WO1997020218A1 - Compositions modulatrices des canaux calciques et procedes associes - Google Patents

Compositions modulatrices des canaux calciques et procedes associes Download PDF

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WO1997020218A1
WO1997020218A1 PCT/US1996/019002 US9619002W WO9720218A1 WO 1997020218 A1 WO1997020218 A1 WO 1997020218A1 US 9619002 W US9619002 W US 9619002W WO 9720218 A1 WO9720218 A1 WO 9720218A1
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syntaxin
channels
peptide
domain
calcium
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Richard W. Tsien
Ilya Bezprozvanny
Richard H. Scheller
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to compounds capable of modulating the activity of N- and Q-type calcium channels, and to methods of screening for such compounds.
  • Mullis, K.B. U.S. Patent No.4,683.202, issued 28 July 1987.
  • Mullis, K.B.. et al. U.S. Patent No 4,683, 195, issued 28 July 1987.
  • Voltage-dependent calcium channels play a central role in the normal functioning of electrically-excitable tissues, such as nervous system tissue and muscle tissue, including skeletal muscle, vascular smooth muscle and cardiac muscle. Depolarization of the cell membrane opens the channels, causing an influx of calcium ions which in turn can activate a variety of cellular responses (e. g. , release of neurotransmitter, hormones, activation of second messenger systems, etc. ), as well as lead to a further depolarization of the cell Abnormal influx of calcium through voltage-dependent calcium channels, or an abnormal cellular response to such an influx, plays a role in a number of pathological conditions affecting the tissues expressing the channels. In cardiovascular tissue, these disorders include anoxic/ischemic heart disease, cardiac arrhythmias, coronary artery disease and cardiomyopathy. In the nervous system, such disorders can include
  • anoxic/ischemic damage stroke
  • epilepsy neuronal death associated with chronic epilepsy and various neurodegenerative diseases, as well as intractable pain.
  • the present invention includes a composition comprising a peptide capable of significantly shifting the steady-state inactivation curve of a voltage-dependent calcium ion channel associated with neurotransmitter release in the hyperpolarized direction.
  • the peptide in the composition has an amino acid sequence derived from the sequence of syntaxin.
  • the peptide preferably contains a first and second domains, where the first domain contains a sequence corresponding to a contiguous region of at least about 14 amino acids from the transmembrane region (TMR) of syntaxin, and the second domain contains a sequence corresponding to a contiguous region of at least about 14 amino acids derived from a region of syntaxin N-terminal to the TMR.
  • TMR transmembrane region
  • Exemplary syntaxin TMRs are provided herein as SEQ ID NO: 8 and SEQ ID NO: 9.
  • the peptide is a natural syntaxin protein missing at least one amino acid at the N-terminus, i.e., containing a deletion of one or more amino acids at the N-terminus.
  • the peptide is a natural syntaxin polypeptide having one or more deletions internally, between the N-terminus and the TMR.
  • the invention includes a method of selectively inhibiting the opening of voltage-dependent calcium ion channels associated with release.
  • the method includes contacting the channels with a peptide having amino acid sequences derived from syntaxin, the peptide comprising a first and second domains, where the first domain contains a sequence corresponding to a contiguous region of at least about 14 amino acids from the transmembrane region (TMR) of syntaxin, and the second domain contains a sequence corresponding to a contiguous region of at least about 14 amino acids derived from a region of syntaxin N-terminal to the TMR.
  • TMR transmembrane region
  • the first domain contains a sequence corresponding to a contiguous region of at least about 20 amino acids from the transmembrane region (TMR) of syntaxin
  • the second domain contains a sequence corresponding to a contiguous region of at least about 20 amino acids derived from a region of syntaxin N- terminal to the TMR .
  • the peptide has an N-terminal portion and a C-terminal portion, the first domain is in the C-terminal portion and the second domain is in the N-terminal portion
  • the second domain is an amphiphilic domain (AD) of syntaxin.
  • the AD may contain at least one helical domain of syntaxin, such as H 1 , H2, and/or H3.
  • the peptide has an amino acid sequence derived from natural syntaxin, such as syntaxin 1A; in another embodiment the peptide is derived from syntaxin 1B. In other embodiments, the peptide is derived from any of the other syntaxin molecules, such as syntaxin 2, syntaxin 3, syntaxin 4 or syntaxin 5. In another general embodiment, the peptide and the channels are from the same species, such as humans.
  • Exemplary peptides of the invention have sequences represented herein as SEQ ID NO: 1
  • channels are N-type calcium channels, such as N-type channels formed of calcium channel subunits ⁇ 1B , ⁇ 3 and ⁇ 2 and those where the channels are Q-type calcium channels, such as Q-type channels formed of calcium channel subunits ⁇ 1A , ⁇ 3 and ⁇ 2 .
  • the invention includes a method of identifying a compound capable of modulating (i. e. , stimulating or inhibiting) the opening of voltage-dependent calcium ion channels involved in neurotransmitter release .
  • the method includes contacting a calcium channel subunit or portion thereof (e.g. , a portion of the subunit that interacts specifically with syntaxin) of a voltage-dependent calcium ion channel involved in neurotransmitter release with a peptide (i) derived from syntaxin and (n) capable of specifically interacting with an N-type or Q-type calcium channel, in the presence and absence of a test compound.
  • a test compound i) derived from syntaxin and (n) capable of specifically interacting with an N-type or Q-type calcium channel
  • the effect of the test compound on the degree of binding between the calcium channel subunit and the peptide is measured by comparing the degree of binding in the presence and in the absence of the compound
  • the peptide contains a sequence of amino acids derived or contained in the transmembrane domain of syntaxin.
  • the compound is selected if the degree of binding in the presence of the compound is significantly different from the degree of binding in the absence of the compound, and the compound is further screened the selected compound for its ability to affect the opening of voltage-dependent calcium ion channels involved in neurotransmitter release as described above.
  • the test compound is identified as effective if it selectively modulates (e.g., inhibits) the opening of the calcium ion channels
  • the identifying includes identifying a selected compound as effective if the compound causes a significant shift in the steady-state inactivation curve of the calcium ion channels (e.g., a
  • the contacting is carried out using a solid support, where one of the interacting pair of proteins (i.e., either the calcium channel subunit or the peptide containing the transmembrane domain of syntaxin, "immobilized” peptide, polypeptide or protein) is immobilized on the solid support and the other of the interacting pair of proteins ("free” peptide, polypeptide or protein) is suspended in a solution that is brought in contact with the derivatized solid support.
  • one of the interacting pair of proteins i.e., either the calcium channel subunit or the peptide containing the transmembrane domain of syntaxin, "immobilized” peptide, polypeptide or protein
  • free free
  • GST glutathione-S-transferase
  • the selected compound is further screened for its ability to affect the opening of voltage-dependent calcium ion channels involved in neurotransmitter release, as described above.
  • the selected compound is identified as effective if it selectively inhibits the opening of the calcium ion channels.
