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WO1997021729A1 - Procedes et compositions pour la modulation de la liberation vesiculaire - Google Patents

Procedes et compositions pour la modulation de la liberation vesiculaire Download PDF

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Publication number
WO1997021729A1
WO1997021729A1 PCT/US1996/019661 US9619661W WO9721729A1 WO 1997021729 A1 WO1997021729 A1 WO 1997021729A1 US 9619661 W US9619661 W US 9619661W WO 9721729 A1 WO9721729 A1 WO 9721729A1
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Prior art keywords
tagmin
snap
amino acid
pins
nsf
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PCT/US1996/019661
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English (en)
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WO1997021729A9 (fr
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Giampietro Schiavo
James E. Rothman
Thomas H. SÖLLNER
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Sloan-Kettering Institute For Cancer Research
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Priority to AU13317/97A priority Critical patent/AU1331797A/en
Publication of WO1997021729A1 publication Critical patent/WO1997021729A1/fr
Publication of WO1997021729A9 publication Critical patent/WO1997021729A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to methods and compositions useful for modulating the release of vesicular contents from cells. Generally, this invention relates to modulating the process whereby intracellular vesicles fuse with the cell membrane to cause the release of the vesicular contents. More particularly, this invention relates to modulating the releases of synaptic vesicles from neurons. In addition, this invention relates to compositions and methods useful for modulating secretion of substances produced in cells. This invention also relates to methods and compositions useful for screening substances which modulate vesicle release.
  • Synaptic vesicle release is believed to result from at least a three step process following the appropriate electrical stimulus.
  • the synaptic vesicle In the first process, docking, the synaptic vesicle is mobilized from a pool of filled vesicles and binds to the presynaptic plasma membrane at active sites.
  • vesicle priming a slow ATP hydrolysis dependent step termed "vesicle priming” occurs. This priming process makes exocytotic synaptic vesicles competent for the third step, fast calcium dependent fusion.
  • the membranes of the synaptic vesicle and plasma membrane fuse in the last step releasing the vesicle contents into the synaptic cleft.
  • the proteins responsible for docking vesicles at the membrane are not yet clear, but may include the GTP- binding protein Rab3 as well as UNC-18 or SNARE proteins
  • ⁇ -SNAP an isoform of ⁇ -SNAP termed ⁇ -SNAP (83% identical to ⁇ -SNAP) is highly expressed in brain 9 ; no special role for ⁇ -SNAP has yet been reported. It has been unclear how, or if, these separate threads of understanding are entwined to account for synaptic transmission.
  • the present invention relates to methods and compositions useful for identifying substances capable of modulating synaptic transmission.
  • a protein complex has been identified which is involved in docking of synaptic vesicles to synaptic membranes in connection with synaptic transmission.
  • This complex is comprised of three proteins ⁇ -SNAP, tagmin, and NSF and the complex, according to this invention, may be used as a target for substances such as drugs which modulate synaptic transmission.
  • Intermediate complexes comprising ⁇ -SNAP and tagmin bind NSF and may also be a target for drugs which modulate synaptic transmission.
  • Another intermediate comprising tagmin, 0-SNAP, and NSF binds o-SNAP to form a quaternary complex which is capable of binding to SNAREs (syntaxin, VAMP and SNAP25) .
  • An aspect of this invention therefore is a cell free protein complex comprising a protein comprising amino acid sequences of ⁇ - SNAP sufficient to bind to tagmin and a protein comprising an amino acid sequence of tagmin capable of binding ⁇ - SNAP.
  • Another aspect of this invention is a cell free complex comprising a protein comprising amino acid sequences of ⁇ -SNAP sufficient to bind to tagmin, an amino acid sequence of tagmin capable of binding ⁇ -SNAP and NSF protein.
  • Tagmin//3-SNAP/NSF complex further comprising oc-SNAP and, optionally the SNAREs.
  • Complexes comprising tagmin and phosphatidylinositol bisphosphate ("PIns-P 2 ”) and phosphatidylinositol trisphosphate (“PIns-P 3 ”) are another aspect of this invention.
  • Another embodiment of this invention is the use of the formation of the cell free /3-SNAP/tagmin, 0-SNAP/tagmin/NSF, or /3-SNAP/tagmin/NSF/oc-SNAP or ⁇ -
  • this invention also relates to assay kits comprising the components to conduct such assays including 0-SNAP, tagmin and optionally NSF, oc-SNAP, SNARE proteins, PIns-P 2 , PIns-P 3 and various reagents for detecting the presence of a protein in the complex. These components should be present in an amount sufficient to detect complex formation in the absence of a test substance.
  • the method of identifying substances capable of inhibiting synaptic transmission comprises combining an amino acid sequence of ⁇ -SNAP capable of binding tagmin and a ⁇ -SNAP binding protein comprising an amino acid sequence of tagmin with a test substance under conditions which allow for ⁇ -SNAP and the ⁇ -SNAP binding protein to form a complex and then detecting the formation of ⁇ - SNAP//3-SNAP binding protein complexes.
  • the formation of a triple complex further comprising NSF or a quaternary complex further comprising oc-SNAP may be assayed in the presence of a test substance.
  • the complex comprising j ⁇ -SNAP/tagmin/NSF/oc-SNAP and at least one SNARE protein may also be used in this invention.
  • substances which are identified which reduce or inhibit the formation of any of the 3-SNAP/tagmin, ⁇ -SNAP/tagmin/NSF, ⁇ -SNAP/tagmin/NSF/oc- SNAP, jS- ⁇ NAP/tagmin/NSF/oc-sNAP/SNARE, tagmin/PIns-P 2 or tagmin/PIns-P 3 complexes may be expected to similarly reduce or inhibit synaptic transmission. Conversely, 5 substances capable of increasing complex formation would be expected to facilitate synaptic transmission.
  • another embodiment of this invention includes assays for detecting substances which modulate synaptic transmission which are specific for neurons or regions of the nervous system based on the
  • tagsmin 15 presence of a particular tagmin isoform. Accordingly, in this embodiment of the invention, assays are conducted using specific isoforms of tagmin which are specific for the region of the nervous system for which synaptic transmission is desired to be modified.
  • This invention also provides methods of
  • the synaptic vesicle membrane protein synaptotagmin (tagmin) is essential for fast, calcium- dependent, neurotransmitter release.
  • the present invention has identified a connection between the observations that polyphosphoinositides are necessary for exocytosis and the finding that the C2B domain of tagmin I binds phosphatidylinositol-4 , 5-bisphosphate (PIns-4 , 5-P 2 ) , its isomer phosphatidylinositol-3, 4-bisphosphate (Plns- 3,4-P 2 ) and phosphatidylinositol-3, 4,5-trisphosphate (PIns-3 , 4, 5-P 3 ) .
