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Mechanically-activated cation channels

A mechanical activation, cationic technology, applied in organic chemistry, compound screening, anti-animal/human immunoglobulin, etc.

Inactive Publication Date: 2013-08-14
IRM有限责任公司，特拉华有限责任公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, only a very small number of MA channels have been identified so far

Method used

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Examples

Experimental program
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preparation example Construction

[0132] Preparation and screening of combinatorial chemical libraries is generally known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al. 84-88(1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemicals include, but are not limited to: peptoids (e.g., PCT Publication No. WO91 / 19735), encoded peptides (e.g., PCT Publication No. WO93 / 20242), random biooligomers (e.g., PCT Publication No. WO92 / 00091 ), benzenediazonium salts (eg, U.S. Pat. No. 5,288,514), diversomers such as hydantoin, benzenediazonium salts, and dipeptides (Hobbs et al., Proc.Nat.Acad.Sci.USA90:6909-6913( 1993)), alkene polypeptides (Hagihara et al., J.Amer.Chem.Soc.114:6568 (1992)), non-peptidic peptidomimetics with glucose scaffolds (Hirschrnann et al., J.Amer.Chem.Soc. 114:9217-9218 (1992)),...

Embodiment 1

[0174] Materials and methods

[0175] This example provides a description of specific materials and methods used in the following examples. Those skilled in the art will readily appreciate that a variety of alternative modifications can be used in the methods described.

[0176] Cell culture and transient transfection: Neuro2A cells were grown in 4.5mg.ml containing -1 Glucose, 10% fetal bovine serum, 50 units.ml -1 Penicillin and 50 μg.ml -1 Streptomycin in Eagle’s Minimum Essential Medium. C2C12 or human] embryonic kidney 293T (HEK293T) cells were grown in 4.5mg.ml -1 Glucose, 10% fetal bovine serum, 50 units.ml -1 Penicillin and 50 μg.ml -1 Streptomycin in Dulbecco's Modified Eagle Medium. Cells were plated in 35 mm dishes or on 12-mm round glass coverslips in 24-well plates and transfected using lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. In Piezo1 overexpression experiments, transfect 500 to 1000ng.ml -1 Cells were recorded 12...

Embodiment 2

[0234] Neuro2A cells express MA currents

[0235]To identify proteins involved in mechanotransduction, cell lines expressing strong MA currents similar to those recorded from primary cells were sought (B. Coste, M. Crest, P. Delmas, JGen Physiol 129, 57 (Jan, 2007)) . Several mouse and rat cell lines (Neuro2A, C2C12, NIH / 3T3, Mia-6, 50B11, F11, PC12) (G.C. McCarter, D.B. Reichling, J.D. Levine, Neurosci Lett 273, 179 (October 8, 1999); B. Coste, M. Crest, P. Delmas, J Gen Physiol 129, 57 (January 2007); L.J. Drew, J.N. Wood, P. Cesare, JNeurosci22, RC228 (June 15, 2002). The Neuro2A (N2A) mouse neuroblastoma cell line expresses the most consistent MA current with appreciable adaptation kinetics ( figure 1 A). In contrast, as a representative MA current recorded in other cell lines, the C2C12 mouse myoblast cell line expresses an MA current with slower deactivation kinetics ( figure 1 B). All N2A MA currents recorded at a holding potential of -80 mV were inactivated at th...

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Abstract

Methods of screening for agents that modulate the activity of a mechanically-activated cation channel are provided. Also provided are compositions and methods for ameliorating pain by antagonizing or inhibiting mechanically-activated cation channels.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 61 / 376,182, filed August 23, 2010. This priority application is hereby incorporated by reference in its entirety. [0003] Statement of Rights to Inventions Made Under Federally Sponsored Research and Development [0004] This invention was made with government support under Grant Nos. DE016927 and NS046303 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the invention [0005] Mechanotransduction, the conversion of mechanical forces into biological signals, plays a key role in physiology. In mammals, embryonic development, touch, pain, proprioception, hearing, regulation of vascular tone and blood flow, flow sensing in the kidney, growth and injury of the lung, bone and muscle homeostasis, and metastasis are all Regulation of mechanotransduction (M. Chalfie, Nat. Rev. Mol. Cell Biol. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B30/04
CPCC12N15/1138C07K16/18C07K2317/34G01N2500/04C12N2310/14C07H21/00G01N33/6872C07K16/28C07K14/705C12N2310/11A61P1/02A61P15/00A61P19/02A61P25/04A61P29/00A61P35/00
Inventor B·科斯特A·帕塔普蒂安
Owner IRM有限责任公司，特拉华有限责任公司
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