Characterization of the Structure and Intermolecular Interactions between the Connexin40 and Connexin43 Carboxyl-terminal and Cytoplasmic Loop Domains

Denis Bouvier,Gaelle Spagnol,Sylvie Chenavas,Fabien Kieken,Heidi Vitrac,Sarah Brownell,Admir Kellezi,Vincent Forge,Paul L Sorgen

doi:10.1074/jbc.m109.039594

Abstract

Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels. The mechanism of heteromeric channel regulation has not been clearly defined. Tissue ischemia leads to intracellular acidification and closure of Cx43 and Cx40 homomeric channels. However, coexpression of Cx40 and Cx43 in Xenopus oocytes enhances the pH sensitivity of the channel. This phenomenon requires the carboxyl-terminal (CT) part of both connexins. In this study we used different biophysical methods to determine the structure of the Cx40CT and characterize the Cx40CT/Cx43CT interaction. Our results revealed that the Cx40CT is an intrinsically disordered protein similar to the Cx43CT and that the Cx40CT and Cx43CT can interact. Additionally, we have identified an interaction between the Cx40CT and the cytoplasmic loop of Cx40 as well as between the Cx40CT and the cytoplasmic loop of Cx43 (and vice versa). Our studies support the "particle-receptor" model for pH gating of Cx40 and Cx43 gap junction channels and suggest that interactions between cytoplasmic regulatory domains (both homo- and hetero-connexin) could be important for the regulation of heteromeric channels.

Highlights

Leading the cardiac tissue to become hypoxic or in the worst case, anoxic
These observations combined with our previous studies identifying that Cx43CT dimerization and the Cx43CT/Cx43CL interaction may be some of the structural changes involved in the pH regulation of Cx43 [23,24,25] have led us to consider that Cx40CT dimerization, Cx40CT/Cx43CT oligomerization, and the Cx40CT/Cx43CL and Cx43CT/Cx40CL interactions are structures involved in pH regulation of homomeric and heteromeric channels involving Cx40
We have biophysically characterized the structure of the CT domain from Cx40 by the use of Nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and circular dichroism (CD)

Summary

Introduction

Leading the cardiac tissue to become hypoxic or in the worst case, anoxic. This metabolic abnormality called ischemia induces intracellular acidification and exerts effects on cardiac contractility and rhythm. Co-expression of Cx43 and Cx40 in the same oocyte was more susceptible to acidification-induced uncoupling than those cells expressing only one connexin isoform [15] These data support that both Cx43 and Cx40 1) follow the particle-receptor hypothesis for pH gating, 2) interact within a heteromeric connexon in a synergistic manner, and 3) have regulatory domains that can interact with a channel formed by another connexin. These observations combined with our previous studies identifying that Cx43CT dimerization and the Cx43CT/Cx43CL interaction may be some of the structural changes involved in the pH regulation of Cx43 [23,24,25] have led us to consider that Cx40CT dimerization, Cx40CT/Cx43CT oligomerization, and the Cx40CT/Cx43CL and Cx43CT/Cx40CL interactions are structures involved in pH regulation of homomeric and heteromeric channels involving Cx40

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Conclusion