  • test compounds suitable for screening using the methods of the present invention include small molecules in a small molecule combinatorial library, and peptides in a peptide combinatorial library.
  • General embodiments of the method include the use of the ⁇ 1B subunit of N-type calcium channels and the ⁇ 1A subunit of Q-type calcium channels.
  • Exemplary calcium channel subunits include fragments of the intact of the ⁇ 1B or ⁇ 1A subunits. where the fragments contain the II/III loop (Sheng, et al. , 1994).
  • the peptide may contain amino acids derived from any of a number of different syntaxin molecules, in various sequence configurations, as described above.
  • Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H and 1I show functional properties of N- type channels in Xenopus oocytes, expressed alone (Figs. 1A and 1G), co-expressed with full-length s1A (a.a. 1-288, Figs. 1B and 1H, SEQ ID NO: 1), or co-expressed with the N- terminal portion of slA (s1A-N; SEQ ID NO:2; a.a. 1-189; Figs. 1C and 1I).
  • Inward Ba 2+ currents were recorded with a "descending staircase" stimulation protocol (Figs. 1D, 1E and 1F).
  • Current traces (Figs. 1A, 1B and 1C) are the last sweep at each holding potential (HP).
  • Figs. 1G, 1H and 1I show peak current amplitudes plotted against time.
  • Figures 2 A, 2B, 2C and 2D show progressive changes in the functional properties of N-type Ca 2+ channels with increasing duration of syntaxin expression and with increasing amount of syntaxin cRNA injected.
  • Figs. 2 A and 2B show the i 80 /i 120 ratio (Fig. 2 A) and ⁇ 120 (Fig. 2B) were determined using the descending staircase protocol at different time points after injection of cRNA for syntaxin (•), for the N-terminal construct s1A-N (v), or an equal volume of water ( o ).
  • the i 80 /i 120 ratio (Fig. 2C) and ⁇ 120 (Fig. 2D) were determined using the descending staircase protocol in oocytes injected with similar volumes of slA cRNA-containing solution, either undiluted ( ⁇ 20 ng/oocyte) or diluted 2-, 4-, 8-, 16- or 32-fold.
  • Figures 3A, 3B, 3C, 3D and 3E show the specificity of syntaxin effect on various types of Ca 2+ channels. Behavior of N-type channels (Fig. 3A), Q-type channels (Fig. 3B) and L-type channels (Fig. 3C). Figs. 3D and 3E show that steady-state inactivation curves of N-type and Q-type Ca channels are shifted in the presence of syntaxin. Voltage- dependent steady-state inactivation curves were assessed with a prepulse protocol (Fig. 3F).
  • Figures 4A and 4B show a comparison of the i 80 /i 120 ratios (Fig. 4B) obtained following co-expression of various syntaxin deletion mutants (Fig. 4A) with N-type Ca 2+ channels.
  • Figure 5 shows an alpha helix amphiphilicity plot, generated using the computer program "DNA STRIDER 1.2" (Christian Marck, Service de Biochemie et de Genetique Moleisme, Department de Biologie Cellulaire et Moleisme, Direction des Sciences de la Vie - CEA - FRANCE), of syntaxin 1A (SEQ ID NO: 1) . Regions falling above the horizontal line at 1 .5 are amphiphilic Bars below the plot show exemplary amphiphilic domain of syntaxin 1A, designated helical domain 1 (H1), helical domain 2 (H2) and helical domain 3 (H3).
  • H1 helical domain 1
  • H2 helical domain 2
  • H3 helical domain 3
  • SEQ ID NO: 1 is the amino acid sequence of rat syntaxin 1A, containing amino acids 1-288 (Bennett, et al. , 1993).
  • SEQ ID NO: 2 is the amino acid sequence of syntaxin mutant S1A-N, containing amino acids 1-189 of rat syntaxin 1A.
  • SEQ ID NO: 3 is the amino acid sequence of syntaxin mutant S 1A-C, containing amino acids 168-288 of rat syntaxin 1A.
  • SEQ ID NO: 4 is the amino acid sequence of syntaxin mutant S 1AM267X, containing amino acids 1-266 of rat syntaxin 1A.
  • SEQ ID NO: 5 is the amino acid sequence of syntaxin mutant S 1AK265X, containing amino acids 1-264 of rat syntaxin 1A.
  • SEQ ID NO: 6 is the amino acid sequence of syntaxin mutant S 1Al195, containing amino acids 1-194 and 267-288 of rat syntaxin 1A.
  • SEQ ID NO: 7 is the amino acid sequence of syntaxin mutant S 1AM215, containing amino acids 1-214 and 267-288 of rat syntaxin 1A .
  • SEQ ID NO: 8 is the amino acid sequence of the transmembrane domain, or region (TMR) of syntaxin 1A, containing amino acids 266-288 of rat syntaxin 1A.
  • SEQ ID NO: 9 is the amino acid sequence of the transmembrane domain, or region (TMR) of syntaxin 1B, containing amino acids 266-289 of rat syntaxin 1B.
  • selective inhibition when used in relation to voltage-dependent calcium ion channels associated with neurotransmitter release, refers to preferential inhibition of voltage-dependent calcium ion channels associated with neurotransmitter release (e. g. , N- type and Q-type channels) over inhibition of other types of calcium channels (e. g. , L-type channels) See Example 3 and Figs 3A-3E for an illustration of such selective inhibition
  • neurotransmitter release refers to voltage-dependent calcium channels that are present at presynaptic terminals and that are considered to be instrumental in mediating the influx of calcium at the presynaptic terminal that causes neurotransmitter release.
  • Exemplary voltage-dependent calcium ion channels associated with neurotransmitter release are N-type and Q-type channels.
  • peptide is understood herein to refer to a peptide or polypeptide chain that is from several to about 300 amino acids in length.
  • calcium channel subunit is understood to be a polypeptide subunit of a calcium channel, or a portion of a polypeptide subunit of a calcium channel (e.g. , the portion containing the II-III loop).
  • the subunit can exist as an isolated polypeptide, or in association with other subunits which together form an intact calcium ion channel.
  • “Inhibiting the opening" of ion channels can be accomplished by plugging the pore of the channel, or by affecting the function of the channel in a manner that inhibits its opening (e.g. , by slowing the rate of opening or by making the open state less energetically favorable).
  • An exemplary method of inhibiting the opening of voltage-dependent ion channels, described herein, is to shift the steady-state inactivation curve of the channel to more hyperpolarized potentials, thereby increasing the percentage of channels that are in the inactivated state and are thus unable to open.
  • the term "significant”, when used with reference to "significantly different” or “significantly shifting”, refers to a difference in a quantifiable parameter between the two groups being compared that is statistically-significant using standard statistical tests.
  • the shift of an steady-state inactivation curve may be quantified using a ratio of currents elicited from different prepulse potentials. If such a ratio is measured for two groups of cells, and differs by an amount such that the standard errors of the means do not overlap, the steady-state inactivation curves from the two groups are said to be significantly shifted with respect to one another.
