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WO1999029847A1 - Canaux calciques potentiel-dependants de type t et leurs methodes d'utilisation - Google Patents

Canaux calciques potentiel-dependants de type t et leurs methodes d'utilisation Download PDF

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Publication number
WO1999029847A1
WO1999029847A1 PCT/US1998/023161 US9823161W WO9929847A1 WO 1999029847 A1 WO1999029847 A1 WO 1999029847A1 US 9823161 W US9823161 W US 9823161W WO 9929847 A1 WO9929847 A1 WO 9929847A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
type calcium
calcium channel
cell
channel
Prior art date
Application number
PCT/US1998/023161
Other languages
English (en)
Inventor
Edward Perez-Reyes
Leanne L. Cribbs
Original Assignee
Loyola University Of Chicago
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Loyola University Of Chicago filed Critical Loyola University Of Chicago
Priority to CA002312477A priority Critical patent/CA2312477A1/fr
Priority to EP98956416A priority patent/EP1036170A1/fr
Publication of WO1999029847A1 publication Critical patent/WO1999029847A1/fr

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Classifications

    • 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

Definitions

  • the nucleic acid For expression in a living cell, the nucleic acid must be introduced into the cell.
  • the present invention provides a vector having a T-type calcium channel nucleic acid of the type described above.
  • Any type of vector suitable for introducing the nucleic acid into a host cell is within the context of the present invention.
  • examples of such vectors include naked DNA and RNA vectors (such as oligonucleotides, plasmids. capped cRNA, etc.).
  • viral vectors such as adeno-associated viral vectors (Berns et al., Annals of the New York Academy of Sciences, 772. 95-104 (1995)), adenoviral vectors (Bain et al..
  • an isolated cell into which the T-type calcium channel nucleic acid has been introduced can be prepared, preferably, such transfection protocols result in a population consisting essentially of such transfected cells.
  • an established cell line consisting essentially of such cells Preferably, for use in high throughput assays, cell lines stably expressing a T-type calcium channel exhibit a current density of at least about 40 pA/pF (e.g.. at least about 45 pA/pF). such as about 50 pA/pF or even 55 pA/pF or higher.
  • a putative drug Once a putative drug is detected, its effect on the electrophysiology of the cell (e.g., single channel conductance, voltage dependency, activation kinetics, inactivation kinetics, and tail current of the cells) can be investigated in detail.
  • the effect of the putative drug on T-type calcium currents is assessed by measuring the various electrophysiological parameters in the presence of various concentrations of the drugs and comparing the data to untreated (or sham-treated) control cells. Cells preferably are maintained in a continuous perfusion chamber during such experiments to facilitate changing solutions.
  • the inventive method of identifying a drug which affects T-type calcium channels can employ any nucleic acid encoding a T-type calcium channel (or derivative thereof), such as those nucleic acids described herein.
  • the method of expressing the T-type calcium channel nucleic acid can also be used in vivo.
  • several neurological and muscular diseases or disorders have implicated mutations affecting native nucleic acids encoding T-type calcium channels.
  • the present invention thus, provides a method of treating a disease or disorder associated with a deficiency in a native T-type calcium channel nucleic acid. The method involves introducing a vector having the T-type calcium channel nucleic acid into cells of a host in which native expression of the nucleic acid is deficient.
  • the vector is introduced into myocardial cells.
  • the vector is introduced into neurons (e.g.. thalamic neurons).
  • the nucleic acid within the vector is expressed to produce active T-type calcium channel.