  • Tagmin operates as a bimodal calcium sensor, switching bound lipids during exocytosis, which is useful in treatment and maintenance of various physiological processes and disorders associated therewith, such as long-term memory and learning.
  • the present invention can also be used to treat various neurological disorders comprising increasing or decreasing the intracellular calcium levels in a patient.
  • the ability to specifically modulate synaptic transmission by identifying drugs which are specific for various isoforms of tagmin provides a means for specifically treating various neurological disorders associated with improper synaptic transmission; i.e. anatomically different areas of the nervous system.
  • Antibodies which are specific for the various complexes are also provided by this invention which may be used for either diagnostic or therapeutic purposes.
  • An object of this invention is to provide compositions and methods for identifying substances useful for deceasing or inhibiting functional docking or priming of vesicles to plasma membranes.
  • Another object of this invention is to provide compositions and methods useful for inhibiting synaptic transmission mediated through docking of synaptic vesicles to SNARES.
  • Another object of this invention is to provide substances useful for treating various neurological or psychiatric disorders. Yet another object of the present invention is to treat various neurological and psychiatric disorders by modulating neurotransmitter release by manipulating the intracellular calcium levels in a recipient with such a disorder. Another object of this invention is to provide assay systems and kits useful for identifying such substances.
  • synaptotagmin is localized to a transport vesicle, binds to a t-SNARE (syntaxin) 14 , is a specialized SNAP receptor (binds /3-SNAP, but not ⁇ -SNAP) , enters a docking and fusion particle, and is released when NSF hydrolyzes ATP.
  • the docking and fusion complexes provided by this invention could serve to link, directly or indirectly, the general process of membrane fusion to calcium entry by attaching a specialized fusion protein (0-SNAP) to a v-SNARE that is also a calcium sensor (synaptotagmin) .
  • a specialized fusion protein (0-SNAP)
  • v-SNARE v-SNARE
  • calcium sensor seaptotagmin
  • the polyphosphoinositols InsP 4 , InsP 5 , and InsP 6 are known to block transmitter release and also block the assembly of the particle by preventing -SNAP from binding to synaptotagmin.
  • Fig. 1 Schematic representation of the formation of protein complexes involved in vesicle docking. Fi .2. Binding specificity of ⁇ -SNAP and ⁇ -SNAP for SNAREs and to tagmin.
  • Fig. 3 Requirements for the assembly of the specialized docking and fusion particle. Immobilized GST-tagmin/ ⁇ - SNAP/NSF complexes and GST were incubated with bovine brain detergent extracts in absence or presence of ⁇ -SNAP or ⁇ -SNAP. Bound syntaxin IA and IB (upper panel) , SNAP-25 (middle panel) and VAMP (lower panel) were analyzed by immunodecoration of Western blots.
  • Fig. 4 Effect of polyphosphoinositols (IHPS) on the binding of ⁇ -SNAP to tagmin.
  • Immobilized GST-tagmin was incubated with ⁇ -SNAP in the presence of different IHPS at increasing concentrations. The results were expressed as percentage of the amount of -SNAP bound to tagmin in the absence of IHPS. (closed squares, InsP 6 ; open triangles, InsP 5 ; closed circle, InsP 4 ; open squares, InsP 3 ) and represented the average of three to five independent experiments.
  • FIG. 5 Mg 2+ ATP dependent disassembly of the specialized docking and fusion particle. Immobilized complexes were treated under conditions that allow Mg 2+ ATP dependent SNARE complex disassembly. This process was monitored by detecting the exposure of VAMP to tetanus toxin following disruption of the SNARE complex (open triangles, samples plus Mg 2+ ATP7S; open squares, samples plus Mg 2+ ATP; closed squares, samples plus Mg 2+ ATP and Ca 2+ ) .
  • FIG. 6 Binding specificity of GST-tag in to liposomes of different compositions.
  • GST-tagmin was incubated with pure l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (PC) or PC containing 1% w/w l-stearoyl-2- arachidonoyl-sn-glycero-3-phospho-D-myo-inositol (PI) or 1% (w/w) l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-D- myo-inositol-4-phosphate (Pins) or 1% (w/w) l-stearoyl-2- arachidonoyl-sn-glycero-3-phospho-D-myo-inositol-4, 5- bisphosphate (Pins 4,5-P 2 ) in the presence or absence of 100 ⁇ M free calcium.
  • PC l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho
  • Liposome binding was quantified by liquid scintillation counting of the [ 3 H]PC used as tracer and expressed as % of total radioactivity used. The unspecific binding of liposomes to the beads was determined by using pure GST and the results were subtracted from the total binding.
  • FIG. 7 The binding of PIns-P 2 on synaptotagmin is localized to the carboxy-terminal domain.
  • GST-tagmin, its amino terminal (C2A) and carboxy-terminal (CB2) domains were incubated with PC and 1, 2-dioleoy-sn-glycero-3- [phospho-L-serine] (PS) (3:1 w/w) (upper panel) or with PC liposomes containing 1% (w/w) of PIns-P 2 (lower panel) .
  • PS 2-dioleoy-sn-glycero-3- [phospho-L-serine]
  • FIG. 8 The binding of tagmin and the tagmin C2B domain to PIns-P 2 , but not the calcium-dependent binding of C2A to PS, is inhibited by inositol 1,2,3,4,5,6- hexakisphosphate. Liposome binding was determined as in Fig. 6.
  • FIG. 9. Synaptotagmin binds to both PIns-P 2 isomers with similar efficiency. Liposomes of PC containing 1% (w/w) of phosphatidylinositol-4, 5-bisphosphate (Pins 4,5-P 2 ) or phosphatidylinositol-3 , 4-bisphosphate (Pins 3,4-P 2 ) were incubated with GST tagmin in the presence or absence of 100 j-iM free calcium as described in Fig. 6. Fig. 10. Synaptotagmin binds specifically to PlnsPs- containing liposomes in a calcium-dependent manner.
  • GST- tagmin was incubated with liposomes containing PC or PC together with distinct phosphoinositide ⁇ (as indicated) in the presence or absence of calcium ions (shaded bars, 2 mM EGTA; filled bars, 100 ⁇ M free Ca 2+ ) . Lipid binding was quantified by liquid scintillation counting of the radioactive PC used as tracer and expressed as % of total radioactivity used.
  • Fig. 11 Calcium dependency of PIns-4,5-P 2 and Plns- 3,4,5-P 3 binding to tagmin.