  • the degree of binding in a protein binding assay may be quantified using standard methods, and means for the degree of binding under different conditions similarly compared for statistically-significant differences.
  • Treating refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.
  • Constant amino acid substitutions are substitutions which do not result in a significant change in the activity (e.g. , channel modulating activity) or tertiary structure of a selected polypeptide. Such substitutions typically involve replacing a selected amino acid residue with a different residue having similar physico-chemical properties. For example, substitution of Glu for Asp is considered a conservative substitution since both are similarlysized negatively-charged amino acids. Groupings of amino acids by physico-chemical properties are known to those of skill in the art and can be found, for example, in Schultz and Schirmer (1979). It will be appreciated in making such conservative amino acid substitutions, however, that in peptides of the invention containing a domain derived from a region of syntaxin including position 244, the identity of that position not be changed from the naturally-occuring amino acid ⁇ valine.
  • “conservative substitutions thereof” refers to sequences that differ from the specific sequence by having conservative amino acid substitutions at one or more positions.
  • first peptide or polypeptide fragment When a first peptide or polypeptide fragment is said to "correspond" to a second peptide or polypeptide fragment, it means that the fragments are essentially co-extensive with one another when the sequences representing the fragments are aligned using a sequence alignment program, such as "MACVECTOR” (IBI, New Haven, CT) .
  • Corresponding peptide or polypeptide fragments typically contain a similar, if not identical, number of residues . It will be understood, however, that corresponding peptide or polypeptide fragments may contain insertions or deletions of residues with respect to one another, as well as some differences in their sequences .
  • a peptide or polypeptide sequence or fragment is "derived" from another peptide or polypeptide sequence or fragment when it has the same sequence of amino acid residues as the corresponding region of the fragment from which it is derived.
  • natural syntaxin is understood to refer to all naturally-occurring members of the syntaxin gene family, including but not limited to, syntaxin 1A, syntaxin 1B, syntaxin 2, syntaxin 3, syntaxin 4 and syntaxin 5.
  • syntaxin is understood to refer to all syntaxins falling under the definition of "natural syntaxin”, as well as syntaxin variants having conservative amino acid substitutions relative to a natural syntaxin .
  • syntaxin can refer to a syntaxin gene, syntaxin cDNA, or syntaxin protein encoded by a syntaxin gene or cDNA, as will be appreciated from the context in which it is used.
  • amphiphilic domain of syntaxin refers to a contiguous sequence of amino acid residues in syntaxin at least about 14 residues in length, preferably at least about 20 residues in length, that has an alpha helix amphiphilicity, as measured by the computer program "DNA STRIDER 1 .2" (Christian Marck, Service de Biochemie et de Genetique Moleisme, Department de Biologie Cellulaire et Moleisme, Direction des Sciences de la Vie - CEA - FRANCE), of greater than about 1 5
  • H1 " , “H2” , and “H3” refer to the first, second, and third helical domains of syntaxin, respectively .
  • H 1 is approximately defined as the region between amino acids 25 and 60
  • H2 is approximately defined as the region between amino acids 68 and 106
  • H3 is approximately defined as the region between amino acids 190-265
  • H1 , H2 and H3 are defined as the polypeptide regions corresponding to the polypeptide regions defined by H1 , H2 and H3 in syntaxin 1A, respectively .
  • a first peptide, polypeptide or protein (e.g., a syntaxin-derived peptide) is said to be "capable of specifically interacting" with a second peptide, polypeptide or protein (e.g., an N-type calcium channel) if (l) the first peptide, polypeptide or protein can bind to the second peptide, polypeptide or protein as determined using, e.g., a standard binding assay, or (n) if the first peptide, polypeptide or protein can alter the function of the second peptide, polypeptide or protein as determined using, e.g., functional assay
  • exemplary functional assays include those employing the Xenopus expression/recording system described herein.
  • These assays may be used, e.g., as described in Example 1 , to show that certain peptides derived from syntaxin (e.g., peptides having sequences represented as SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7) are capable of specifically interacting with N-type calcium channels as evidenced by functional effects on the channels (e.g., shifting the steady-state inactivation curve of the channels in the hyperpolarized direction).
  • syntaxin e.g., peptides having sequences represented as SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7
  • modulator when used with reference to calcium channels or calcium channel function refers to a compound which affects the function of the calcium channel.
  • a modulator may be an inhibitor, an antagonist, an agonist, or a compound which enhances the effects of other agonists or antagonists.
  • Voltage-dependent calcium (Ca 2+ ) channels provide an interface between the electrical signals carried by nerve cells and intracellular messengers (Hille, 1992).
  • a single opening of a Ca 2+ channel can allow hundreds or thousands of Ca 2+ ions to flow into a neuron, contributing to rises in [Ca 2+ ], that control such varied functions as transmitter release, excitability, metabolism and gene expression .
  • the diversity of Ca 2+ channel types revealed by a combination of electrophysiology and molecular biology (Tsien, et al., 1988; Bean, 1989; Hess, 1990; Miller and Fox, 1990; Llinas, et al., 1992; Snutch and Reiner, 1992), mirrors their multiplicity of function.
  • the pore-forming subunit of calcium channels is termed the ⁇ subunit.
  • the pore-forming subunit of calcium channels is termed the ⁇ subunit.
  • ⁇ subunit The pore-forming subunit of calcium channels.
  • isoforms of the ⁇ , subunit are expressed in mammalian brain.
  • the ⁇ 1A and ⁇ IB subunits are both expressed in many parts of the brain, including the hippocampus.
  • the ⁇ 1B Ca 2+ channel subunit generates an N-type ( ⁇ -conotoxin-GVIA-sensitive) Ca 2+ channel, while expression of the ⁇ 1A Ca 2+ channel subunit in Xenopus oocytes yields a novel phenotype, also found in cerebellar granule neurons, labeled 'Q-type' .
  • Channels encoded by ⁇ 1A subunits (Sather, et al., 1993; Stea, et al., 1994) are major mediators of Ca 2+ entry, and together with N-type channels, support synaptic transmission at hippocampal CA3-CA1 synapses (Wheeler, et al., 1994a,b).
  • ⁇ 1A channels The biophysical and pharmacological properties of ⁇ 1A channels were characterized using macroscopic and single channel recordings from Xenopus oocytes injected with ⁇ 1A cRNA to determine the degree of similarity of such expressed channels to traditional P-type channels (Sather, et al., 1993). These studies found that ⁇ 1A currents activate and inactivate more rapidly and display steeper voltage-dependence of gating than ⁇ 1C currents.
  • ⁇ 1A channels were largely insensitive to dihydropyridines (DHPs) and FPL 64176 (2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester), but responded to the cone snail peptide ⁇ -CTx-MVIIC (SNX-230), a potent and relatively selective inhibitor
  • DHPs dihydropyridines
  • FPL 64176 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester
  • SNX-230 cone snail peptide
  • ⁇ 1A channels in oocytes were ⁇ 10 2 -fold less sensitive to ⁇ -Aga-IVA
  • ⁇ 1A channels were not inhibited by Bay K 8644 and inactivated much more rapidly than P-type Ca 2+ channels .
