Identification of a Novel Peptide That Interferes With the Chemical Regulation of Connexin43

Abstract

Abstract-The carboxyl-terminal domain of connexin43 (Cx43CT) is involved in various intra-and intermolecular interactions that regulate gap junctions. Here, we used phage display to identify novel peptidic sequences that bind Cx43CT and modify Cx43 regulation. We found that Cx43CT binds preferentially to peptides containing a sequence RXP, where X represents any amino acid and R and P correspond to the amino acids arginine and proline, respectively.A biased "RXP library" led to the identification of a peptide (dubbed "RXP-E") that bound Cx43CT with high affinity. Nuclear magnetic resonance data showed RXP-E-induced shifts in the resonance peaks of residues 343 to 346 and 376 to 379 of Cx43CT. Patch-clamp studies revealed that RXP-E partially prevented octanol-induced and acidificationinduced uncoupling in Cx43-expressing cells. Moreover, RXP-E increased mean open time of Cx43 channels. The full effect of RXP-E was dependent on the integrity of the CT domain. These data suggest that RXP-based peptides could serve as tools to help determine the role of Cx43 as a regulator of function in conditions such as ischemia-induced arrhythmias. (Circ Res. 2006;98:1365-1372.)Key Words: Cx43CT Ⅲ connexin Ⅲ particle-receptor interaction Ⅲ gap junctions C onnexins are integral membrane proteins that oligomerize to form intercellular channels called gap junctions. The most abundant gap junction protein in a number of mammalian systems is connexin43 (Cx43). Our previous work has suggested that regulation of Cx43 channels results from the association of the carboxyl-terminal (CT) domain, acting as a gating particle, and a separate region of the connexin molecule, acting as a receptor for the gating particle. 1,2 Additional studies have shown that this intramolecular interaction can be modulated by other intermolecular interactions in the microenvironment of the gap junction plaque. 3 Thus, the emerging picture of a gap junction plaque is that of a macromolecular complex in which proteins act in concert to modulate intercellular communication. At the center of these interactions is the CT domain, which acts as a substrate for a number of kinases, 4 a ligand for noncatalytic proteins, and a gating particle to modify coupling between cells. 5 As a key player in the regulation of gap junctions, CT presents itself as a target of chemical 6,7 or genetic manipulation intended to modify function. 8 Here, we sought to disrupt the regulation of Cx43 by chemical means. Our rationale was based on the knowledge that Cx43CT is capable of interacting with other proteins. We reasoned that this "stickiness" of Cx43CT can be used to "adhere" peptidic sequences to it. We further speculated that the interaction of Cx43CT with small peptides can modify the behavior of the gap junction channel. This rationale was supported by previous work showing that peptides can modify both the chemical and voltage-gating behavior of Cx43. 6,7 In the present study, we used a highthroughput phage display screening to find peptidic sequences that bind Cx43CT. Furth...

Pharmacological modulation of connexin‐formed channels in cardiac pathophysiology

“…Reduces atrial defibrillation upon oral administration RXP-E Ser-Asp-Asp-Leu-Arg-Ser-Pro-Gln-Leu-His-AsnGlu-Glu-Glu-Ser-Ala-Val-Pro-Phe-Tyr-Ser-His-Ser-HisMet-Val-Arg-Arg-Lys-Pro-Arg-Asn-Pro-Arg Prevents acidification-induced uncoupling (Shibayama et al, 2006) CyRP-71…”

“…Nuclear magnetic resonance data showed that RXP-E induced a shift in the resonance peaks of amino acids D376 to D379 and amino acids N343 to K346 of Cx43. The latter two amino acids are part of the a-helical domain of the carboxyterminal domain of Cx43 (Shibayama et al, 2006). These two amino acid stretches are involved in the pH-dependent dimerization of the carboxyterminal domain (Sorgen et al, 2004).…”

“…These two amino acid stretches are involved in the pH-dependent dimerization of the carboxyterminal domain (Sorgen et al, 2004). RXP-E partially prevented acidification-induced uncoupling by increasing the stability of the open state without altering the unitary conductance (Shibayama et al, 2006). In a next step, the RXP-E peptide was fused to a cytoplasmic transduction peptide (CTP) (Kim et al, 2006) to facilitate cellular uptake of the peptide (Lewandowski et al, 2008).…”

Differentiation of Human Atrial Myocytes From Endothelial Progenitor Cell-Derived Induced Pluripotent Stem Cells

“…Recently, a new class of pharmacophores (RXP-E) have been developed that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block [329,330]. While these compounds do not alter Cx40 conductance, we postulate that enhanced Cx43 conductance will overcome the effect of disease causing mutations in Cx40 and furthermore will demonstrate the capacity of iPSC based disease models for drug-discovery.…”

Roles of Connexins in Atherosclerosis and Ischemia-Reperfusion Injury

“…In 2006, Shibayama et al [143] identified by phage display a series of RXP peptides (sequence of arginine, any amino acid, proline) capable of binding to the CT of Cx43. One of these peptides, RXP-E, prevented heptanol-and acidosis-induced closure of Cx43 gap junction channels in transfected cells and neonatal cardiomyocytes via an significant prolongation of the single channel open time [143,144].…”