  • GST-tagmin beads were simultaneously incubated with two populations of PC liposomes either containing PIns-4,5-P 2 or Plns-3 ,4 ,5-P 3 at variable Ca 2+ concentrations (open circles, PIns-4,5-P 2 ; closed squares, PIns-3 ,4,5-P 3 ) .
  • the PInsPs binding site on tagmin is localized to the C2B domain.
  • This invention relates to the process of synaptic release. More specifically, this invention relates to methods and compositions useful for modulating 5 docking of synaptic vesicles to membranes based on the formation of specific protein complexes which, according to the invention, mediate functional docking of synaptic vesicles.
  • the mechanism identified for docking may be modelled in vitro in cell free systems according to the 10 invention by detecting the formation of various protein complexes.
  • compositions of this invention are cell free protein complexes comprised of proteins involved in binding of synaptic vesicles to plasma membrane associated
  • the complexes of this invention comprise tagmin and 0-SNAP ( Figure 2) and may optionally contain one or more additional components such as, for example NSF, oc-SNAP, SNAREs, ( Figure 3) polyphosphoinositols such as, for example, inositol
  • IP 6 1,2,3,4,5,6 hexakis phosphate
  • IP 5 inositol 1,3,4,5,6 pentakisphosphate
  • polyphosphoinositides such as, for example, phosphatidylinositol-4 , 5-bisphosphate (Plns- 4,5-P 2 ) or phosphatidylinositol-3, 4 , 5 trisphosphate (Plns- 3,4,5-Pj) .
  • __ tagmin also referred to in the art as synaptotagmin or p65
  • 0-SNAP soluble NSF attachment protein
  • Yet another complex of this invention comprises tagmin and PIns-P 2 , with or without 0-SNAP or tagmin and PIns-P 3 , with or without 0-SNAP.
  • Another such complex of the invention comprises tagmin, -SNAP and NSF (N-ethylmaleimide- sensitive fusion protein) .
  • Still another complex of the invention comprises a quaternary complex of ⁇ - SNAP/tagmin/NSF and oc-SNAP.
  • Another complex comprises the quaternary complex bound to one or more SNARE proteins. The formation of these complexes in vitro provide the basis for assays useful for identifying substances which modulate synaptic transmission and would therefore be useful as therapeutics or diagnostic reagents.
  • tagmin refers to any amino acid sequence which comprises at least, the portion of the carboxy terminal region of tagmin which binds ⁇ -SNAP.
  • Other amino acid sequences which are sufficiently homologous to this portion of the amino acid sequence of tagmin and which retain the ability to bind /3-SNAP are also suitable for use in this invention.
  • Native tagmin isolated from synaptic vesicles a ⁇ , for example, the isolation of tagmin I as described in S ⁇ dhof et al., Neuron 2:1475-1481 (1989)
  • recombinant tagmin obtained from cells which have been transformed with a gene encoding tagmin, or a fragment thereof are suitable for use in this invention.
  • Recombinant tagmin, or fragments thereof may be expressed either directly or as a fusion protein attached to additional amino acids. Such methods of expressing protein are known in the art and are also described herein in the examples.
  • cDNA cloning and the construction of vectors for expression of tagmins or specific amino acid sequences of various tagmins fused to giutathione S-transferase (GST) has previously been described in Ullrich et al., Neuron. 13:1281-1291 (1994) and Li et al., Nature, 375:594-599 (1995) .
  • Tagmin exists in different isoforms which are distributed throughout the nervous system. Ullrich et al., Neuron 13:1281-1291 (1994) .
  • synaptotagmins I to VIII are localized at the level of the nervous system.
  • Synaptotagmins I and II are the two more abundant isoforms and are generally distributed in the rostral and caudal brain regions respectively.
  • Synaptotagmins III and IV are reported to be coexpressed in neurons expressing synaptotagmins I and/or II, but also exhibit differences in regional distribution. Whereas synaptotagmin IV is reported to be distributed throughout the nervous system, synaptotagmin III exhibits pronounced regional differences. Synaptotagmin III is expressed at appreciable levels in the cortex, hippocampus and olfactory bulb and at higher levels in neurons of the basal ganglia and thalamus. Other regions exhibiting appreciable expression of synaptotagmin III include neurons of the midbrain, caudal brainstem, motor nuclei agnocellular part of the red nucleus and the gigantocellular neurons of the reticular formation.
  • tagmin In addition to using the entire tagmin protein, smaller amino acid sequences of tagmin may be used which retain the ability to bind ⁇ -SNAP. Specific amino acids may be substituted provided the ability to bind /3-SNAP is retained. Based on results described herein, the preferred region of tagmin for use in this invention is the carboxy terminal (C2B) region as this is the region which binds ⁇ -SNAP.
  • C2B carboxy terminal
  • fragments comprising the entire cytoplasmic domain, the C2B domain, amino acids 79-421, or 248-421 retain the ability to bind ⁇ -SNAP and are preferred. More preferred is the amino acid region of 248-421.
  • Preferred amino acid sequence for other tagmin isotypes are as follows: tagmin II, amino acid residues about 249 to 422; tagmin III, amino acid residues about 405-588; tagmin IV, amino acid residues about 263-425; tagmin V, amino acid residues about 116 to 279; tagmin VI, amino acid residues about 338-511; tagmin VII, amino acid residues about 242 to 403; and tagmin, VIII amino acid residues 177 to 255.
  • the amino acid sequences recited above are based on rat tagmin isoforms but would substantially correspond to the tagmin isoforms of other species as well.
  • the amino acid sequences of the proteins for use in this invention may be modified by substituting amino acids provided they retain the binding characteristics necessary to assess complex formation.
  • ⁇ -SNAP is a brain specific 34Kd NSF binding protein capable of binding to SNAP receptors.
  • the amino acid sequence of bovine jS-SNAP is known. Whiteheart et al., Nature, 362:353-355 (1993) which is incorporated herein by reference.
  • ⁇ -SNAP may be used in accordance with this invention as the full length protein.
  • a peptide corresponding to amino acids 25 to 45 of ⁇ -SNAP inhibits the interaction between ⁇ -SNAP and tagmin and for this reason, the amino terminal end of /3-SNAP is thought to interact directly with tagmin.
  • This preferred peptide could be used to modulate the -SNAP/tagmin interaction sequence for use in this invention.
  • native 3-SNAP or a recombinant form may be used.
  • NSF is a protein which has been described by Wilson et al., Nature, 339:355-359 (1989) .
