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...

Abstract-Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing Ϸ20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH i 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP-RXP-E-treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart. (Circ Res. 2008;103:519-526.)Key Words: Cx43CT Ⅲ particle-receptor interaction Ⅲ gap junctions Ⅲ connexin43 Ⅲ rotigaptide C onnexins (Cxs) are integral membrane proteins that oligomerize to form intercellular channels called gap junctions (GJs). The most abundant GJ protein in a number of mammalian systems is Cx43. GJs allow passage of ions and small molecules between cells and are regulated by a variety of chemical interactions between the Cx molecule and the microenvironment. As such, GJs act as active filters to control passage of intercellular messages and modulate function.Our previous work has suggested that regulation of Cx43 results from the association of the carboxyl-terminal (CT) domain, acting as a gating particle, and a separate region of the Cx molecule acting as a receptor for the gating particle. 1,2 Additional studies have shown that this intramolecular particle-receptor interaction can be modulated by other intermolecular interactions in the microenvironment of the GJ plaque. 3-5 Based on the particle-receptor model, we reasoned that regulation of Cx43 could be disrupted by the binding of exogenous molecules to regions of the gating particle requi...