  • an nucleic acid having a sequence antisense to a sequence encoding a T-type calcium channel (or a portion thereof) can be expressed within a cell. The presence of an antisense sequence can down-regulate the expression of native T-type calcium channel genes by hybridizing to T-type channel mRNA within the cell.
  • the present invention is useful to treating disorders associated with over-expression of T-type calcium channels.
  • This example demonstrates the cloning and characterization of putative T-type calcium channels.
  • Figures 1 A- IE indicate this conservation between the proteins.
  • the conservation of charged residues, particularly in the S4 domains, is consistent with the role of the ⁇ lG, ⁇ lH, and all proteins as ion channels.
  • two of the glutamates associated with ion specificity in other calcium channels have been replaced with aspartate. suggesting altered ion selectivity.
  • Strikingly, ⁇ lG, ⁇ lH, and all display only low homology to sequences linking the membrane-spanning regions within each domain, and even less homology between the intracellular loops linking domains.
  • neither ⁇ lG, ⁇ lH. nor all possesses sequences known to bind ⁇ subunits or Ca 2+ ions.
  • This example demonstrates the production of cell lines stably expressing the cloned ⁇ l G. ⁇ l H. and al l proteins.
  • the internal pipette solution contained the following: 55 mM CsCl, 75 mM CsSO , 10 mM MgCl 2 , 0.1 mM EGTA, 10 mM HEPES, pH adjusted to 7.2 with CsOH.
  • the external Tyrodes solution was the following: 140 mM NaCl, 6 mM KC1, 2 mM CaCl 2 , 10 mM glucose, 5 mM HEPES, pH 7.4.
  • the recording solution contained the following: 10 mM BaCl 2 solution (or 2 mM CaCl 2 ), 140 mM tetraethylammonium (TEA) chloride, 5 mM CsCl.
  • Figures 2A-2E depict data obtained from these experiments using cells injected with ⁇ lG ( Figure 2A). ⁇ lH ( Figure 2B). and all ( Figure 2C) and ⁇ lE ( Figure 2D). These data indicate that cells expressing ⁇ lG. ⁇ lH. and al l exhibit T-type calcium current, while oocytes expressing ⁇ lE as well as uninjected oocytes ( Figure 6A) do not.
  • tail current for each of the cloned channels is between about 1 ms and about 10 ms following repolarization to a membrane potential from about -80 mV to about -60 mV in a solution with a barium concentration of from about 10 mM to about 40 mM.
  • Single channel conductance is complicated by the low probability of channel opening at negative potentials when the driving force is large. Thus, single channel conductance was measured similarly for measurements of tail currents to enhance channel opening at negative potentials.
  • Single channels were measured with standard depolarizing bath and pipette (1 15 mM BaCl 2 , 1 mM EGTA, and 10 mM HEPES, pH 7.4) solutions (Lacerda et al., Biophys. J., 66. 1833-43 (1994)). Data were analyzed with TRANSIT (VanDongan, Biophys J., 70, 1303-15 (1996)).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un acide nucléique isolé ou sensiblement purifié codant une protéine comprenant au moins un domaine d'un canal calcique de type T, ainsi que des cellules et des lignées cellulaires exprimant ces acides nucléiques. La présente invention concerne également un canal calcique de type T isolé ou sensiblement purifié et une molécule anticorps isolée ou sensiblement purifiée reconnaissant un épitope sur une protéine de canal calcique de type T.
PCT/US1998/023161 1997-12-05 1998-10-30 Canaux calciques potentiel-dependants de type t et leurs methodes d'utilisation WO1999029847A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA002312477A CA2312477A1 (fr) 1997-12-05 1998-10-30 Canaux calciques potentiel-dependants de type t et leurs methodes d'utilisation
EP98956416A EP1036170A1 (fr) 1997-12-05 1998-10-30 Canaux calciques potentiel-dependants de type t et leurs methodes d'utilisation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US98580997A 1997-12-05 1997-12-05
US08/985,809 1997-12-05

Publications (1)

Publication Number Publication Date
WO1999029847A1 true WO1999029847A1 (fr) 1999-06-17

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/023161 WO1999029847A1 (fr) 1997-12-05 1998-10-30 Canaux calciques potentiel-dependants de type t et leurs methodes d'utilisation

Country Status (3)