  • the results of these experiments demonstrated that the ⁇ 1A subunit is capable of generating
  • Q-type current was > 2-fold larger than any another component (43 % of the total current).
  • Q-type current strongly resembled ⁇ 1A Ca 2+ channels expressed in Xenopus oocytes in pharmacology, voltage- and time-dependence.
  • Q-type current was partially blocked (up to 50%) by high doses (100/200 nM) of ⁇ -Aga-IVA .
  • Block of transmission was not seen at 30 nM ⁇ -Aga-IVA, but only at much higher agatoxin concentrations (200-1000 nM). Block was also seen with ⁇ -CTx-MVIIC .
  • the pharmacology resembled that of the ⁇ 1A Ca 2+ channel subunit expressed in Xenopus oocytes (Sather, et al ., 1993) and the Q-type Ca 2+ channel current in cerebellar granule neurons (Randall and Tsien, 1995) .
  • Voltage-dependent Ca 2+ channels respond to a sustained membrane depolarization by undergoing a series of conformational changes that control channel opening and eventual closing (Hille, 1992).
  • the closing of the channel termed inactivation, is important in determining how much Ca 2+ entry occurs as a consequence of single or multiple action potentials. In this way, Ca 2+ channel inactivation helps govern the impact of electrical activity on diverse cellular events such as transmitter release, contraction, modulation of membrane excitability, metabolism, and gene expression.
  • Inactivation may also be characterized using a "steady-state inactivation curve" (Hille, 1992).
  • This curve which is typically plotted as the fraction of channels available to open v. holding or prepulse voltage, reflects the distribution of calcium channels between closed states from which they can open v. inactivated states from which they cannot open. It is measured by holding the membrane at the "prepulse" voltage for a sufficiently long period to allow at least a large majority of the channels to reach equilibrium with respect to closed and inactivated states, and then pulsing the membrane voltage to a potential at which opening of any channels that are available to open can be detected.
  • Rapid and reliable synaptic transmission depends upon close proximity between voltage-gated calcium channels and neurotransmitter-containing vesicles in the presynaptic terminal (Llinas, et al., 1981 ). It has long been recognized that a local Ca 2+ rise conveys the essential signal from Ca 2+ channels to the exocytotic mechanism (Katz, 1969). The proteins involved in the exocytotic events have only recently been identified, however. One of these proteins is syntaxin.
  • Syntaxin is a 35 kDa presynaptic membrane protein (Bennett, et al., 1992; Yoshida, et al., 1992; Bennett, et al., 1993) that is thought to play a key role in synaptic vesicle docking and fusion (Sollner, et al., 1993) and that has specific interactions with (i.e. , binds to) N-type Ca 2+ channels (Bennett, et al., 1992; Yoshida, et al., 1992; Horikawa, et al., 1993; Leveque, et al., 1994; Sheng, et al., 1994).
  • the domain structure of syntaxin includes putative helical domains (e.g. , H3 domain, amino acids ⁇ 190-265) with propensity to interact with other proteins (Chapman, et al., 1994; Kee, et al., 1995) and a putative transmembrane domain (Bennett, et al., 1993) (amino acids 266-288; SEQ ID NO: 8).
  • Example 1 The effects of coexpression of syntaxin and N-type calcium channels on the electrophysiological properties of the expressed channels were examined as detailed in Example 1.
  • the functional interaction between syntaxin and N-type Ca 2+ channels was demonstrated by a standardized experimental protocol in which inward Ba 2+ currents were evoked by a series of 50 ms test pulses to 0 mV from one of three holding potentials In the first set of 10 pulses the holding potential was -60 mV, in the second set of 10 pulses it was -80 mV and in the last set of 20 pulses it was -120 mV. The peak of the current-voltage relationship of the current evoked by the last pulse in each set was plotted .
  • This protocol was used to compare the behavior of N-type channels ( ⁇ 1B ⁇ 3 ⁇ 2 ) expressed by themselves (control, sham re-injection with water), co-expressed with full- length syntaxin 1A (S1A, a a 1-288, SEQ ID NO: 1), or co-expressed with the N-terminal two-thirds of syntaxin 1A (S1A-N, a a 1-189, SEQ ID NO: 2).
  • Figs 1A-1I The results of the experiments are shown in Figs 1A-1I.
  • peak current amplitudes obtained during the course of the procedure (Figs. 1G, 1H and 1I) are shown beneath current records evoked by the last pulse from each holding potential (Figs 1A, 1B and 1C).
  • the results show that in comparison to oocytes injected with water or with the N-terminal portion of syntaxin (S1A-N), coexpression of full-length syntaxin with N-type channels decreased their availability at -60 mV and -80 mV relative to that at -120 mV.
  • Figs . 2A and 2B show the time course of changes in the i 80 /i 120 ratio (Fig 2A) and the time constant ⁇ 120 (Fig. 2B).
  • injection of water (control) or s1A-N failed to affect channel availability (as in Fig. 1)
  • injection of cRNA encoding s1a caused a gradually developing decrease in the relative size of the current at -80 mV and an increase in ⁇ 120 .
  • some effect of full-length syntaxin was apparent as early as 14 hr after injection of its cRNA, it took - 2 days for these effects to reach their maximal extent. This is consistent with the expected time course of appearance of proteins expressed in oocyte plasma membrane.
  • Example 3 presents experiments which compare the effects of syntaxin coexpression on Q- and L-type calcium channels with the effects on N-type channels described with respect to Example 1 .
  • Q-type channels were expressed by injection of oocytes with cRNAs for the subunit combination ⁇ 1A ⁇ 3 ⁇ 2 .
  • the oocytes were divided into 3 groups for later reinjection with water (control), or cRNAs encoding s1A or s1A-N.
  • the properties of the resulting currents were evaluated 4-5 days after the second cRNA injection.
  • L-type channels failed to respond in any detectable way to co-injection of si A cRNA when studied with a protocol appropriate to their voltage-dependence of inactivation (40/100).
  • a protocol appropriate to their voltage-dependence of inactivation 40/100.
  • the value of i 40 /i 100 in tne presence of syntaxin was not different than that found in control or with the N-terminal segment of syntaxin.
  • the three types of Ca2 + channels display an overall pattern of responsiveness to syntaxin that corresponds to their degree of involvement in evoked synaptic transmission at CNS nerve terminals .
  • syntaxin binding favors inactivated rather than available channel states in both Q-type and as N-type calcium channels.
  • channel inactivation strengthens the syntaxin interaction.
  • a series of syntaxin mutants was evaluated to identify the structural determinants involved in the interaction with N-type channels.
  • the mutants were co-expressed with N- type channels in oocytes, and the properties of the resulting currents were evaluated 3-4 days after cRNA injection by means of the standard 60/80/120 protocol as described in Example 4.
  • the mutants are illustrated schematically in Fig . 4A .