  • the amino acid sequences of NSF from various different animal species are known. Methods of recombinantly expressing NSF have also been described. Wilson et al., Nature. 339:355-359 (1989) and Wilson and Rothman, Meth. Enzymol.. 219:309-318 (1990) .
  • NSF may be expressed recombinantly using any of the expression systems available to those in the art including, for example E. coli.
  • native NSF purified from natural sources may be used.
  • the species from which the various proteins are derived is not significant as there is substantial homology across species and complexes can be formed using proteins from different species.
  • ⁇ - SNAP and ⁇ -SNAP amino acid sequences are bovine
  • NSF is a hamster sequence
  • tagmin is a rat sequence.
  • Such therapeutics are useful for treating or diagnosing various neurological or psychiatric disorders for which decreasing or inhibiting synaptic transmission generally or in specific regions of the nervous system is desirable.
  • neurological disorders for which decreasing or inhibiting synaptic transmission would be expected to be therapeutic include epilepsy which is characterized by improper electrical discharge in one or more brain regions; and diseases such as Huntington's, in which pathology is related to the action of an excitatory neurotransmitter.
  • Bipolar anic- depressive disorder which is currently treated with lithium is another disease for which decreasing synaptic transmission through inhibition of complex formation would be expected to be therapeutic.
  • Pathologies of the neuromuscular junction or pathologies associated with motor neuron function may also be related to the physiology of these complexes.
  • the assays of this invention for identifying substances which modulate synaptic transmission may be carried out using various protocols known to those in the art for measuring binding of molecules to proteins.
  • any assay capable of detecting relative amounts of complex formation in the presence and absence of test substances is suitable.
  • one of the complex components is immobilized on a solid support, e.g., agarose beads or immobilized antibodies which is then combined in an appropriate buffer with the other components of the complex in the presence and absence of test substance.
  • the solid support is then separated from the unbound components and the formation of the complex is assessed by detecting the presence of the various components bound to the solid support.
  • the presence of the components in the complex may be detected either by direct analysis of the complex bound to the solid support or following dissociation of the complex and analysis of the liberated components. Presence of 0-SNAP, tagmin, NSF or oc-SNAP in any of the complexes may be detected, for example by using specific antibodies. Any method known in the art for detecting the presence of a specific antibody may be used in the assays of this invention including the use of radioactive isotopes, chemiluminescence or enzymatic action.
  • each form of complex ( ⁇ -SNAP/tagmin; ⁇ -SNAP/tagmin/NSF; ⁇ -SNAP/tagmin/NSF/oc- SNAP; or ⁇ -SNAP/tagmin/NSF/oc-SNAP/SNARE) is tested separately with test substances to determine the effect on complex formation and on the capability to bind PIns-P 2 or PIns-P 3 .
  • test substances to determine the effect on complex formation and on the capability to bind PIns-P 2 or PIns-P 3 .
  • the present invention In addition to identifying substances which influence formation of the binary, tertiary, quaternary, or pentameric complexes, the present invention also identifies the formation of other complexes which are involved in docking of vesicles to plasma membrane to cause vesicle release. According to the model provided by this invention, prior to vesicle release, the tertiary tagmin//3-SNAP/NSF complex interacts with ⁇ -SNAP and is then capable of binding SNAREs to cause vesicle docking.
  • binding of ⁇ -SNAP to the tertiary tagmin/jS-SNAP/NSF complex to form the quaternary tagmin/ '-SNAP/NSF/ ⁇ -SNAP provides another site useful for identifying potentially therapeutic substances and for modulating synaptic transmission.
  • Binding of the tagmin/p'-SNAP/NSF/ ⁇ -SNAP complex to a SNARE complex, i.e. docking, provides yet another site for identifying substances which may be useful to inhibit synaptic transmission.
  • the complexes of this invention are also useful as immunogens to raise antibodies (polyclonal or monoclonal) specific for each form of complex ( ⁇ -
  • SNAP/tag in; ⁇ -SNAP/tagmin/NSF; 0-SNAP/tagmin/NSF/ ⁇ -SNAP; and ⁇ -SNAP/tagmin/NSF/ ⁇ -SNAP/SNARE) .
  • antibodies are conformation-specific in that they recognize the functional form of the various complexes of this invention. These antibodies may then be used as specific substances for decreasing synaptic release or as diagnostics for detecting the presence of intracellular complexes.
  • the blood brain barrier would prevent passage of peripherally administered antibodies.
  • the blood brain barrier may be more permeable allowing passage of such peripherally administered antibodies.
  • large molecules such as antibodies may be administered directly into spinal fluid or intrathecally.
  • the requirement of several proteins to effect docking significantly increases the likelihood that one or more neurological diseases results from improper expression or function of one of these proteins leading to improper complex formation.
  • the antibodies which are provided by this invention may be used to analyze tissue for the presence of appropriate complexes. Binding of tagmin and j8-SNAP/tagmin Complexes to Membrane Phospholipids
  • an inositol-containing phospholipid, phosphatidylinositol-4 , 5-bisphosphate, the isomer phosphatidylinositol-3 ,4-bisphosphate and phosphatidylinositol-3,4, 5-trisphosphate present as minor constituents in cellular membranes are compatible with tagmin/j3-SNAP complexes.
  • PIns-P 2 refers to either of the phosphatidyl inositol biphosphates
  • PIns-P 3 refers to any of the phosphatidylinositol triphosphates.
  • polyphosphatidylinositol may also be suitable for use with this invention and may vary based on the length of the fatty acid chain, for example C 16 to C 24 and location and number of double bonds or the degree and position of phosphorylation of the inositol ring, although monophosphates appear inactive.
  • the present invention also relates to a novel calcium sensor and switch important in regulating the priming step of synaptic neurotrans ission.
  • Calcium has been identified as the signal for altering the affinity of various inositol polyphosphates.
  • PIns-4 naturally occurring phosphatidylinositol bisphosphates, PIns-4 , 5-P 2 or PIns-3,4-P 2 , only moderately increase the interaction of tagmin with the liposomes (Fig. 10A) .
  • Plns- 3,4,5-P 3 strongly promotes the binding of liposomes to tagmin.
  • Fig. 10 shows that promotes the binding of liposomes to tagmin.
  • phosphatidylinositol Pins
  • PIns-4-P phosphatidylino ⁇ itol-4-phosphate
  • Tagmin contains two C2 domains homologous to the calcium and acidic phospholipid binding domain of protein kinase c. 29 * 30
  • the more amino-terminal C2 domain (C2A) binds PS (25 % w/w) in a calcium-dependent manner with a Kd of 3-6 ⁇ M. 27 - 28
  • the more carboxy-terminal C2 domain (C2B) binds InsPP and interacts with both PIns-P 2 and PIns-P 3 .
