Key Words: plakophilin-2 Ⅲ intercalated disc Ⅲ arrhythmogenic right ventricular cardiomyopathy Ⅲ cardiac desmosomes A high-resolution image of the site of end-end contact between cardiomyocytes reveals an electron-dense organization called "the intercalated disc." Its classic definition involves 3 structures: desmosomes and adherens junctions, providing mechanical coupling; and gap junctions, allowing electric/metabolic synchronization between cells. Recent studies show that other molecules, not directly involved in intercellular coupling, also reside preferentially at the intercalated disc. Among them is Na V 1.5, the major ␣ subunit of the cardiac sodium channel. 1 Here, we ask whether Na v 1.5 and the desmosomal protein plakophilin-2 (PKP2) coexist in the same molecular complex and whether loss of PKP2 expression affects (1) the amplitude and kinetics of the sodium current and (2) action potential propagation in a monolayer of cardiomyocytes. Our data demonstrate a functional crosstalk between a protein defined in the context of intercellular junctions (PKP2) and another protein that is fundamental to the electrical behavior of the single myocyte.

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.

Background Brugada syndrome (BrS) primarily associates with loss of sodium channel function. Previous studies showed features consistent with sodium current (INa) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (AC; or arrhythmogenic right ventricular cardiomyopathy, ARVC). Experimental models showed correlation between loss of expression of desmosomal protein plakophilin-2 (PKP2), and reduced INa. We hypothesized that PKP2 variants that reduce INa could yield a BrS phenotype, even without overt structural features. Methods and Results We searched for PKP2 variants in genomic DNA of 200 patients with BrS diagnosis, no signs of AC, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L and MOG1. We identified 5 cases of single amino acid substitutions. Mutations were tested in HL-1-derived cells endogenously expressing NaV1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased INa and NaV1.5 at site of cell contact. These deficits were restored by transfection of wild-type PKP2 (PKP2-WT), but not of BrS-related PKP2 mutants. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) from a patient with PKP2 deficit showed drastically reduced INa. The deficit was restored by transfection of WT, but not BrS-related PKP2. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related INa deficiency to reduced number of channels at the intercalated disc, and increased separation of microtubules from the cell-end. Conclusions This is the first systematic retrospective analysis of a patient group to define the co-existence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell–cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.

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Regulation of cell-cell communication by the gap junction protein connexin43 can be modulated by a variety of connexin-associating proteins. In particular, c-Src can disrupt the connexin43 (Cx43)-zonula occludens-1 (ZO-1) interaction, leading to down-regulation of gap junction intercellular communication. The binding sites for ZO-1 and c-Src correspond to widely separated Cx43 domains (ϳ100 residues apart); however, little is known about the structural modifications that may allow information to be transferred over this distance. Here, we have characterized the structure of the connexin43 carboxyl-terminal domain (Cx43CT) to assess its ability to interact with domains from ZO-1 and c-Src. NMR data indicate that the Cx43CT exists primarily as an elongated random coil, with two regions of ␣-helical structure. NMR titration experiments determined that the ZO-1 PDZ-2 domain affected the last 19 Cx43CT residues, a region larger than that reported to be required for Cx43CT-ZO-1 binding. The c-Src SH3 domain affected Cx43CT residues Lys-264 -Lys-287, Ser-306 -Glu-316, His-331-Phe-337, Leu-356 -Val-359, and Ala-367-Ser-372. Only region Lys-264 -Lys-287 contains the residues previously reported to act as an SH3 binding domain. The specificity of these interactions was verified by peptide competition experiments. Finally, we demonstrated that the SH3 domain could partially displace the Cx43CT-PDZ-2 complex. These studies represent the first structural characterization of a connexin domain when integrated in a multimolecular complex. Furthermore, we demonstrate that the structural characteristics of a disordered Cx43CT are advantageous for signaling between different binding partners that may be important in describing the mechanism of channel closure or internalization in response to pathophysiological stimuli.Gap junction channels serve to directly interconnect the cytoplasm of neighboring cells, allowing the passage of moderately small ions, metabolites, and signaling molecules. Mammalian gap junction channels are formed by as many as 21 different connexin proteins (1). Of these, connexin43 (Cx43) 1 is the most completely characterized in terms of channel gating properties (2-4), phosphorylation sites (5-7), mechanisms of pH sensitivity (8 -11), and overall molecular structure (12). Cx43 is the most abundant gap junction protein in various tissues, including heart and brain. Cx43 null mice have been extensively investigated, with important differences being found as compared with wild types with regard to numerous processes, including cardiac developmental abnormalities, electrical synchrony in the heart, spreading depression in brain, as well as global gene expression changes in heart and astrocytes (13)(14)(15)(16)(17)(18)(19)(20).Connexin molecules are tetraspan membrane proteins, with both amino and carboxyl termini within the cytoplasm. Although the structure of the membrane-spanning portions of Cx43 has been solved to a resolution of about 7.5 Å (in the membrane plane) using electron crystallography (12), a constr...

pH-induced closure of connexin43 (Cx43) channels involves interaction of the Cx43 carboxyl-terminal (Cx43CT) with a separate "receptor" domain. The receptor location and structure and whether the interaction is directly intramolecular are unknown. Here we show resonant mirror technology, enzyme-linked sorbent assays, and nuclear magnetic resonance (NMR) experiments demonstrating pH-dependent binding of Cx43CT to region 119 -144 of Cx43 (Cx43L2), which we propose is the receptor. NMR showed that acidification induced ␣-helical order in Cx43L2, whereas only a minor modification in Cx43CT structure was detected. These data provide the first demonstration of chemically induced structural order and binding between cytoplasmic connexin domains.

Abstract-Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts.We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins is an inherited disease that presents with sustained monomorphic ventricular tachycardia and sudden cardiac death. The disease is characterized by progressive fibrofatty infiltration of the myocardium, most prominent in the free wall of the right ventricle. 1 Recent studies have linked ARVC with mutations in proteins of the cardiac desmosome, 2 a component of the intercalated disc essential for mechanical coupling between cardiac cells. 3 It is estimated that as many as 70% of the mutations linked to familial ARVC are in the gene coding for plakophilin (PKP)2, 4 a 98-kDa desmosomal protein. PKP2 interacts with plakoglobin, desmoplakin, and the desmosomal cadherins via its amino terminal ("head") domain. [5][6] Loss of PKP2 destabilizes the desmosome, 7 and its genetic deletion in mice leads to rupture of the myocardial wall during the embryonic stage. 7 Loss of desmosomal integrity could lead to disruption of mechanical function in hearts afflicted with ARVC; yet, the latter does not directly explain the highly arrhythmogenic nature of the disease, particularly in cases in which lifethreatening arrhythmias occur in the absence of severe displacement of myocardium with fatty or fibrous tissue. 8 Recently, Saffitz and colleagues proposed that disruption of mechanical coupling may lead to loss of gap junctionmediated electrical communication between cells. 8 -10 This hypothesis awaits confirmation in a cellular model in which protein expression can be manipulated and intercellular communication can be assessed directly.Here, we used small interfering (si)RNA technology to silence PKP2 expression in neonatal cardiac cells, and we explored the effect of loss of PKP2 expression on the distribution and function of gap junctions. Our studies focused primarily on 2 cell populations: cardiac myocytes and epicardium-derived cells (EPDCs). Although the importance of cardiac myocytes in the context of ARVC and arrhythmias seems self-evident, a possible role for EPDCs in ARVC has not been described. Yet, as progenitors of the cardiac fibroblast cell lineage, the function of EPDCs deserves atte...