  • the carboxyl-terminal transmembrane domain (amino acids 266-288, SEQ ID NO: 8) is shaded.
  • the average i 80 /i 120 ratios for the various mutants are shown in Fig. 4B.
  • s1A-N SEQ ID NO: 2
  • co-expression of a carboxyl terminal construct (s1A-C, a a 168-288, SEQ ID NO: 3) had nearly the same inhibitory effect on N-type channel availability as syntaxin itself. This suggests that the N-terminal two-thirds portion of syntaxin is apparently not required for syntaxin's action on Ca 2+ channel gating, although the presence of the domain may alter some aspects of the effect.
  • the C-terminal portion of syntaxin consists in turn of two distinct domains the H3 domain (a.a. 191-265), a region of high ⁇ -helical content and amino acid conservation (Kee, et al. , 1995), and a putative transmembrane domain, or region (TMR, SEQ ID NO: 8; a.a. 266-288, Bennett, et al., 1993).
  • the H3 domain has been implicated in protein-protein interactions between syntaxin and several other key proteins involved in the formation of a fusion complex, such as SNAP-25, ⁇ -SNAP, VAMP and n-secl (Kee, et al., 1995).
  • At least one alpha-helical amphiphilic domain e.g. , H 1 , H2 and/or H3 is involved in the interaction of a synataxin-derived peptide of the invention with a calcium channel responsible for neurotransmitter release.
  • Such a peptide or peptides is administered to an individual suffering from a disease or disorder which could benefit from inhibition of neurotransmitter release.
  • a disease or disorder which could benefit from inhibition of neurotransmitter release.
  • Examples of such conditions and/or disease include
  • anoxic/ischemic heart disease cardiac arrhythmias, coronary artery disease cardiomyopathy, anoxic/ischemic brain damage (stroke), epilepsy, neuronal death associated with chronic epilepsy and various neurodegenerative diseases, as well as intractable pain.
  • cardiac arrhythmias cardiac arrhythmias
  • coronary artery disease cardiomyopathy anoxic/ischemic brain damage (stroke)
  • epilepsy neuronal death associated with chronic epilepsy and various neurodegenerative diseases, as well as intractable pain.
  • Peptides of the invention may be administered by any of a number of methods known in the art, including oral, nasal insufflation, intraocular, parenteral, and anal and/or vaginal suppository administrations.
  • a preferred method of delivery to sites outside the blood-brain barrier is via hposomes (e.g, fusogenic hposomes) loaded with the selected peptide using standard known methods.
  • the hposomes may further be constructed to contain a targeting moiety or hgand, such as an antigen, an antibody, or a virus on their surface to facilitate delivery to the appropriate target sites (Betageri, G.V., et al.
  • the liposomes are then delivered to the target tissue using standard methods.
  • the liposomes can be delivered to the heart by mtracardiac injection or intracardiac catheter.
  • the hposome preparation may be lyophilized for long-term storage according to methods known in the art.
  • the compounds may be introduced by injection into either the epidural or subarachnoid space of the central nervous system (CNS) Epidural injection is routinely and safely performed, e.g., for administering local anesthetic to women in childbirth.
  • the peptides may be suspended in a sterile carrier or buffer suitable for epidural injection (e.g., preservative-free saline), and administered to the individual, e.g., as an epidural bolus injection or as a continuous, constant-rate epidural infusion.
  • the peptides may also be injected directly into cerebrospinal fluid (i e , into the subarachnoid space). Injection into the subarachnoid space of the spinal cord may be accomplished using a lateral cervical (C1-C2) puncture or, preferably, a lumbar puncture Alternatively, the peptide may be introduced into the cerebrospinal fluid by lntracerebro-ventricular (ICV) injection . As discussed above, the peptide is suspended in a sterile buffer compatible with cerebrospinal fluid (e.g., sterile physiological preservative-free saline solution) and injected using standard medical techniques for such injections, e.g. , techniques employing mtracerebroventricular catheters (see, e.g., Buchsbaum, et al. , 1991; Bryan, et al., 1982) .
  • a sterile buffer compatible with cerebrospinal fluid e.g., sterile physiological preservative-free
  • the methods may be practiced using an implantable pump or drug delivery device.
  • implantable or body-mountable pumps useful in delivering compound at a regulated rate are known in the art .
  • One such pump described in U .S. Patent 4,619,652, is a body-mountable pump that can be used to deliver compound at a tonic flow rate or at periodic pulses.
  • Prolonged administration may also be effected by depot or sustained release formulations known in the art.
  • the peptide is combined with slow release polymers such as poly-2-hydroxyethylmethacrylate or methylenevinylacetate copolymer for implantation at the target site .
  • the peptide can be combined with a topical ointment and applied directly to the surface of the skin (e.g., for surface pain) .
  • the peptides are delivered to the target sites at regular intervals for the duration of treatment, which is determined by the attending physician.
  • a therapeutic peptide may be delivered once to several times daily in bolus injections containing between about 1 and about 100 ⁇ g peptide.
  • the peptide is contained in an encapsulant (e.g., liposomes), the amount of the suspension delivered is adjusted so that the selected pharmaceutically-effective amount of peptide is delivered.
  • the dose is determined in part based on the bioavailability and pharmacokinetics of the particular peptide employed using standard pharmacokinetics principles known in the art. Methods for preparing such dosages are known or will be apparent to those skilled in the art, for example, see Remington's Pharmaceutical Sciences (1980).
  • Peptides of the invention can be translated into bioavailable low molecular weight organic drug lead compounds that can be tested for their ability to inhibit calcium channels involved in neurotransmitter release.
  • a peptide mimetic e.g., a small organic compound
  • pharmacophonc groups that are responsible for activity are identified through structure-function analyses such as "alanine scanning” (e.g., Beck-Sickmger and Jung, 1995; Goldberg, et al. , 1993, Nagashima. et al.
  • the present invention includes methods of identifying syntaxin-derived Ca 2+ channel modulatory compounds which are capable of stimulating or inhibiting the opening of voltage-dependent calcium ion channels involved in neurotransmitter release.
  • the method includes contacting a calcium channel subunit of a voltage-dependent calcium ion channel involved in neurotransmitter release with a peptide derived from syntaxin, in the presence and absence of a test compound.
  • the effect of the test compound on the degree of binding between the calcium channel subunit and the peptide is measured by comparing the degree of binding in the presence and in the absence of the compound.
  • the calcium channel subunit may be only a portion of a complete calcium channel subunit; for example, it may be a peptide containing only that portion of the subunit that interacts with syntaxin (e.g. , the II/III loop; Sheng, et al.).
  • syntaxin e.g. , the II/III loop; Sheng, et al.
  • the compound is selected if the degree of binding in the presence of the compound is significantly different from the degree of binding in the absence of the compound, e.g. , if the degree of binding in the presence of the compound is significantly lower or significantly higher.
  • the selected compound is further screened for its ability to affect the opening of voltage-dependent calcium ion channels involved in neurotransmitter release as described above.
