Impaired Cx43 gap junction endocytosis causes morphological and functional defects in zebrafish

Abstract

Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of connexins of only 1-5 hours, resulting in constant endocytosis and biosynthetic replacement of gap junction channels has remained puzzling. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43∆256-289) and a zebrafish model ( cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx 43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that Cx43 gap junction endocytosis is an essential aspect of gap junction function and when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Interrogation of Carboxy-Terminus Localized GJA1 Variants Associated with Erythrokeratodermia Variabilis et Progressiva

“…This turned out to be the case for the P283L variant, but not the T290N variant. This finding is generally in keeping with a recent study in culture cells and zebrafish where a ∆256–289 mutant caused dysregulated Cx43 gap junction endocytosis resulting in a longer Cx43 protein half-life, elevated levels of Cx43, more numerous and larger gap junction plaques, and increased GJIC that coincided with severe cardiovascular defects [ 38 ]. Thus, we deduced that the P283L variant has a significant impact on the fate of Cx43.…”

“…Since the EKVP-associated variants occur in a motif of Cx43 that is the binding site for several members of the interactome and a domain known to regulate Cx43 internalization and degradation ( Table 1 ) [ 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ], we next sought to assess the resident time of wildtype and Cx43 variants within gap junctions at the cell surface. When brefeldin A (BFA) was used to reversibly block protein secretion at the level of the ER/Golgi apparatus, the clearing of gap junctions composed of wildtype and Cx43 variants was tracked over a seven-hour treatment ( Figure 7 A,B).…”

“…Cx43 undergoes continuous and rapid turnover with a half-life of 1–3 h [ 48 , 57 , 58 , 59 ], and changes in connexin half-life kinetics may have linkages to the onset of developmental abnormalities and disease [ 38 , 60 , 61 , 62 ]. It is also known that the C-terminal domain of Cx43 has a large interactome that includes binding sequences for NEDD4, an E3-ubiquitin ligase, and AP-2, that are likely all involved with Cx43 internalization [ 31 ].…”

“…We can only speculate on how prolonged Cx43 residency within functional gap junctions might disrupt the homeostasis of the epidermis. The fact that delayed Cx43 removal leads to cardiovascular defects in zebrafish suggests that potentially too much GJIC can lead to a pathology [ 38 ]. In the skin, dysregulated control of Cx43 turnover may change the delicate balance between keratinocyte proliferation and differentiation.…”

“…Due to the steric properties of proline residues, the P283L amino acid substitution may have altered the accessibility and functionality of proximal Cx43 binding sequences. Interestingly, the P283 site occurs in close proximity to the binding sites of Hsc70 and the µ2 subunit of the AP-2 adapter complex, which have been reported to be involved in the turnover of Cx43 [ 36 , 37 , 38 ]. Two MAPK phosphorylation sites that regulate NEDD4 binding are also found upstream at residues S279 and S282 [ 31 , 32 , 36 , 39 ].…”