  • mice PIns-P 2 binds to recombinant and native tagmin and is inhibited by PIns-P 6 with an apparent K d of about 5 ⁇ M (Fig. 13) .
  • the intracellular concentration of PIns-P 6 i ⁇ estimated to be in the low micromolar range, which is unlikely to interfere in vivo with the binding of tagmin to the polyphosphoinositides described herein.
  • Presynaptic injection of higher concentrations of InsPP causes a reversible blockade of neurotransmitter release 21 .
  • the present findings indicates that InsPP also acts by competing for the binding of tagmin to PIns-4,5-P 2 and/or PIns-3 ,4 , 5-P 3 .
  • Binding of PIns-4,5-P 2 and PIns-3,4,5-P 3 to tagmin is highly specific, as shown by 1) lack of tagmin binding to liposomes containing Pins and PIns-4-P; 2) saturable binding of PIns-4,5-P 2 to tagmin with 1:1 stoichiometry; 3) the competitive inhibition of the above binding reactions by the soluble inositol derivative InsP 6 ; and 4) the switch in specificity from PIns-3,4, 5-P 3 to PIns-4,5-P 2 as a function of free calcium ion.
  • tagmin switches its specificity from PIns-3,4, 5-P 3 to PIns-4,5-P 2 .
  • Many proteins of diverse function are known to contain C2 domains 29 or are known to bind InsPP. 31 Therefore, they, too, have switchable lipid binding specificity, although the switch may be thrown by mechanisms other than calcium binding.
  • This switch (and the binding to PS) is important in the rapid triggering of exocytosis that occurs after calcium levels rise. It is noted that the C2B domain of tagmin is known to bind /3-SNAP and NSF in a Ca + - independent fashion and assemble with them and ⁇ -SNAP and SNAREs into a putative docking and fusion particle for exocytosis. 35
  • Modulation of PIns-P 2 and tagmin binding is another mode of modifying synaptic transmission according to this invention, as PIns-P 2 is involved in secretion of synaptic vesicles. Binding of PIns-P 2 to tagmin also occurs to the carboxy-terminal portion a ⁇ with ⁇ -SNAP and is inhibited by polyphosphoinositols (see Figure 8) . Tagmin/jS-SNAP complex binds to PIns-P 2 with the same efficiency as the pure tagmin.
  • Another aspect of this invention is therefore assays which detect changes in PIns-P 2 binding to the cell free tagmin complexes of this invention and the method of decreasing synaptic transmission by decreasing or inhibiting PIns-P 2 binding to complexes comprising tagmin, jS-SNAP or any of the other proteins which participate in docking to SNAREs.
  • Substances identified which decrease or inhibit PIns-P 2 and tagmin interactions may be useful to treat neurological or psychiatric diseases in which it is desirable to reduce synaptic transmission.
  • Lithium a well known drug for the treatment of bipolar manic- depressive di ⁇ orders, acts by altering the inositol homeostasis and in particular by reducing the PIns-P 2 levels in the membrane.
  • This invention provides an assay for identifying other such substances which may be useful as adjuncts or in place of traditional therapies for bipolar depression which rely on lithium.
  • the phosphatidylinositol pathway which causes phosphorylation at position 3 i ⁇ ⁇ ignificantly different from that resulting in phosphorylation at position ⁇ 4 and 5.
  • Enzymes for the synthesis and degradation of these two different PIns-P 2 species are completely separated and non-overlapping in the cell. Accordingly, by targeting modulation of enzymes specific for one PIns-P 2 isomer over the other, this invention provides another means of specifically modulating synaptic transmission.
  • One embodiment of this invention relates to a method of treatment to improve synaptic transmission by modulating formation of tagmin-containing complexes, wherein calcium sensitivity of PIns-P 2 /tagmin and Plns- P 3 /tagmin complex formation is modulated by the addition of external substances.
  • Examples of "external substances" which may be used in this invention include several preferred categories of substances.
  • a first preferred category of external substances encompasses those that act by altering intracellular calcium concentrations, such as, for example, calcium channel blockers, including phenylalkylamines, dihydropyridines, benzothiazepines and diphenylpiperazines.
  • Other calcium channel blockers are known in the art, such as those described by Goodman and Gilman's The Pharmacological Basic of Therapeutics 8 th Eds. ((Eds. Goodman Gilman et al.) Pergamon Press 1990 p. 774- 783) .
  • a second preferred category of external substances encompasses those that inhibit or enhance mobilization of calcium from intracellular calcium stores.
  • Such substances include, for example, thapsigargin, inositol 1,4,5 trisphosphate ("IP 3 ”) and 2 , 5-di (tert-butyl) -1, 4- benzohydro quinone.
  • IP 3 inositol 1,4,5 trisphosphate
  • IP 6 5-di (tert-butyl) -1
  • 4- benzohydro quinone Other such compounds are known in the art.
  • a third preferred category of external substances useful in this method of the invention encompass substances which inhibit or enhance the synthesis of polyphosphoinositides and their soluble analogues including IP 6 and its derivatives, for example, lithium which reduces the level IP 3 and of polyphosphoinositides.
  • the assay system ⁇ and kits of this invention comprise the reagents necessary to conduct the various binding assays described above which may be used to identify substances which reduce or inhibit complex formation.
  • a typical assay kit of this invention would comprise 0-SNAP or an amino acid sequence of jS-SNAP capable of binding to tagmin, one or more containers each comprising an isoform of tagmin or an amino acid sequence of tagmin capable of binding ⁇ -SNAP, and optionally containers comprising separately one or more of NSF, ⁇ -SNAP, SNAREs complex, labelled specific antibodies, PIns-P 2 , PIns-P 3 and one or more polyphosphoinositols to serve as controls to compare against test substances.
  • Component ⁇ of the kits or assay systems may be provided in appropriate buffers, or may be lyophilized for later reconstitution and use.
  • a preferred buffer is HEPES-KOH (20 mM) , 100 mM KCI, dithiothreitol (0.1 mM) at pH 7.6.
  • the region of the amino acid sequence of tagmin extending from about residue 248 to about 421 or the entire cytoplasmic domain (amino acids 78-421) are particularly preferred for use in this invention because, as shown below, this region contains the binding site for ⁇ -SNAP and are stable in solution.
  • a preferred buffer for the binding reaction is HEPES-KOH (20mM, pH 7.6), potassium acetate (150 mM) , glycerol (1%), octylglycoside (0.6 mM) and dithiothreitol (0.1 mM) .