  • test compound is identified as effective if it selectively modulates (e.g. , stimulates or inhibits) the opening of calcium ion channels involved in neurotransmitter release using a functional (i.e. , physiological) assay.
  • the calcium channel subunit and the syntaxin-derived peptide may be produced either recombinantly or synthetically.
  • polynucleotide sequences encoding, e.g. , a selected calcium channel subunit or portion thereof are cloned into an expression plasmid to produce corresponding polypeptides.
  • Exemplary plasmids suitable for this method include p-GEX-2T and p-GEX-4T (Pharmacia Biotech, Piscataway, NJ) as well as pQE-9 (Qiagen, Chatsworth, CA).
  • the pGEX plasmids are designed for inducible, high level intracellular expression of genes or gene fragments as fusions with Schistosoma japonicum glutathione S-transferase (GST; Smith and Johnson). They contain a tac promoter for chemically-inducible expression, the GST gene, a thrombin protease recognition site, a multiple cloning site, an ampicillin resistance gene, a pBR322 ori, and an internal lac Iq gene.
  • Another exemplary plasmid is pGEX-KG (Guan and Dixon). which was derived from the pGEX-2T plasmid (Pharmacia Biotech) by incorporation of an EcoRI fragment encoding a nine amino-acid glycine-rich linker (Guan and Dixon).
  • Plasmids containing the desired sequences can be transformed into appropriate strains of E. coli and fusion protein production can be induced by the addition of IPTG (isopropyl-thio galactopyranoside). Solubilized recombinant fusion protein can then be purified from cell lysates of the induced cultures using glutathione agarose affinity chromatography according to standard methods (e.g., Ausubel, et al., 1992).
  • Isolated recombinant polypeptides produced as described above may be purified by standard protein purification procedures. These procedures may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, lsoelectric focusing, gel electrophoresis and affinity chromatography.
  • calcium channel subunit and/or syntaxin proteins or polypeptides can be isolated from selected cells by affinity-based methods, such as by using appropriate antibodies. Further, calcium channel subunit and/or syntaxin peptides may be chemically synthesized using methods known to those skilled in the art.
  • the contacting is carried out using a solid support, where one of the pair of interacting proteins (i.e., either the calcium channel subunit or the peptide derived from syntaxin) is immobilized on the solid support (i.e., the solid support is "derivatized” with the protein or peptide), and the other of the interacting pair of proteins is suspended in a solution that is brought in contact with the derivatized solid support.
  • the peptide, polypeptide or protein that is immobilized on the solid support is referred to as the "immobilized” peptide, polypeptide or protein
  • the peptide, polypeptide or protein that is suspended in solution is referred to as the "free” peptide, polypeptide or protein.
  • An exemplary solid-support-based method for assaying the degree of binding between two peptides or polypeptides employs protein fusions between the proteins of interest (i.e., a calcium channel subunit or a peptide containing the transmembrane domain of syntaxin) and glutathione-S-transferase (GST).
  • the proteins or polypeptides used in the assays are produced in bacteria as fusions between glutathione-S- transferase (GST) and the recombinant protein (i.e., a calcium channel subunit or a peptide containing the transmembrane domain of syntaxin).
  • Bacterial cell lysates from clones containing the respective constructs are prepared and passed over agarose beads derivatized with glutathione, resulting in the attachment of the GST portions of the fusions to the glutathione on the agarose beads.
  • the immobilized polypeptide is left attached to the beads, while the free polypeptide is cleaved from the beads (e.g., with a protease such as thrombin) prior to the assay and suspended in a solution to be contacted with the derivatized beads.
  • the unbound free protein was washed off, and free protein that bound to the immobilized protein is detected using any of a number of methods known in the art (Ausubel, et al. , 1992, Calakos, et al., 1994, Sheng, et al., 1994).
  • the amount of binding can be detected using a Western blot approach with antibodies directed against the free protein.
  • the free protein can be labeled, e.g., with 125 I, prior to the assay, and the amount of free protein bound to the immobilized protein detected using standard methods.
  • a partially-purified (e.g., by the GST methods above) syntaxin polypeptide may be attached to the bottoms of wells in a multiwell plate (e.g., 96-well plate) by introducing a solution containing the polypeptide into the plate and allowing the polypeptide to bind to the plastic. The excess peptide-containing solution is then washed out, and a blocking solution (containing, for example, bovine serum albumin (BSA)) is introduced to block non-specific binding sites. The plate is then washed several more times and a solution containing a labelled calcium channel subunit is added. The solution is then washed off as above, and the amount of bound calcium channel subunit is detected .
  • a blocking solution containing, for example, bovine serum albumin (BSA)
  • a solution containing the test compound may be incubated with the immobilized polypeptide or with the free polypeptide prior to the assay.
  • the test compound may be included in the solution in which the free polypeptide is suspended. The effect of the test compound on the extent of binding between the immobilized and free polypeptides is measured, and a compound is selected if the degree of binding in the presence of the compound is significantly different from the degree of binding in the absence of the compound.
  • different wells may contain, for example, different test compounds, different concentrations of the same test compound, or different concentrations of the interacting proteins.
  • the wells may also be pre-coated with substance(s) that enhance attachment of the protein to be immobilized and/or decrease the level of non-specific binding .
  • the wells may be derivatized to contain glutathione and may be pre-coated with BSA, to promote attachment of the immobilized protein in a known orientation with the binding site(s) exposed.
  • the selected compound is further screened for its ability to affect the opening of voltage-dependent calcium ion channels involved in neurotransmitter release, as described above.
  • the selected compound is identified as effective if it selectively modulates (e.g., stimulates or inhibits) the opening of the calcium ion channels. Modulation of the opening of calcium ion channels may be assayed using, for example, the electrophysiological approaches detailed herein. Alternatively or in addition, modulation may be assayed using optical techniques with calcium-sensitive dyes. For example, a synaptosome preparation, as detailed in Example 5, may be used to evaluate the effectiveness of selected test compounds on calcium influx as measured using, e.g., Fura-2.
  • modulatory compounds described herein may stimulate, inhibit, or otherwise augment the function of N- and Q-type calcium channels.
  • inhibitory compounds described herein peptides having sequences identified as SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO : 6 and SEQ ID NO: 7 shift the steady-state inactivation curve of calcium channels in the hyperpolarized direction, decreasing the probability that a given channel will open (i.e. , inhibiting the opening of the channel) .
  • Other compounds identified according to the methods of the present invention may be effective at stimulating the opening of calcium channels, for example, by shifting the inactivation curve in the depolarized direction.
  • a variety of different compounds may be screened using methods of the present invention. They include peptides, macromolecules, small molecules, chemical and/or biological mixtures, and fungal, bacterial, or algal extracts . Such compounds, or molecules, may be either biological, synthetic organic, or even inorganic compounds, and may be obtained from a number of sources, including pharmaceutical companies and specialty suppliers of libraries (e.g., combinatorial libraries) of compounds.
