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

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.

Characterization of the Structure and Intermolecular Interactions between the Connexin 32 Carboxyl-terminal Domain and the Protein Partners Synapse-associated Protein 97 and Calmodulin

“…The ability to form the ␣-helical structure as observed for the CT domains from the ␣ (Cx43) (12), ␤ (Cx32), and ␥ (Cx45) 3 subdivisions suggests this transitioning from a intrinsically disordered to ␣-helical structure is a general mechanism for connexins to facilitate a biological reaction, such as modulating protein-protein interactions. However, we did not observe a change in the Cx32CT structure when bound to the SAP97 GUK domain.…”

“…CT domains, in particular, contain multiple sites for protein-protein interactions that play essential roles in channel localization and regulation (12)(13)(14). The cytoplasmic domains of transmembrane proteins are often intrinsically disordered, including the gap junction proteins Cx43CT and Cx40CT from the ␣ subdivision (12).…”

“…CT domains, in particular, contain multiple sites for protein-protein interactions that play essential roles in channel localization and regulation (12)(13)(14). The cytoplasmic domains of transmembrane proteins are often intrinsically disordered, including the gap junction proteins Cx43CT and Cx40CT from the ␣ subdivision (12). Intrinsically disordered proteins play important roles in regulation, signaling, and control pathways, where binding to multiple partners via high-specificity/low-affinity interactions is facilitated via a disordered to ordered conformational transition (15)(16)(17).…”

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

“…It is conceivable that such dimers alter the required conformational changes from the closed to the open state of Cx43 hemichannels leading to various substates. It is known that the intermolecular interactions of the C terminus and cytoplasmic loop domains of Cx43 are essential for the hemichannel activity (57).…”

Expression and cellular distribution of cyclin-dependent kinase 4 (Cdk4) and connexin 43 (Cx43) in porcine oocytes before and after in vitro maturation

“…These observations suggest that Cx43 may be differentially localised in porcine oocytes dependent on the stage of maturation. Bouvier et al (2009) described an interaction between Cx43 and Cx40, which led to the formation of connexin heterodimers with one protein loop anchored in the cytoplasm. It is suggested that Cx43 forms a heterodimer structure with Cx40 in porcine oocytes, which is manifested by a distinct cytoplasmic distribution of Cx43 after IVM.…”