Document Detail


Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26.
MedLine Citation:
PMID:  21478159     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.
Authors:
So Nakagawa; Xiang-Qun Gong; Shoji Maeda; Yuhua Dong; Yuko Misumi; Tomitake Tsukihara; Donglin Bai
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2011-04-08
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  286     ISSN:  1083-351X     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2011 Jun 
Date Detail:
Created Date:  2011-05-30     Completed Date:  2011-08-01     Revised Date:  2013-06-30    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  19672-81     Citation Subset:  IM    
Affiliation:
Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
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MeSH Terms
Descriptor/Qualifier:
Animals
Asparagine / chemistry,  genetics,  metabolism
Connexins / chemistry*,  genetics,  metabolism*
Gap Junctions / chemistry*,  genetics,  metabolism*
HeLa Cells
Humans
Mice
Models, Molecular*
Grant Support
ID/Acronym/Agency:
//Canadian Institutes of Health Research
Chemical
Reg. No./Substance:
0/Connexins; 0/connexin 32; 127120-53-0/connexin 26; 7006-34-0/Asparagine
Comments/Corrections

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