  • Tagmin/p'-SNAP/NSF complex is stable in the presence of Ca 2+ /Mg 2+ ATP (right panel) .
  • GST fusion proteins of different tagmin constructs or GST alone were incubated with ⁇ -SNAP (lanes 2, 4, 7) or ⁇ -SNAP (lanes 3, 5, 7-13) with (lanes 4-7, 10-13) or without (lanes 1-3, 8, 9) addition of NSF.
  • Three samples, identical to the one in lane 5, were further incubated in the presence of ATP7S (lane 11) , ATP (lane 12) or ATP + Ca 2+ (lane 13) .
  • c) ⁇ - SNAP and jS-SNAP binding to the SNARE complex were isolated from bovine brain detergent extract and incubated with increasing amounts of ⁇ -SNAP (closed squares) or ⁇ -SNAP (open sguares) .
  • METHODS GST-fu ⁇ ion proteins containing the cytoplasmic domain of synaptotagmin I (aa. 79-421; 4 ⁇ g) or either the amino-terminal (GST-p65,. 3 ; aa. 96-265; 3 ⁇ g) or the carboxy-terminal (GST-p65 3 . 5 ; aa. 248-421; 3 ⁇ g) parts, were immobilized on GSH-agarose beads.
  • 3 ⁇ g of His 6 - ⁇ -SNAP 3 or 3 ⁇ g of His 6 -/3-SNAP were diluted in 250 ⁇ l of 20 mM HEPES-KOH pH 7.6, 150 mM Acetate-K, 1% glycerol, 5 ⁇ M EDTA, 0.8% (w/v) octyl- ⁇ -D-glucopyranoside, 0.4 mg/ml ovalbumin, spun at 15,000 x g for 5 min and the supernatant was incubated with beads for 1 h at 4°C.
  • the beads were washed twice with 250 ⁇ l of the same buffer without ovalbumin. Some samples were further incubated for 1 h at 4°C with 5 ⁇ g of recombinant NSF 3 in 250 ⁇ l of 20 mM HEPES-KOH pH 7.6, 150 mM Acetate-K, 1 mM MgCl 2 , 5 ⁇ M EGTA, 0.5 mM ADP, 1% glycerol, 0.8% (w/v) octyl- ⁇ -D- glucopyranoside, 0.4 mg/ml ovalbumin. The beads were then washed in the same buffer without ovalbumin and samples analyzed in SDS-PAGE.
  • the stability of the complex was tested by incubating the complex-bound beads for 30 min at 37°C in 20 mM HEPES-KOH pH 7.6, 150 mM Acetate-K, 1 mM MgCl 2 , 1% glycerol, 0.5% (w/v) Triton X-100 (buffer A) containing 0.5 mM adenosine-5'-[ ⁇ -thio]triphosphate (ATP7S)/1 mM EGTA, 0.5 mM ATP/1 mM EGTA, or 0.5 mM ATP/300 ⁇ M free Ca 2+ .
  • buffer A containing 0.5 mM adenosine-5'-[ ⁇ -thio]triphosphate (ATP7S)/1 mM EGTA, 0.5 mM ATP/1 mM EGTA, or 0.5 mM ATP/300 ⁇ M free Ca 2+ .
  • NSF Since NSF is known to bind to membranes in a SNAP-dependent manner," 12 we tested whether NSF might bind to the ⁇ -SNAP-tagmin complex. NSF binds efficiently to the tagmin- ⁇ -SNAP complex (lane 5) , while ⁇ -SNAP neither substitutes for 0-SNAP (lane 4) nor interferes with this binding (lane 7) .
  • NSF bound through ⁇ -SNAP to SNAREs hydrolyses ATP, releasing NSF and ⁇ -SNAP, with disruption of the SNARE complex 7 .
  • complexes of NSF and 0-SNAP with tagmin are stable in the presence of Mg 2+ -ATP, with (lane 13) or without (lane 12) calcium (0.3 mM) , and these complexes form in the absence or presence (0.3 mM) of calcium (not shown) .
  • the lack of release of NSF with Mg 2+ -ATP suggests that -SNAP and NSF are bound to tagmin differently than in 20S particles, 3,7 and implies that the tag ⁇ in/0-SNAP/NSF "triple" complex should be stable in vivo under conditions in which ample ATP is present.
  • Tagaya and colleagues 13 have reported that NSF is present on synaptic vesicles, and is not released by Mg 2+ -ATP; the tagmin/ ⁇ -SNAP/NSF triple complex may explain their findings.
  • RESULTS GST, GST-tagmin, GST-tagmin/ ⁇ -SNAP and GST-tagmin/ -SNAP/NSF complexes, bound to GSH-agarose beads were prepared as described in Fig. 2.
  • the complexes were resuspended in 20 mM HEPES-KOH pH 7.6, 150 mM Acetate-K, 1% glycerol, 0.8% (w/v) octyl- ⁇ -D-glucopyranoside, (Buffer C) containing 1 mM MgCl 2 , 0.5 mM ADP, 1 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol and mixed in a final volume of 500 ⁇ l with bovine brain extract (250 ⁇ g of protein) prepared as described 3 by using octyl-3-D-glucopyranoside as detergent.
  • ⁇ -SNAP (4 ⁇ g) diluted in buffer C containing 0.4 mg/ml ovalbumin and previou ⁇ ly ⁇ pun at 15,000 x g for 5 min was added.
  • the samples (250 ⁇ l final volume) were incubated for 1 h at 4°C, washed twice with 250 ⁇ l of buffer C and then analyzed by SDS-PAGE.
  • the ⁇ -SNAP bound to GST-tagmin was determined by scanning of the Coomassie Blue R250 stained gel as described before (see legend Fig. 2) .
  • NSF entering the possible docking and fusion particle bound in the "triple" complex with ⁇ -SNAP and tagmin can hydrolyze ATP to release VAMP as shown by cleavage by tetanus toxin 22 (Fig. 5, open squares) .
  • ATP7S does not substitute for ATP (open triangles)
  • an ATP- hydrolysis deficient mutant of NSF E329Q/D604Q double mutant
  • the particle-containing beads (15 ⁇ l per sample) were then added to 235 ⁇ l of pre-warmed buffer A supplemented with 400 nM of the recombinant catalytic subunit of tetanus toxin 22 and containing alternatively 0.5 mM ATP7S/I mM EGTA, 0.5 mM ATP/1 mM EGTA, or 0.5 mM ATP/300 ⁇ M free Ca 2+ (obtained by buffering with 1 mM EGTA) . At the indicated times samples were chilled on ice and centrifuged at 5,000 x g for 1 min.
