Redefining The Interaction Domain Of Cx43CT With RXP-E And Cx43CL

Abstract

Connexins are integral membrane proteins that oligomerize to form intercellular gap junction channels. These channels allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. Many factors have been identified to regulate the channel gating of the major cardiac connexin, connexin43 (Cx43) and our laboratory has focused on pH-regulation, which is a cause of ischemia-induced arrhythmias. Our previous studies have suggested that regulation of Cx43 channels results from the association of the carboxyl-terminal domain (Cx43CT), acting as a gating particle, and the cytoplasmic loop domain (Cx43CL), acting as a receptor for the gating particle. Recently, we have identified a synthetic peptide, RXP-E (30-mer peptide containing a RXP sequence specifically recognized by Cx43CT, where X represents any amino acid, and R and P correspond to arginine and proline) that interacts with the Cx43CT and can prevent closure channel. The question remains as to whether this peptide is involved in the disruption of the Cx43CT/Cx43CL interaction. Using NMR, we investigated the changes in the structure of Cx43CT with RXP-E and identified the binding site in the first half of the Cx43CT. The identification of this new specific binding domain was furthermore confirmed by mutagenesis. Additionally, we studied the interaction of Cx43CT with Cx43CL and identified a similar binding region as RXPE, confirming the potentiality of our peptide to act as a “scaffold”, thereby holding the channels in its open state and reducing life threatening arrhythmias.