  • an identified active compound is a peptide
  • the peptide may be utilized to aid in the discovery of orally-active small molecule mimetics.
  • a peptide having the sequence (SEQ ID NO :1 , SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 7) is effective at inhibiting the opening of calcium channels.
  • the structure of any of such peptides or polypeptide may be determined using, for example, NMR, and may be used to select the types of small molecules screened.
  • methods of the present invention are well suited for screening libraries of compounds in multi-well plates (e.g., 96-well plates), with a different test compound in each well
  • the methods may be employed with combinatorial libraries .
  • a variety of combinatorial libraries of random-sequence oligonucleotides, polypeptides, or synthetic oligomers have been proposed (Kramer, et al. , 1993, Houghten, 1985, 1994, Houghten, et al. , 1986, 1991 , 1992, Ohlmayer, et al. , 1993, Dooley, et al. , 1993a- 1993b, Eichler, et al. , 1993, Pinilla, et al.
  • Combinatorial libraries of oligomers may be formed by a variety of solution-phase or solid-phase methods in which mixtures of different subunits are added stepwise to growing oligomers or parent compound, until a desired oligomer size is reached (typically hexapeptide or heptapeptide).
  • a library of increasing complexity can be formed in this manner, for example, by pooling multiple choices of reagents with each additional subunit step (Houghten, et al., 1991).
  • the library may be formed by solid-phase synthetic methods in which beads containing different-sequence oligomers that form the library are alternately mixed and separated, with one of a selected number of subunits being added to each group of separated beads at each step (Furka, et al., 1991 ; Lam, et al., 1991 , 1993; Zuckermann, et al. ; Sebestyen, et al.).
  • library compounds with desired effects on the binding of a calcium channel subunit to a syntaxin peptide can be determined by conventional means, such as iterative synthesis methods in which sublibraries containing known residues in one subunit position only are identified as containing active compounds.
  • iterative synthesis methods in which sublibraries containing known residues in one subunit position only are identified as containing active compounds.
  • restriction enzymes and DNA modifying enzymes were obtained from New England Biolabs (Beverly, MA), Boehringer Mannheim (Indianapolis, IN) or Promega Corp. (Madison, WI), and other chemicals were purchased from Sigma (St. Louis, MO) or United States Biochemical (Cleveland, OH).
  • Xenopus oocytes were isolated essentially as described by Zaeaux, et al. (1989).
  • Female Xenopus laevis were obtained from NASCO (Fort Atkinson. WI) and maintained at room temperature (20-22°C). The frogs were anesthetized by immersing for 20 minutes in 0.1-0.2 % ethyl m-aminobenzoate (tricaine; MS-222; Sigma Chemical Co. , St. Louis, MO). A small incision was made on the abdomen and the ovarian lobes were removed into OR2 solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 5 mM HEPES, adjusted to pH 7.6 with NaOH).
  • stage V and VI oocytes were then transferred to ND96 solution (96 mM NaCl, 2 mM KCl, 1 .8 mM CaCl, 1 mM MgCl 2 , 5 mM HEPES adjusted to pH 7.6 with NaOH) and maintained at 18°C until used for injection with cRNA (typically a few minutes to a few hours).
  • cRNA typically a few minutes to a few hours.
  • rat syntaxin s1A The coding sequence of rat syntaxin s1A (Bennett, et al ., 1992, GenBank Accession Number M95734) was isolated using PCR (Mullis, 1987, Mullis, et al ., 1987) with primers containing restriction enzyme recognition sites (5' primer had a HindIII site, 3' primer had an EcoRl site), the PCR product was digested with the enzymes, blunted on the 5' end and introduced into the SmaI/EcoRI sites of the pGEMHE Xenopus oocyte expression vector (Liman, et al ., 1992) .
  • the N-terminal construct (s1A-N) was generated in the same way, but with a stop codon replacing the triplet encoding Gln-190 of si A (Bennett, et al ., 1992)
  • cRNA encoding calcium channel subunits was synthesized using cDNA clones encoding human brain ⁇ 1B (Ellinor, et al ., 1994), rabbit skeletal muscle ⁇ 2 / ⁇ (Mikami, et al ., 1989), and rabbit brain ⁇ 3 (Hullin, et al ., 1992) as templates.
  • Capped cRNA was synthesized in vitro as described (Bezprozvanny and Tsien, 1995) and tested by in vitro translation with a rabbit reticulocyte lysate system (Promega Corp ) prior to injection into oocytes . The efficiency of translation was monitored with 35 S-Met and a "FUJIBAS-2000" phosphoimager.
  • the plasmid was linearized with NheI, 2-5 ⁇ g of the linearized plasmid were suspended in 50 ⁇ l of a standard transcription buffer (Ausubel, et al ., 1992; Sambrook, et al.
  • RNA was purified by extractions with phenol/chloroform (1 :1 , vol/vol) and chloroform, ethanol precipitated with aminonium acetate, and resuspended in 20 ⁇ l diethyl pyrocarbonate (DEPC)-treated water.
  • DEPC diethyl pyrocarbonate
  • Oocytes prepared as above were microinjected with approximately 20 ng cRNA ( ⁇ 50 nl of a solution containing ⁇ 0 4 ⁇ g/ ⁇ l cRNA prepared as above and diluted if necessary with DEPC-treated water) using a Drummond Nanoject (Fisher Scientific, Pittsburgh, PA) pressure injector.
  • Injection pipets were fashioned using a standard patch pipet puller (Narashige, Japan) set to produce pipets with long ( ⁇ 1.5 - 2 cm) tips. The pipets baked at > 150°C for 4-5 hours to inactivate RNases, and the tips were broken by poking a ChemWipe. The pipets were backfilled with mineral oil and tips were filled with solution containing the RNA to be injected.
  • Injected oocytes were maintained in ND96 solution supplemented with 2.5 mM sodium pyruvate, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin (Pen-Strep mixture obtained from Sigma, cat # P0781) at 18°C. D Recording Calcium Channel Currents
  • Calcium channel subunit cRNAs ( ⁇ 1B , ⁇ 3 and ⁇ 2 ) were premixed in roughly equimolar ratios and injected into freshly isolated Xenopus laevis oocytes as described above . Four to five days later, oocytes were divided into several groups and reinjected with syntaxin s1A cRNA, s1A-N cRNA or an equal volume of water (control). Expressed Ca 2+ channel currents were evaluated 2-4 days after the second cRNA injection unless indicated otherwise. Two-microelectrode voltage clamp recordings were carried out using an Oocyte Clamp OC-725A voltage clamp (Warner Instruments, New Haven, CT).
  • Intracellular Ag/AgCl electrodes were constructed with an initial input impedance of 0.5 to 2 M ⁇ in 3 M K Cl. Unless indicated otherwise, the oocyte recording solution contained 5 mM external Ba 2+ as charge carrier (5 mM Ba(OH) 2 , 2 mM KOH, 85 mM tetraethylaminonium and 10 mM HEPES, pH adjusted to 7 4 with methanesulfonic acid).