  • priming includes the NSF-ATPase-dependent disruption of the proposed synaptic docking and fusion particle, producing a latent fusogenically-active state awaiting calcium. If this assignment is correct, then, like priming, disruption of the docking and fusion particle should be independent of calcium, as we observed (Fig. 5) .
  • This view in no way contradicts the additional need for ATP during the priming stage to maintain a pool of phosphatidylinositol-4, 5-bispho ⁇ phate (PtdIns-4 ,5-P 2 ) for later fusion after calcium activation 26 .
  • the protein components that might constitute the latest fusogenically- active state that be produced when ATP is hydrolyzed by NSF are unknown but could be satisfied by tagmin which possesses a calcium binding site and an affinity for ⁇ - SNAP.
  • Liposomes (175 ⁇ g/ml) were made by pure l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) or PC and 1, 2-dioleoy- ⁇ n-glycero-3-[pho ⁇ pho-L- ⁇ erine] (PS) (3:1 w/w) or PC containing 1% w/w l-stearoyl-2- arachidonoyl-sn-glycero-3-phospho-D-myo-inositol (PI) or
  • l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-D- myo-inositol-4-phosphate PIns-P
  • PIns-P l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-D- myo-inositol-4-phosphate
  • PIns-P 1-stearoyl- 2-arachidonoyl-sn-glycero-3-phospho-D-myo-inositol-4 ,5- bisphosphate
  • Pins , 5-P 2 bisphosphate
  • Lipids were purchased from Avanti Polar Lipids (PC, PS and PI) or Boehringer Mannheim GmbH (PIns-P and PIns-P 2 ) . Pho ⁇ pholipids were dried by a gentle flow of argon on a 4 ml glass vial and then the residual traces of solvents 5 were eliminated under vacuum for 15 minutes at room temperature. The lipid film were resuspended in 20 mM HEPES-KOH pH 7.6, 100 mM KCI mM DTT (buffer A) by vigorou ⁇ stirring for 10 minutes and the top of the solution were layered with argon.
  • Liposomes were prepared either by 0 sonication in a Branson cup sonicator (intensity setting 7, duty cycle 50%, 8 minutes) 28 followed by liposomes purification on Sephadex G-25 M (Pharmacia) or by freeze- thawing (5 times) and extrusion (19 times) through a 100 nm pore carbonate membrane (Avestin, Ottawa, Canada) 42 . 5 Large aggregates were eliminated by centrifugation at
  • the liposomes are stored at 4°C and are used within one week.
  • the PIns-P 2 binding site is localized to the carboxy-terminal domain (C2B) and accounts for the entire PIns-P 2 binding of synaptotagmin.
  • C2A the amino terminal domain
  • the amino terminal domain, C2A show ⁇ barely detectable binding activity which i ⁇ not distinguishable from the fusion protein alone (GST) (Fig. 7, lower panel) .
  • the functionality of the C2A domain is demonstrated from the calcium-dependent binding of this region of synaptotagmin (Fig. 7, upper panel).
  • Liposomes (175 ⁇ g of lipid/ l) were made from pure l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (PC) or PC and in addition 1% (w/w) of 1- stearoyl-2-arachidonoyl-sn-glycero-3-phospho-D-myo- inositol (Pins) or l-stearoyl-2-arachidonoyl-sn-glycero-3- phospho-D-myo-inositol-4-phosphate (PIns-4-P) or 1- stearoyl-2-arachidonoyl-sn-glycero-3-phospho-D-myo- inositol-4 , 5-bi ⁇ phosphate (PIns-4 , 5-P 2 ) or 1,2 dipalmitoyl sn-glycero-3-phospho-D-myo-inositol -3-phospho-D
  • Lipids were purchased from Avanti Polar Lipids (PC and Pins) or Boehringer Mannheim GmbH (PIns-4-P and PIns-4,5- P 2 ) or Matreya Inc (PIn ⁇ -3,4-P 2 ) .
  • PIns-3 ,4 , 5-P 3 was synthesized as described before 41 and its identity was confirmed by ⁇ - and 3l P-NMR. Lipids were dried by a gentle flow of argon, dissolved in 200 ⁇ l of 100 % ethanol and then kept under vacuum for 30 min. at room temperature.
  • the homogeneous lipid film was then resuspended in 20 M HEPES-KOH pH 7.6, 100 mM KCI, 0.2 mM dithiothreitol (DTT) (buffer A) by vigorous stirring for 10 minutes, after overlaying the top of the solution with argon.
  • Liposomes were prepared either by sonication 28 followed by liposome purification on Sephadex G-25 M (Pharmacia) or by extrusion 42 . Large aggregates were eliminated by centrifugation at 15,000g for 10 minutes.
  • Purified GST-fusion proteins containing the cytoplasmic domain of tagmin 1 (aa. 79-421; 15 ⁇ g) or GST alone (8 ⁇ g) were immobilized on 20 ⁇ l 50% glutathione-agarose beads
  • FIG. 10A shows the binding specificity of GST- tagmin to liposomes.
  • GST-tagmin wa ⁇ incubated with liposomes containing PC or PC together with distinct phosphoinositides (as indicated) in the presence or absence of calcium ions (shaded bars, 2 mM EGTA; filled bars, 100 ⁇ M free Ca 2+ ) .
  • Lipid binding was quantified by liquid scintillation counting of the radioactive PC used as tracer and expressed as % of total radioactivity used. Specific binding was calculated by ⁇ ubtracting the non ⁇ specific lipid interaction of GST (2,4 ⁇ 0.7%) from individual samples. Similar results were obtained both with small and large unilamellar vesicles, thus suggesting that size and curvature of the liposome do not influence the binding.
  • PIn ⁇ -4,5-P 2 and PIns-3, 4 , 5-P 3 were incorporated into separate liposome populations, labeled either with a t 14 C] or with a [ H]-PC tracer. Binding of each vesicle type to tagmin-containing bead ⁇ wa ⁇ determined in a mixed incubation a ⁇ a function of free calcium concentration (Fig. 11) .
  • GST-tagmin bead ⁇ were ⁇ imultaneously incubated with two populations of PC liposomes either containing PIns-4,5-P 2 or PIns-3 , 4 , 5-P, at variable Ca 2+ concentration ⁇ (See Figure 11, open circle ⁇ , PIns-4,5-P 2 ; closed squares, PIns-3, 4 ,5-P 3 ) .
