Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution. INTRODUCTIONThe gap junction (GJ) is a plaque-like aggregate of intercellular channels that facilitates cytoplasmic interchange of ions, second messengers, and other molecules Ͻ1 kDa between cells (Goodenough et al., 1996). Frequent and variably sized GJ channel aggregates couple most cells in animal tissues. In excitable organs such as the heart and brain, GJs show distinctive organizational patterns that configure extended intercellular pathways for stable and long-term propagation of action potential (Lo, 2000). The channels comprising individual GJ plaques are composed of proteins encoded by the connexin family of genes (Willecke et al., 2002). Assembly of GJs from connexin monomers is thought to proceed in a multistep process Goodenough, 1991, 1993). First, six connexins oligomerize into a hemichannel, called a connexon, followed by trafficking to the plasma membrane. Subsequently, a connexon docks with a second connexon from the apposed membrane of an adjacent cell to form an intercellular channel. In a process that occurs either simultaneous with or after this docking step, channels aggregate to form the functional organelle of cell-cell communication-the GJ plaque.The gating of single channels within a GJ plaque is regulated by various stimuli, including voltage, pH, and phosphorylation (Saez et al., 2003). Intercellular communication is also regulated at the level of the plaque as a whole by factors that affect the abundance, size, and cellular distribution of GJ channel aggregates (Hall and Gourdie, 1995;Bukauskas et al., 2000;Spach et al., 2000;Johnson et al., 2002;Lauf et al., 2002). Irregularities in the extent and geometry of gap junctional contacts have been implicated in cardiac and neural electrophysiological pathologies in humans, incl...

Abstract-The intercellular geometry of connexin43 (Cx43) gap junctional coupling is key to coordinated spread of electrical activation through the ventricle of the mammalian heart. A progressive redistribution of electrical and mechanical junctions into intercalated discs occurs during postnatal development. Breakdown of disc-localized pattern in the adult heart, to recapitulate immature distributions, is thought to be key to the genesis of conduction disturbance and arrhythmia. Recently, ZO-1 (a PDZ-MAGUK protein), has been suggested to have a role in generating coupling geometries between myocytes. We therefore investigated the codistribution of ZO-1 with Cx43 and N-cadherin in the adult rat ventricle using quantitative immunoconfocal and immunoelectron microscopy. These analyses indicated that, whereas ZO-1 and Cx43 codistribute within discs, only low to moderate point-by-point colocalization of Cx43 and ZO-1 is found within these domains compared with the relatively high level of colocalization between N-cadherin and ZO-1. By contrast, levels of association between Cx43 and ZO-1 increased rapidly and significantly (PϽ0.001) after partial or complete enzymatic dissociation of myocytes from intact ventricle-a treatment known to induce gap junction endocytosis. Coimmunoprecipitation using Cx43-and ZO-1-specific antibodies confirmed that significantly (PϽ0.03) increased ZO-1 is precipitated relative to Cx43 in freshly dissociated myocytes as compared with intact ventricle. On immunoblots, decreases in Cx43 relative mobility, consistent with increased phosphorylation, were observed following myocyte dissociation. The increased ZO-1-Cx43 association that occurs after remodeling of myocyte intercellular contacts indicates the possibility of unanticipated roles for ZO-1 in gap junction turnover during cardiac development and disease processes.