  • 5 mM external Ba 2+ as charge carrier 5 mM Ba(OH) 2 , 2 mM KOH, 85 mM tetraethylaminonium and 10 mM HEPES, pH adjusted to 7 4 with methanesulfonic acid).
  • N-type calcium channels expressed in Xenopus oocytes were studied as follows.
  • the channels were either expressed alone (Figs 1A and 1G), co-expressed with full-length syntaxin s 1 A (SEQ ID NO:1 ; Figs. 1B and 1H), or co-expressed with the N-terminal portion of s1A (s1A-N, SEQ ID NO: 2; Figs 1C and 1I) as described above.
  • Test pulses 50 ms in duration to 0 mV, which is the peak of the N-channel I-V relationship (Ellinor, et al. , 1994, Bezprozvanny and Tsien, 1995), were applied at 0. 1 Hz from successively more negative holding potentials (HPs).
  • Oocytes already expressing N-type Ca 2+ channels were reinjected with cRNA encoding si A as described above to determine if alterations in channel properties increased with time, as expected for progressively-increasing s1A expression .
  • Results of the experiments are illustrated in Figs 2A, 2B, 2C and 2D.
  • Figs 2C and 2D show the i 80 /i 120 ratio (Fig 2C) and ⁇ 120 (Fig 2D) determined using the descending staircase protocol in oocytes injected with similar volumes of s 1A cRNA-containing solution, either undiluted ( ⁇ 20 ng/oocyte) or diluted 2-, 4-, 8-, 16- or 32-fold.
  • Zero concentration corresponds to the data obtained with a control batch of oocytes injected with water .
  • cRNA for Q-type channels was made with an ⁇ 1A cDNA template derived from rabbit brain (Mori, et al ., 1991) and cRNA for L-type channels was made with an ⁇ 1c cDNA clone derived from rabbit heart (Mikami, et al., 1989) .
  • L-type Ca 2+ channel currents are smaller and express more slowly that either N- or Q-type channel currents, oocytes injected with message for L-type channels were given more time (total of 5-8 days) before reinjection with s1A and s1A-N cRNA.
  • recordings from L-type channels were carried out with 40 mM external Ba 2+ solution and a test potential of +20 mV
  • FPL 64176 (2 ⁇ M) was present to enhance the L-type currents. Results obtained in the presence and absence of FPL 64176 were not significantly different and were averaged together.
  • Oocytes expressing ⁇ 1A ⁇ 3 ⁇ 2 (Q-type channels) or ⁇ 1C ⁇ 3 ⁇ 2 (L-type channels) were reinjected with water (control) or cRNAs encoding s1A or s1A-N as described above.
  • Inactivation properties of the Q-type currents were evaluated 4-5 days after the second injection and those of L-type currents were evaluated 5-8 days after the second injection. Because inactivation of Q- and L-type channels takes place over a less negative range of potentials than N-type channels (Bezprozvanny and Tsien, 1995), the descending staircase protocol was changed from 60/80/120 to 40/60/120 for Q-type channels and to 40/100 for L-type channels.
  • Figs 3 A, 3B, 3C, 3D and 3E compare syntaxin effects on the properties of N-type channels (Fig 3A -data from experiments described in Example 1 , Fig 3D), Q-type channels (Figs 3B and 3E) and L-type channels (Fig 3C).
  • the peak currents for the most negative holding potential ranged between 5-15 ⁇ A for N-type, 5-25 ⁇ A for Q-type, and 0 5-2 5 ⁇ A for L-type.
  • L-type channels (a 1C ⁇ 3 ⁇ 2 ) failed to respond in any detectable way to co-injection of s 1A cRNA (Fig. 3C).
  • syntaxin was examined over a wide range of holding potentials to determine if the inhibitory effects on N- and Q-type channels were accentuated as channels became increasingly inactivated. If this were the case, the voltage-dependence of channel availability would be expected to shift toward more negative potentials.
  • Voltage-dependent steady-state inactivation curves were assessed with a prepulse protocol (Fig. 3F).
  • the oocyte membrane was held for 30 s at various potentials (V prepulse ) between -20 mV and -140 mV, returned to -80 mV for 20 ms, and then depolarized with a 50 ms test pulse to 0 mV to evoke inward Ba 2+ current.
  • the voltage-dependence of the channels is assessed in oocytes re-injected with water (control), with cRNA for s1A or with cRNA for s1A-N
  • Fig 4A and the effects of coexpression of the constructs on the i 80 /i 120 ratio of N-type calcium channels are shown in Fig . 4B.
  • a carboxy-terminal segment (S1A-C, SEQ ID NO: 3; a.a. 168-288) had nearly the same inhibitory effect on channel availability as full-length syntaxin.
  • This segment includes a putative helical domain, H3 (a.a. 191-265), important for interactions with several key presynaptic proteins (Chapman, et al., 1994, Kee, et al., 1995, McMahon and Sudhof, 1995, Hayashi, et al., 1995).
  • Syntaxin-derived Ca 2+ channel modulatory compounds are tested in rat brain synaptosome preparations (e.g., Fontana and Blaustein, 1993, Taghatela, et al. , 1990) to evaluate their effect on Ca 2+ influx through native Ca 2+ channels.
  • the calcium indicator dye Fura-2 is used to monitor Ca 2+ influx in a spectrofluorimeter. Test compounds are introduced to the synaptosome preparation and the Fura-2 signal from such test samples is compared to signal from control samples Compounds which are effective at shifting the voltage-dependence of calcium channel inactivation in the hyperpolarized direction are expected to result in decreased calcium influx and accordingly lower Fura-2 signal.
  • Compounds which are effective in the synaptosome assay may be further tested in cells expressing predominantly N-type or Q-type native calcium channels.
  • the calcium current is measured using the whole-cell patch-clamp technique (Randall and Tsien, 1995)
  • Sympathetic neurons are particularly useful because they can form synapses very close to the cell body, with good access for agents introduced by whole cell dialysis.
  • Test agents are introduced either intracellularly or extracellularly and the effects of the agents are evaluated using electrophysiological approaches as described above.

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Abstract

Composition dérivée de la syntaxine, contenant un peptide capable de déplacer dans le sens de l'hyperpolarisation la courbe d'inactivation à l'équilibre d'un canal calcique potentiel-dépendant, procédé permettant d'inhiber sélectivement l'ouverture des canaux calciques potentiel-dépendants associés à la libération de neurotransmetteurs, et procédé permettant d'identifier un composé capable de moduler l'ouverture desdits canaux.
PCT/US1996/019002 1995-12-01 1996-11-27 Compositions modulatrices des canaux calciques et procedes associes Ceased WO1997020218A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999033981A3 (fr) * 1997-12-31 1999-11-04 Incyte Pharma Inc Proteines contenant des sequences-signaux humaines
WO2002061431A3 (fr) * 2001-01-30 2003-06-05 Herbert Young Gaisano Nouvelle cible de medicament dans le recepteur de sulfonyluree

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