br Mechanisms of gap junction internalization disaggregation

Mechanisms of gap junction internalization: disaggregation, endocytosis and annular gap junctions It has been clearly shown that gap junction internalization can occur by a distinctive mechanism where one cell internalizes an entire gap junction via formation of double membrane structure termed an annular junction [19], [20], [21], [22], [23], [24], [25], [26] (see Fig. 1) or “connexisome” [79]. This process requires that the plasma membranes of both LLY507 undergo repair by a mechanism which is not presently well understood or investigated. However, there is some controversy as to whether this is the primary or sole means of disassembling and regulating gap junctions. The appearance of annular junctions, easily visualized by electron microscopy, varies widely across cell and tissue types and conditions. As discussed above, live cell imaging shows gap junctions to be fluid, dynamic structures. Thus, it seems likely that connexins can also be internalized through less severe means, such as endocytosis. In one study, treatment of cells with glycyrrhetinic acid, a compound which inhibits gap junction communication, led to disruption of the arrangement of connexin channels in the gap junction as shown by freeze fracture microscopy [38]. In this case, while channel size and morphology appeared normal, the channels were found in a disordered arrangement with irregular spacing, rather than being in a densely packed array. In a separate study, increased phosphorylation and interaction with both Src [80] and PKC [81] was observed in response to treatment with glycyrrhetinic acid. These data together have led us to speculate that phosphorylation and channel packing/organization cooperate to direct the timing and route of gap junction internalization either through an endocytic process or formation of annular junctions. Attempts to clearly map these functions have proven challenging due to the number of kinases involved in Cx43 regulation. What does seem clear is that gap junction disassembly can be signaled through multiple pathways; these redundancies argue for the importance of gap junction regulation, especially under conditions involving tissue or cellular remodeling.
Importance of connexin43 in cardiac tissue Gap junctions play a critical role in impulse propagation in the heart and are localized to a distinct and specialized structure referred to as the intercalated disc (ID) [1], [82]. In fact, dysregulation of gap junctions is a signature of many cardiac pathologies [83]. Cx43, Cx40, and Cx45 can all be found in heart and are expressed in a spatially distinct manner with Cx43 being the dominant gap junction component in the ventricle or working myocardium (reviewed in [84]). Cardiomyocytes are coupled from end to end through the collection of proteins and subcellular structures which form the ID [1], [82]. The ID is a highly organized hub containing gap junctions, mechanical junctions and ion channels that coordinate and regulate the synchronous beating of the heart. Recent developments in high resolution and 3D imaging are beginning to elucidate just how important and complex these interactions are and highlight the importance of connexins in generating and maintaining function at the ID [40].
Conclusions and future research Gap junctions are increasingly being understood to be dynamic membrane domains providing for interaction of a variety of kinases, channels and structural proteins. The assembly and disassembly of gap junctions is highly regulated by a sequence of protein kinase activation and Cx43 phosphorylation events. We know that disassembly of gap junctions occurs in response to a variety of stimuli but depending on the cell type or tissue, the phosphorylation events and kinetics driving disassembly can be variable. This is likely a result of the proteins interacting at and around the gap junction. While annular junction or “connexisome” formation is well documented in specific cell types and during autophagy, it is not clear that it can account for all types of gap junction turnover. In Fig. 1 we present a model whereby an alternate disassembly process occurs in subregions of the gap junction where channels undock and assume less dense channel packing then undergo an endocytic process (i.e., upon src activation or glycyrrhetinic acid treatment). The fact that there is so much complexity and redundancy in regulating gap junctions certainly argues for their importance in allowing rapid responses both at the intracellular and intercellular levels.