  • PC liposomes 350 ⁇ g of lipid/ml containing either 1% (w/w) of PIns-4,5-P 2 and 0.1 ⁇ Ci/ml of l-palmitoyl-2-[l- 14 C]palmitoyl-sn-glycero-3-phospho- choline (55 mCi/m ol, Amersham) or 1% (w/w) of PIns-3,4,5- P 3 and 1 ⁇ Ci/ml of 1, 2-dipalmitoyl-sn-glycero-3-pho ⁇ pho- [N-methyl- 3 H]-choline (66 Ci/mmol, Amersham) were prepared as above.
  • the two liposome populations were pre-mixed at a 1:1 ratio (final lipid concentration 175 ⁇ g/ml) and free Ca 2+ concentrations ranging from 1 nM to 300 ⁇ M in the presence of 0.5 mM free Mg 2+ were obtained by adding the suitable amount of calcium or magnesium chloride to 2 mM EGTA in buffer A. GST-tagmin beads were then incubated with the mixed liposomes for 30 min. at room temperature and washed in buffer A containing the corresponding amount of free Ca 2+ . [ 3 H]/[ 14 C] radioactivity as ⁇ ociated with the pellet wa ⁇ then determined by scintillation counting.
  • Tagmin loses the capacity to bind PIns-3 ,4, 5-P 3 between about 0.1 and 1 ⁇ M free calcium. It gains the capacity to bind PIns-4 , 5-P 2 progressively above about 1 ⁇ M free calcium with maximum binding reached only in the 100 ⁇ M range.
  • the calcium dependency of the switch of specificity was not significantly affected by altering the concentration of PIns-4,5-P 2 or PIns-3,4, 5-P 3 in the liposome.
  • magnesium 0.5-1.0 mM was required for the complete switch.
  • EXAMPLE 8 Tagmin domains and Polyphosphoinositides binding specificity.
  • the PlnsPs binding site on tagmin is localized to the C2B domain.
  • the PIns-4,5-P 2 and the PIns-3 ,4 , 5-P 3 -directed binding of lipo ⁇ ome ⁇ occur with the C2B domain of tagmin, with an efficiency similar or higher to the full length tagmin cytoplasmic domain (Fig.l2B and C) .
  • EXAMPLE 9 Binding Kinetics of Micellar PIns-4,5-P 2 to Recombinant and Native Tagmin.
  • GST-tagmin was incubated in the presence of saturable amount of PIns-4,5-P 2 (6 ⁇ M) with increa ⁇ ing amount of InsP 6 in the presence of 2 mM EGTA, as shown in Figure 13B.
  • PIns-4,5-P 2 was solubilized at a final concentration of 20 ⁇ M (20 Ci/Mole) in buffer A containing 0.8 % (w/v) OG in the presence of 2 mM EGTA and sonicated as above.
  • panels C) and D) native tagmin (5 ⁇ g/sa ple) was immunoprecipitated from octyl- ⁇ - D-glucopyranoside (OG) extract of bovine brain cortex 35 with an anti-tagmin monoclonal antibody (M48) 33 covalently coupled to protein G-Sepharose Fa ⁇ t Flow (Pharmacia) 3 .
  • Fig. 13D beads were analyzed by SDS-PAGE and the proteins were stained with Coomassie Blue R.
  • Competition experiments were carried out by incubating GST-tag in-containing beads with saturable amount of [ 3 H]- PIns-4,5-P 2 (6 ⁇ M) premixed with increasing concentrations of InsP 6 (1 nM-1 mM) in buffer A containing 0.8 % OG, 2 mM EGTA for 30 minutes at 4°C. After washing with the same buffer, the amount of radioactivity was determined and results expressed as % of the binding in absence of InsP 6 .
  • Radiolabeled PIns-4,5-P 2 was dispersed in octyl- ⁇ -D-glucopyranoside (OG) micelles using a vast excess of detergent to ensure that there would be at most one molecule of PIns-4 ,5-P 2 per detergent micelle.
  • OG octyl- ⁇ -D-glucopyranoside
  • At saturation almost exactly one mole of PIns-4 , 5-P 2 wa ⁇ bound per mole of tagmin (Fig. 13A) , with an apparent Kd of about 1 ⁇ M.
  • the binding of micellar PIns-4,5-P 2 monomers was independent of calcium. In ⁇ P 6 competes with this binding, with a Ki of about 10 ⁇ M.
  • Fig. 13B Full length native tagmin, immunopurified from brain cortex with an antibody directed against its first C2 homology domain 33 (Fig. 13D) shows the saturable and stoichiometric binding properties for PIns- , 5-P 2 (Fig. 13C) as was observed with the recombinant protein. InsP 6 inhibits the interaction of micellar PIns-4,5-P 2 monomers with the native protein, but less effeciently than observed with the recombinant tagmin (Ki > 100 ⁇ M) .

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Abstract

L'invention concerne des procédés et des compositions qui contribuent à moduler la transmission synaptique. Les compositions décrites sont des complexes à base de combinaisons diverses de protéines (par exemple, tagmine, protéine de fusion sensible à N-éthylmaléimide (NSF), protéines solubles d'attachement de NSF (β-SNAP, α-SNAP) et récepteurs de SNAP (SNARE)) intervenant dans la jonction et la fusion des vésicules synaptiques aux membranes. On décrit par ailleurs des complexes à base de tagmine et de phospholipides spécifiques (phosphatidyl inositols) et qui entrent dans la composition des membranes impliquées dans le processus de secrétion. Ces complexes sont utiles pour l'identification d'autres substances pouvant moduler la transmission synaptique.
PCT/US1996/019661 1995-12-13 1996-12-12 Procedes et compositions pour la modulation de la liberation vesiculaire WO1997021729A1 (fr)

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WO1999004265A3 (fr) * 1997-07-17 1999-08-26 Ludwig Inst Cancer Res Acides nucleiques et polypeptides associes au cancer
US6686147B1 (en) 1998-07-15 2004-02-03 Ludwig Institute For Cancer Research Cancer associated antigens and uses therefor
KR101472088B1 (ko) 2012-07-12 2014-12-16 광주과학기술원 시냅스 소낭 막 융합 억제제의 스크리닝 방법 및 이를 위한 조성물
WO2014169087A3 (fr) * 2013-04-10 2014-12-04 The Trustees Of Columbia University In The City Of New York Traitement de l'atrophie musculaire spinale proximale
EP3415909A1 (fr) 2017-06-16 2018-12-19 Euroimmun Medizinische Labordiagnostika AG Diagnostic d'une maladie neuro-autoimmune
DE102018004759A1 (de) 2017-06-16 2018-12-20 Euroimmun Medizinische Labordiagnostika Ag Diagnose einer Neuroautoimmunkrankheit
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