disruption of the spatial order of electromechanical junctions at myocyte-intercalated disks (ICDs) is a poorly understood characteristic of many cardiac disease states. Here, in vitro and in vivo evidence is provided that zonula occludens-1 (ZO-1) regulates the organization of gap junctions (GJs) and adherens junctions (AJs) at ICDs. We investigated the contribution of ZO-1 to cell-cell junction localization by expressing a dominant-negative ZO-1 construct (DN-ZO-1) in rat ventricular myocytes (VMs). The expression of DN-ZO-1 in cultured neonatal VMs for 72 h reduced the interaction of ZO-1 and N-cadherin, as assayed by colocalization and coimmunoprecipitation, prompting cytoplasmic internalization of AJ and GJ proteins. DN-ZO-1 expression in adult VMs in vivo also reduced N-cadherin colocalization with ZO-1, a phenomenon not observed when the connexin-43 (Cx43)-ZO-1 interaction was disrupted using a mimetic of the ZO-1-binding ligand from Cx43. DN-ZO-1-infected VMs demonstrated large GJs at the ICD periphery and showed a loss of focal ZO-1 concentrations along plaque edges facing the disk interior. Additionally, there was breakdown of the characteristic ICD pattern of small interior and large peripheral GJs. Continuous DN-ZO-1 expression in VMs over postnatal development reduced ICD-associated Cx43 GJs and increased lateralized and cytoplasmic Cx43. We conclude that ZO-1 regulation of GJ localization is via an association with the N-cadherin multiprotein complex and that this is a key determinant of stable localization of both AJs and GJs at the ICD.N-cadherin; connexin-43; postsynaptic density 95/Drosophila disk large/zonula occludens-1; heart THE INTERCALATED DISK (ICD) is a specialized domain of electromechanical coupling between myocytes (6, 31). The disk incorporates three types of intercellular junctions: adherens junctions (AJs), desmosomes, and gap junctions (GJs). AJs and desmosomes provide for cadherin-mediated adhesion between myocytes and serve as a platform for cytoskeletal attachment. GJs are aggregates of cell-to-cell channels that mediate the exchange of small molecules (Ͻ1,000 Da) between cells and in the myocardium conduct the ion transients responsible for the intercellular propagation of the cardiac action potential. Based on early electron microscopic studies, the ICD was perceived more as a locus at which multiple junctional types were found rather than as an integrated unitary structure in its own right. However, the disk is now recognized as a functional unit specialized for electromechanical coupling in which AJs, GJs, and desmosomes, together with cytoskeletal proteins, are interlinked via a web of protein-protein interactions (31, 32).Over postnatal ventricular development, the remodeling of electromechanical junctions from lateral, side-by-side contacts between myocytes results in a preferential accumulation of AJs, desmosomes, and GJs at ICDs in the adult (2, 13, 16). The average size of GJs also increases during the postnatal growth of the ventricle (16,36). A distinct population ...

Intercellular connectivity mediated by gap junctions (GJs) composed of connexin43 (Cx43) is critical to the function of excitable tissues such as the heart and brain. Disruptions to Cx43 GJ organization are thought to be a factor in cardiac arrhythmias and are also implicated in epilepsy. This article is based on a presentation to the 4th Larry and Horti Fairberg Workshop on Interactive and Integrative Cardiology and summarizes the work of Gourdie and his lab on Cx43 GJs in the heart. Background and perspective of recently published studies on the function of Cx43-interacting protein zonula occludens-(ZO)-1 in determining the organization of GJ plaques are provided. In addition how a peptide containing a PDZ-binding sequence of Cx43, developed as part of the work on cardiac GJ organization is also described, which has led to evidence for novel and unexpected roles for Cx43 in modulating healing following tissue injury.

The gap junction (GJ) is an aggregate of intercellular channels that facilitates cytoplasmic interchange of ions, second messengers, and other molecules of less than 1000 Da between cells. In excitable organs such as heart and brain, GJs configure extended intercellular pathways for stable and long-term propagation of action potential. In a previous study in adult rat heart, we have shown that the Drosophila disks-large related protein ZO-1 shows low to moderate colocalization at myocyte borders with the GJ protein Cx43. In the present study, we detail a protocol for characterizing the pattern and level of colocalization of ZO-1 with Cx43 in cultures of neonatal myocytes at the level of individual GJ plaques. The data indicate that ZO-1 shows on average a partial 26.6% overlap (SD = 11.3%) with Cx43 GJ plaques. There is a strong positive correlation between GJ plaque size and area of ZO-1 colocalization, indicating that the level of associated ZO-1 scales with the area of the GJ plaque. Qualitatively, the most prominent colocalization occurs at the plaque perimeter. These studies may provide insight into the presently unknown biological function of ZO-1 interaction with Cx43.