Direct Association of the Gap Junction Protein Connexin-43 with ZO-1 in Cardiac Myocytes

Abstract

The gap junction protein connexin-43 is normally located at the intercalated discs of cardiac myocytes, and it plays a critical role in the synchronization of their contraction. The mechanism by which connexin-43 is localized within cardiac myocytes is unknown. However, localization of connexin-43 likely involves an interaction with the cytoskeleton; immunofluorescence microscopy showed that in cardiac myocytes, connexin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 and ␣-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitopetagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with ␣-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity binding assays with deleted ZO-1 and connexin-43 fusion proteins. Immunoblot analysis of associated proteins showed that the C-terminal domain of connexin-43 binds to the Nterminal domain of ZO-1. The role of this linkage in gap junction formation was examined by a dominant-negative assay using the N-terminal domain of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin-43-expressing cells resulted in redistribution of connexin-43 from cell-cell interfaces to cytoplasmic structures; this intracellular redistribution of connexin-43 coincided with a loss of electrical coupling. We therefore conclude that the linkage between connexin-43 and ␣-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.Gap junctions are aggregates of channels at cell-cell interfaces (1-3). Each channel is formed through the docking of two hemichannels located in opposing cell membranes, and each hemichannel is composed of a connexin homohexamer. By permitting the direct exchange of ions and small molecules between cells, these channels play a major role in a wide variety of cellular processes, including embryogenesis, cellular differentiation and development, and electrical coupling. In heart, gap junctions are a prominent feature of intercalated discs, which connect myocytes in an end-to-end orientation; the coupling provided by gap junctions serves to synchronize the activity of cells, thus providing an isochronous front for the wave of excitation that sweeps through ventricular muscle (4, 5).How gap junctions are localized at the intercalated discs in cardiac myocytes is unknown, but a component of gap junctions, connexin-43, may interact with specific elements of the cytoskeleton that restrict its diffusion in the plane of the membrane. Recent studies indicate that a number of membrane proteins are anchored by cytoskeletal elements such as PSD-95/SAP90 (6), ankyrin (7), and ␣-spectrin (8). ZO-1, which has been identified at vertebrate tight junctions (9, 10), is thought to play a role in tissue compartmentalization and in maintaining the apical-basolateral polarity of epithelial cells. In cardiac myocytes, ZO-1 appears at the intercalated discs in the i...

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N-cadherin haploinsufficiency affects cardiac gap junctions and arrhythmic susceptibility

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Defective Epidermal Barrier in Neonatal Mice Lacking the C-Terminal Region of Connexin43

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