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Material for screening for compound capable of acting on ion channel, and use thereof

一种离子通道、化合物的技术，应用在含细胞材料领域，能够解决配体优化困难等问题

Active Publication Date: 2013-07-31
NAGOYA CITY UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Additionally, in the case of targeted ion channel screens, ligand optimization is often difficult, resulting in high throughput coupled with the need for assay precision.
[0007] However, as previously described, conventional fluorescent membrane potential assays for measuring small changes in membrane potential in cells have problems with accuracy and applicability, while automated patch-clamp methods have problems with efficiency and cost

Method used

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  • Material for screening for compound capable of acting on ion channel, and use thereof
  • Material for screening for compound capable of acting on ion channel, and use thereof
  • Material for screening for compound capable of acting on ion channel, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] (Generation of IFM motif mutants at Nav1.5 channel inactivation sites)

[0108] The hydrophobic amino acid sequence Ile-Phe-Met (IFM motif) present in the inactive III-IV linker region controlling the Nav1.5 channel was all mutated to Gln. The mutated amino acid sequence (motif) is shown below.

[0109] hNav1.5 amino acid sequence (only the region containing the targeted mutated IFM is shown) (SEQ ID NO: 1)

[0110] 1470-IDNFNQQKKKLGGQD IFM TEEQKKYYNAMKK-1500

[0111] (The underlined part of IFM is mutated to QQQ)

[0112] Inactivation-inhibited mutagenesis was generated by using pcDNA3.1 / Nav1.5 as a template with the specific PCR primers shown below and the Quik Change Site-Directed Mutagenesis Kit (Stratagene). Mutant Nav-QQQ, the pcDNA3.1 / Nav1.5 was obtained by subcloning human Nav1.5 (GenBank accession number: NM_198056.2) into pcDNA3.1(+) (Invitrogen) . The Big Dye Terminator Ver.3.1 Cycle Sequencing Kit (Big Dye Terminator Ver.3.1Cycle Sequencing Kit) (Appl...

Embodiment 2

[0117] (cell culture and gene insertion)

[0118] Human embryonic kidney cells (HEK293 cells) were purchased from the Health Sciences Research Resource Bank (HSRRB). 10% FBS (Gibco) was added to D-MEM medium (Wako Pure Chemical Industries, Ltd.) containing 100 U / ml penicillin (Wako Pure Chemical Industries, Ltd.) and 100 μg / ml streptomycin (Meiji Seika Co., Ltd.) , then at 37 °C in CO 2 cultivated in. Lipofectamine2000 reagent (Invitrogen) was used to insert pcDNA / Kir2.1 by subcloning human Kir2.1 (NM_00891.2) into pcDNA3.1(+) (Invitrogen) bleomycin-resistant cells were then cloned to produce constant expression of Kir2 .1 cells (HEK-Kir). Additionally, the inactivated mutant Nav-QQQ (HEK-Kir-mutated Nav) was inserted into cells constantly expressing Kir2.1 using the same method, and then experiments were performed 24 to 72 hours later.

[0119] (Electrophysiology experiment)

[0120] Amperometric measurements were performed using the patch clamp method established by Ha...

Embodiment 3

[0131] hERG- HEK cells and hERG-Kir mutant Nav cells were then performed 24 hrs to 72 hrs later for electrophysiological experiments. According to Example 2, electrophysiological experiments were performed on native HEK cells, hERG-HEK cells and hERG-HEK mutant Nav cells, and then hERG channel currents and action potentials were measured. The hERG K ion channel exhibits inward rectification, and when expressed in cells for screening, the hERG K ion channel functions to suppress cell death by shortening an action potential generated by electrical stimulation or the like.

[0132] The hERG channel inhibitor nifekalant was administered to hERG-HEK cells obtained by transiently expressing hERG channels in native HEK cells, and the potential of the cell membrane was measured before and after the administration. The current diagram is shown in Figure 7 , while the current-voltage curve is shown in Figure 8 middle. Such as Figure 7 and 8 As shown in , hERG channel currents w...

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Abstract

Disclosed is a screening system which targets an ion channel and has excellent efficiency. Specifically disclosed is a material for use in the screening of a compound capable of acting on a target ion channel, which comprises a cell that carries at least one first DNA encoding a voltage-dependent Na ion channel that cannot be inactivated, wherein a K ion channel in the cell is so activated that the depth of the resting membrane potential is increased gradually in the negative direction.

Description

technical field [0001] Cross References to Related Applications [0002] This application claims priority from Japanese Patent Application No. 2010-147255 filed on Jun. 29, 2010, the contents of which are incorporated herein by reference. [0003] The present invention relates to materials for screening compounds acting on ion channels and uses thereof, more particularly, the present invention relates to cell-containing materials useful for screening compounds acting on ion channels, including membrane transporters, and the use of said materials screening method. Background technique [0004] Ion channels have important functions in physiology. Discovery of agonists and inhibitors acting on ion channels by targeting these ion channels is expected to provide useful drugs. A known example of a method for evaluating screening systems for drugs targeting such ion channels, such as voltage-dependent ion channels, is the fluorescent membrane potential assay, which uses a voltag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12M1/34C12N15/09
CPCG01N2500/10C12Q1/025G01N33/6872G01N2510/00C07K14/705
Inventor 今泉祐治藤井将人大矢进山村寿男
Owner NAGOYA CITY UNIVERSITY
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