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Method of modulating activity of calcium channels in cardiac cells and reagents therefor

A calcium channel and channel technology, which can be used in non-central analgesics, testing pharmaceutical preparations, chemical instruments and methods, etc., and can solve problems such as toxicity and calcium overload.

Inactive Publication Date: 2004-07-28
AUSTRALIEN NAT UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the long run, increased cAMP levels can become toxic and cause calcium overload

Method used

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  • Method of modulating activity of calcium channels in cardiac cells and reagents therefor
  • Method of modulating activity of calcium channels in cardiac cells and reagents therefor
  • Method of modulating activity of calcium channels in cardiac cells and reagents therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0164] A 20-mer peptide that modulates cardiac RYR2 calcium channel activity

[0165] Materials and methods

[0166] Material

[0167]Chemical and biological reagents were purchased from Sigma-Aldrich (Castle Hill, Australia). The DHPR II-III cyclic peptide (SEQ ID Nos: 1-7) was synthesized by Applied Biosystems 430A peptide synthesizer, and purified by HPLC, mass spectrometry and NMR with a purity of ≥98%. Peptides were prepared as ~2 mM stock solutions in water and frozen in 20 [mu]l aliquots. The exact mother liquor concentration was determined by Auspep Pty Ltd using acid hydrolysis followed by standard PTC (phenylthiocarboxamido) method and analyzed by reverse phase HPLC.

[0168] peptide

[0169] The specific peptides used for the study were:

[0170] 1. 20-mer peptide of DHPR II-II cytoplasmic loop (SEQ ID NO: 2):

[0171] Thr Ser Ala Gln Lys Ala Lys Ala Glu Glu Arg Lys Arg Arg Lys Met Ser Lys Gly Lea

[0172] 2. Peptide NB (N-...

Embodiment 2

[0189] A 20-mer peptide that modulates cardiac RyR2 calcium channel activity

[0190] result

[0191] When the concentration is 10 -7 M cis Ca 2+ , the addition of a 20-mer peptide (SEQ ID NO: 2) to the cytoplasmic (cis) side of the channel observed an increase in the activity of myocardial RyRs. Cs around 450pS at +40mV or -40mV double layer potential + The ability to conduct and block channels at the end of the assay with 30 μM ruthenium red identified single channels as RyRs.

[0192] Figure 2 shows recordings from an experiment in which cardiac RyRs were strongly activated by a 20-mer peptide (SEQ ID NO: 2) at -40 mV and Figure 3 at +40 mV. Before peptide addition, channel activity consisted of brief intermittent openings (Fig. 2, panel A; Fig. 3, panel A). Channel opening increased at -40 mV within 10 s of addition of 65 nM peptide (SEQ ID NO: 2).

[0193] The opening of the second channel in the bilayer was accompanied by an incr...

Embodiment 3

[0203] Functional Analysis of DHPR 20-MER Fragment Derivatives and Analogs

[0204] Materials and methods

[0205] peptide

[0206] Four peptides were tested in this series of experiments and they are:

[0207] (i) native DHPR 20-mer peptide (SEQ ID NO: 2);

[0208] (ii) Ser in the peptide of SEQ ID NO: 2 687 (residue 17) was substituted with an alanine residue to generate SEQ ID NO: 8;

[0209] (iii) Arg in the peptide of SEQ ID NO: 2 688 (residue 18) is replaced by the D isomer to generate SEQ ID NO: 9; and

[0210] (iv) Ser in the peptide of SEQ ID NO: 2 687 Mutation to alanine, Arg 688 Substitution to the D isomer yielded SEQ ID NO:10.

[0211] from cardiac SR Ca 2+ Determination of release

[0212] Add cardiac SR vesicles (50 μg protein) to a cuvette to a final volume of 2 ml solution containing (in mM): 100, KH 2 PO 4 (pH=7); 4, MgCl 2 ; 1, Na 2 ATP; 0.5, antipyrylazo III. Use Cray50 or Cray100 spectrophotometer to monitor ex...

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Abstract

The present invention relates generally to novel peptides that are capable of modulating the activity of calcium channels in cardiac cells. More specifically, the present invention provides a method of modulating the activity of a cardiac calcium channel comprising contacting a cardiac ryanodine receptor (RyR2) with an amount of a fragment of a dihydropyridine receptor (DHPR) polypeptide sufficient to modulate the activity of said RyR2, and determining the activity of said calcium channel. The inventive method is useful for the treatment of a range of disorders and diseases associated with cardiac dysfunction, particularly those diseases and disorders involving reduced cardiac output and / or aberrant excitation-contraction coupling, calcium overload, or calcium leakage, in cardiac cells.

Description

field of invention [0001] The present invention generally relates to novel peptides that modulate calcium channel activity in cardiac cells. More specifically, the present invention provides a method of modulating the activity of a cardiac calcium channel comprising: exposing a cardiac ryanodine receptor (RyR2) to an amount of a dihydropyridine receptor (DHPR) sufficient to modulate said RyR2 activity polypeptide fragments, and assay the calcium channel activity. The methods of the invention are useful in the treatment of a range of disorders and diseases associated with abnormal cardiac function, in particular diseases involving reduced cardiac output and / or abnormal excitation-contraction coupling, calcium overload or calcium leakage in cardiac cells and Disorder treatment. Background of the invention [0002] Bibliographic details of publications referenced in this document are collected at the end of this specification. Reference to prior art herein, including referen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00A61K38/17A61P9/00A61P9/04A61P9/06A61P9/10G01N33/50A61P29/00A61P31/04A61P43/00C07K14/435C07K14/705G01N33/15G01N33/566G01N33/68G01N33/94
CPCG01N33/566G01N2800/32G01N33/6893G01N2500/00A61K38/00G01N33/9453C07K14/705A61P29/00A61P31/04A61P43/00A61P9/00A61P9/04A61P9/06A61P9/10A61K38/16
Inventor A·F·杜尔汉提M·G·卡萨罗托
Owner AUSTRALIEN NAT UNIV
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