Country Link
EP (1) EP1036170A1 (fr)
CA (1) CA2312477A1 (fr)
WO (1) WO1999029847A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000070044A3 (fr) * 1999-05-13 2001-05-17 Univ Johns Hopkins Variants episses de la sous-unite alpha des canaux calcium t dans le cerveau humain
WO2001002561A3 (fr) * 1999-07-02 2001-06-28 Neuromed Tech Inc Nouveaux canaux calciques mammiferes et sondes, lignees cellulaires et procedes correspondants
US6309858B1 (en) 1998-09-29 2001-10-30 Syntex (U.S.A.) Llc T-type calcium channel variants; compositions thereof; and uses
WO2001059446A3 (fr) * 2000-02-11 2002-03-14 Biofocus Discovery Ltd Analyse amelioree
WO2002101035A1 (fr) * 2001-05-10 2002-12-19 Mochida Pharmaceutical Co., Ltd. Proteine du canal cation dependant du potentiel
EP1224218A4 (fr) * 1999-10-26 2003-06-25 Ortho Mcneil Pharm Inc Adn codant un canal calcium humain de type t alpha1g-c
EP0973889B1 (fr) * 1997-02-28 2005-10-26 Neuromed Technologies Inc. SOUS-UNITES ALFA-1i DE CANAUX A CALCIUM HUMAINES ET SONDES, LIGNEES CELLULAIRES ET PROCEDES ASSOCIES
WO2005108575A1 (fr) * 2004-05-10 2005-11-17 Neuromed Pharmaceuticals Ltd. Compositions a base de variants d'epissage des canaux calciques de type t, et methodes associees
US7297504B1 (en) 1997-02-28 2007-11-20 Neuromed Pharmaceuticals Ltd. Methods for identifying agonists and antagonists of human T-type calcium channels
JP2008501633A (ja) * 2004-02-11 2008-01-24 ユニバーシティ オブ ヴァージニア パテント ファウンデーション 癌の診断及び治療のためのＣａｖ３アイソフォーム及びそのδ２５スプライスバリアントの阻害
AU2011203315B2 (en) * 2004-02-11 2012-01-12 University Of Virginia Patent Foundation Inhibiting CAV3 isoforms and the delta25B splice varients for the diagnosis and treatment of cancer
US9427429B2 (en) 2010-03-01 2016-08-30 Tau Therapeutics Llc Cancer diagnosis and imaging

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004083A1 (fr) * 1991-08-15 1993-03-04 The Salk Institute Biotechnology/Industrial Associates, Inc. Compositions de canaux calciques humains et procedes
WO1995004144A1 (fr) * 1993-07-30 1995-02-09 Neurex Corporation Adn codant une sous-unite alpha-1e de canal calcique humain

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993004083A1 (fr) * 1991-08-15 1993-03-04 The Salk Institute Biotechnology/Industrial Associates, Inc. Compositions de canaux calciques humains et procedes
WO1995004144A1 (fr) * 1993-07-30 1995-02-09 Neurex Corporation Adn codant une sous-unite alpha-1e de canal calcique humain

