1 fusion proteins immobilized on glutathione–Sepharose 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 2–3 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
Concentration–effect 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Synaptotagmin 1 contains two C-terminal C2 domains (C2A and C2B) that bind Ca2+ and phospholipids,15 17 which is a function essential to its role as a Ca2+ sensor in synaptic vesicle exocytosis.12 Fusion proteins encompassing these domains coupled to an N-terminal GST tag were prepared and purified by affinity chromatography for analysis of Ca2+/phospholipid binding as a functional assay. The purified recombinant fusion proteins attached to glutathione–Sepharose were used in binding assays with radiolabelled liposomes as described.15 All three fusion proteins bound mixed phospholipid liposomes composed of PS/PC (Fig. 2A) or PI/PC (Fig. 2B). Binding was mediated by the C2 domains since no interaction occurred between PS/PC liposomes (Fig. 3) or PI/PC liposomes (data not shown) and the GST tag alone. The C2A domain showed Ca2+-dependent binding to PS/PC and PI/PC liposomes, and the C2AB showed Ca2+-dependent binding to PI/PC liposomes (Fig. 2) as described.15 Binding of C2B to PS/PC and PI/PC and of C2AB to PS/PC was Ca2+-independent (Fig. 2). The magnitude of PS/PC liposome binding was greatest to the C2B domain, with slightly less binding to the C2A and tandem C2AB domains. Binding of PI/PC was almost twofold greater to the C2A domain than to the C2B or C2AB domains.



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Fig 2 Anaesthetic effects on phospholipid binding to C2 domains of synaptotagmin 1. Recombinant rat GST-synaptotagmin 1 fusion proteins containing the first (C2A), second (C2B) or both (C2AB) C2 domains were immobilized on glutathione–Sepharose beads. Beads were incubated with [3H]phospholipid liposomes composed of: (A) phosphatidylserine and phosphatidylcholine (PS/PC); or (B) phosphatidylinositol and phosphatidylcholine (PI/PC); in the absence or presence of Ca2+ 0.2 mM, halothane 1.26 mM, and/or isoflurane 1.04 mM, as indicated. Beads were washed and the bound phospholipids were quantified by liquid scintillation counting. Data are shown for a single representative experiment (n=3) as mean (SD) of triplicate determinations. There was no significant effect by either anaesthetic on binding (ANOVA).

 


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Fig 3 Calcium concentration-dependence of PS/PC binding to C2A domain of synaptotagmin 1 with and without (A) halothane and (B) isoflurane. Recombinant rat GST-synaptotagmin 1 C2A domain fusion protein immobilized on glutathione–Sepharose beads was incubated with [3H]-labelled PS/PC liposomes in the presence of the concentrations of Ca2+ indicated in the absence or presence of halothane (A) or isoflurane (B). Data are shown for a single representative experiment (n=3) as mean (SD) of triplicate determinations. Data were fitted to sigmoidal concentration–effect curves for determination of Emax and EC50 values, which did not differ between experimental groups (ANOVA).

 
Halothane and isoflurane inhibit neurotransmitter release from isolated nerve terminals at clinically relevant concentrations.2 18 19 Neither anaesthetic at concentrations of ~3 times MAC (0.35 mM for halothane and isoflurane in rat20) had a discernible effect on the interaction of synaptotagmin 1 C2 domains with PS/PC or PI/PC liposomes in the absence or presence of a saturating Ca2+ concentration (Fig. 2; P<0.05 by ANOVA with Newman–Kuels post hoc test). The possibility that volatile anaesthetics might affect the Ca2+ sensitivity of synaptotagmin 1 binding to phospholipids was investigated by determining the effects of halothane and isoflurane on the Ca2+ dependence of C2A domain interactions with PS/PC liposomes (Fig. 3). There was no significant effect of halothane or isoflurane at 0.5 or 1.0 mM on the Ca2+-dependence of GST-C2A binding.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Neurotransmitter release is triggered by nerve terminal depolarization leading to activation of voltage-gated ion channels, Ca2+ influx leading to increases in intracellular Ca2+, which then activates fusion/endocytosis of neurotransmitter-containing synaptic vesicles with the presynaptic plasma membrane.6 7 Previous studies have implicated synaptic vesicle function as a target for general anaesthetics. Chemically evoked release of glutamate and GABA from isolated rat cortical nerve terminals is inhibited by halothane and isoflurane.2 1719 21 These findings have recently been confirmed by demonstrations that isoflurane inhibits action potential-evoked exocytosis in cultured rat hippocampal neurons22 and in rat calyceal terminals.23 Collectively, these results support a presynaptic mechanism for inhibition of evoked synaptic vesicle exocytosis by volatile general anaesthetics without significant effects on vesicular endocytosis. Possible targets for inhibition of exocytosis by volatile anaesthetics include presynaptic ion channels, receptors, and the vesicle fusion machinery itself.

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 evidence23–25 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.


    Acknowledgments
 
This work was supported by National Institutes of Health grants GM 58055 and GM 61925. We thank Dr R. Jahn for helpful discussions.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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