Asparagine 175 of Connexin32 Is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Connexin26

So Nakagawa,Xiang-Qun Gong,Shoji Maeda,Yuhua Dong,Yuko Misumi,Tomitake Tsukihara,Donglin Bai

doi:10.1074/jbc.m110.204958

Abstract

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.

Highlights

Communication between cells is important for tissue and organ homeostasis in multicellular organisms
Considering that mammalian cells typically express more than one connexin and almost every connexin is present in multiple cell types, heterotypic gap junction channels might be very important in intercellular communication in vivo
Our results indicate that hydrogen bonds (HBs) involving asparagine 175 of Cx32 at the docking interface are critical in forming functional heterotypic gap junction channels with Cx26

Summary

Introduction

Communication between cells is important for tissue and organ homeostasis in multicellular organisms. Direct intercellular signaling is mediated by gap junction channels composed of connexins (Cxs).. Direct intercellular signaling is mediated by gap junction channels composed of connexins (Cxs).3 These channels cluster in specialized regions of the plasma membrane, which allow passage of ions and small molecules, including second messengers, amino acids, nucleotides, and other metabolites, between adjacent cells [1, 2]. Functional studies in paired Xenopus oocytes and transfected cell lines expressing different connexins demonstrate that heterotypic gap junction channels can be formed only between compatible Cxs, suggesting that the docking is selective [9, 14]. Because the tertiary structure data were lacking, point mutation studies alone were not able to yield enough insights; the molecular structural basis underlying hemichannel docking selectivity remains to be elucidated

Results

Discussion

Conclusion