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CRIBBS LL ET AL: "Cloning and characterization of alpha1H from human heart, a member of the T-type Ca2+ channel gene family.", CIRC RES, JUL 13 1998, 83 (1) P103-9, UNITED STATES, XP002093640 *
DZHURA IO ET AL: "Characterization of hypothalamic low-voltage-activated Ca channels based on their functional expression in Xenopus oocytes.", NEUROSCIENCE, FEB 1996, 70 (3) P729-38, UNITED STATES, XP002093638 *
EMBL DATABASE Accession number q18840 WILSON R. ET AL. 1996 *
ERTEL S I ET AL: "Low-voltage-activated T-type Cachannels", TRENDS IN PHARMACOLOGICAL SCIENCES, vol. 18, no. 2, February 1997 (1997-02-01), pages 37-42, XP004055849 *
NOONEY JM (REPRINT) ET AL: "Identifying neuronal non-L Ca2+ channels - more than stamp collecting?", TRENDS IN PHARMACOLOGICAL SCIENCES, 10-1997, 18, 363-371, XP002093637 *
PEREZ-REYES E ET AL: "Molecular characterization of a neuronal low-voltage-activated T-type calcium channel [see comments]", NATURE, FEB 26 1998, 391 (6670) P896-900, ENGLAND, XP002093639 *
WILSON R ET AL: "2.2 MB OF CONTIGUOUS NUCLEOTIDE SEQUENCE FROM CHROMOSOME III OF C ELEGANS", NATURE, vol. 368, 3 March 1994 (1994-03-03), pages 32 - 38, XP002910426 *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7501263B2 (en) 1997-02-28 2009-03-10 Neuromed Technologies, Inc. Nucleic acids encoding mammalian T-type calcium channels
US7297504B1 (en) 1997-02-28 2007-11-20 Neuromed Pharmaceuticals Ltd. Methods for identifying agonists and antagonists of human T-type calcium channels
EP0973889B1 (fr) * 1997-02-28 2005-10-26 Neuromed Technologies Inc. SOUS-UNITES ALFA-1i DE CANAUX A CALCIUM HUMAINES ET SONDES, LIGNEES CELLULAIRES ET PROCEDES ASSOCIES
US7517672B2 (en) 1997-02-28 2009-04-14 Neuromed Pharmaceuticals Ltd. Nucleic acids encoding mammalian T-type calcium channels
US7157243B1 (en) 1997-02-28 2007-01-02 Neuromed Pharmaceuticals Ltd. DNA encoding mammalian T-type calcium channels
US6309858B1 (en) 1998-09-29 2001-10-30 Syntex (U.S.A.) Llc T-type calcium channel variants; compositions thereof; and uses
US6589787B2 (en) 1998-09-29 2003-07-08 Syntex (U.S.A.) Llc T-type calcium channel variants; compositions thereof; and uses
US6893842B2 (en) 1998-09-29 2005-05-17 Syntex (U.S.A.) Llc T-type calcium channel variants; compositions thereof; and uses
WO2000070044A3 (fr) * 1999-05-13 2001-05-17 Univ Johns Hopkins Variants episses de la sous-unite alpha des canaux calcium t dans le cerveau humain
EP1780278A3 (fr) * 1999-07-02 2007-06-27 Neuromed Technologies, Inc. Nuveaux canaux calciques mammiferes et sondes, lignees cellulaires et procedes correspondants
WO2001002561A3 (fr) * 1999-07-02 2001-06-28 Neuromed Tech Inc Nouveaux canaux calciques mammiferes et sondes, lignees cellulaires et procedes correspondants
JP2003504020A (ja) * 1999-07-02 2003-02-04 ニューロメド テクノロジーズ， インコーポレイテッド 新規な哺乳動物カルシウムチャネルならびに関連するプローブ、細胞株および方法
EP1224218A4 (fr) * 1999-10-26 2003-06-25 Ortho Mcneil Pharm Inc Adn codant un canal calcium humain de type t alpha1g-c
WO2001059446A3 (fr) * 2000-02-11 2002-03-14 Biofocus Discovery Ltd Analyse amelioree
WO2002101035A1 (fr) * 2001-05-10 2002-12-19 Mochida Pharmaceutical Co., Ltd. Proteine du canal cation dependant du potentiel
EP1730190A4 (fr) * 2004-02-11 2008-07-23 Univ Virginia Inhibition d'isoformes de cav3 et variants d'epissage $g(d)25b pour diagnostic et traitement de cancers
JP2008501633A (ja) * 2004-02-11 2008-01-24 ユニバーシティ オブ ヴァージニア パテント ファウンデーション 癌の診断及び治療のためのＣａｖ３アイソフォーム及びそのδ２５スプライスバリアントの阻害
JP2011132255A (ja) * 2004-02-11 2011-07-07 Univ Of Virginia Patent Foundation 癌の診断及び治療のためのＣａｖ３アイソフォーム及びそのδ２５スプライスバリアントの阻害
AU2005211764B2 (en) * 2004-02-11 2011-07-21 University Of Virginia Patent Foundation Inhibiting CAV3 isoforms and the delta25B splice varients for the diagnosis and treatment of cancer
AU2011203315B2 (en) * 2004-02-11 2012-01-12 University Of Virginia Patent Foundation Inhibiting CAV3 isoforms and the delta25B splice varients for the diagnosis and treatment of cancer
AU2011203315B8 (en) * 2004-02-11 2012-02-16 University Of Virginia Patent Foundation Inhibiting CAV3 isoforms and the delta25B splice varients for the diagnosis and treatment of cancer
WO2005108575A1 (fr) * 2004-05-10 2005-11-17 Neuromed Pharmaceuticals Ltd. Compositions a base de variants d'epissage des canaux calciques de type t, et methodes associees
US9427429B2 (en) 2010-03-01 2016-08-30 Tau Therapeutics Llc Cancer diagnosis and imaging

Also Published As

Publication number Publication date
CA2312477A1 (fr) 1999-06-17
EP1036170A1 (fr) 2000-09-20

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