Jegan Iyyathurai

Regulation of connexin- and pannexin-based channels by post-translational modifications

Connexin (Cx) and pannexin (Panx) proteins form large conductance channels, which function as regulators of communication between neighbouring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signalling, survival and death processes. The functional properties of gap junctions and hemichannels are modulated by different physiological and pathophysiological stimuli. At the molecular level, Cxs and Panxs function as multi‐protein channel complexes, regulating their channel localisation and activity. In addition to this, gap junctional channels and hemichannels are modulated by different post‐translational modifications (PTMs), including phosphorylation, glycosylation, proteolysis, N‐acetylation, S‐nitrosylation, ubiquitination, lipidation, hydroxylation, methylation and deamidation. These PTMs influence almost all aspects of communicating junctional channels in normal cell biology and pathophysiology. In this review, we will provide a systematic overview of PTMs of communicating junction proteins and discuss their effects on Cx and Panx‐channel activity and localisation.

Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond

Intercellular communication in primary bovine corneal endothelial cells (BCECs) is mainly driven by the release of extracellular ATP through Cx43 hemichannels. Studying the characteristics of Ca2+-wave propagation in BCECs, an important form of intercellular communication, in response to physiological signaling events has led to the discovery of important insights in the functional properties and regulation of native Cx43 hemichannels. Together with ectopic expression models for Cx43 hemichannels and truncated/mutated Cx43 versions, it became very clear that loop/tail interactions play a key role in controlling the activity of Cx43 hemichannels. Interestingly, the negative regulation of Cx43 hemichannels by enhanced actin/myosin contractility seems to impinge upon loss of these loop/tail interactions essential for opening Cx43 hemichannels. Finally, these molecular insights have spurred the development of novel peptide tools that can selectively inhibit Cx43 hemichannels, but neither Cx43 gap junctions nor hemichannels formed by other Cx isoforms. These tools now set the stage to hunt for novel physiological functions for Cx43 hemichannels in primary cells and tissues and to tackle disease conditions associated with excessive, pathological Cx43-hemichannel openings.

Negatively charged residues (Asp378 and Asp379) in the last ten amino acids of the C-terminal tail of Cx43 hemichannels are essential for loop/tail interactions

Connexin 43 (Cx43)-hemichannel activity is controlled by intramolecular interactions between cytoplasmic loop and C-terminal tail. We previously identified the last 10 amino acids of the C-terminal tail of Cx43 as essential for Cx43-hemichannel activity. We developed a cell-permeable peptide covering this sequence (TAT-Cx43CT). In this study, we examined the critical molecular determinants in TAT-Cx43CT to restore Cx43-hemichannel activity. Using amino acid substitutions in TAT-Cx43CT, we identified the two aspartate (Asp378 and Asp379) and two proline (Pro375 and Pro377) residues as critical for TAT-Cx43CT activity, since TAT-Cx43CT(DD/AA) and TAT-Cx43CT(PP/GG) did not overcome the inhibition of Cx43-hemichannel activity induced by thrombin, micromolar cytoplasmic Ca(2+) concentration or truncation of Cx43 at M(239). Consistent with this, we found that biotin-Cx43CT(DD/AA) was much less efficient than biotin-Cx43CT to bind the purified CL domain of Cx43 in surface plasmon resonance experiments. In conclusion, we postulate that Asp378 and Asp379 in the C-terminal part of Cx43 are essential for loop/tail interactions in Cx43 hemichannels, while Pro375 and Pro377 may help to properly coordinate the critical Asp residues.

Connexins: substrates and regulators of autophagy

Connexins mediate intercellular communication by assembling into hexameric channel complexes that act as hemichannels and gap junction channels. Most connexins are characterized by a very rapid turn-over in a variety of cell systems. The regulation of connexin turn-over by phosphorylation and ubiquitination events has been well documented. Moreover, different pathways have been implicated in connexin degradation, including proteasomal and lysosomal-based pathways. Only recently, autophagy emerged as an important connexin-degradation pathway for different connexin isoforms. As such, conditions well known to induce autophagy have an immediate impact on the connexin-expression levels. This is not only limited to experimental conditions but also several pathophysiological conditions associated with autophagy (dys)function affect connexin levels and their presence at the cell surface as gap junctions. Finally, connexins are not only substrates of autophagy but also emerge as regulators of the autophagy process. In particular, several connexin isoforms appear to recruit pre-autophagosomal autophagy-related proteins, including Atg16 and PI3K-complex components, to the plasma membrane, thereby limiting their availability and capacity for regulating autophagy.

Structural and adhesive properties of the long polar fimbriae protein LpfD from adherent-invasive Escherichia coli.

Crohns disease (CD) is an inflammatory bowel disease characterized by an exaggerated immune response to commensal microbiota in the intestines of patients. Metagenomic studies have identified specific bacterial species and strains with increased prevalence in CD patients, amongst which is the adherent-invasive Escherichia coli (AIEC) strain LF82. AIEC strains express long polar fimbriae (LPF), which are known to target Peyers patches in a mouse CD model. Here, the recombinant production of a soluble, self-complemented construct of the LpfD protein of E. coli LF82 is reported and it is demonstrated that it forms the adhesive tip subunit of LPF. The LpfD crystal reveals an N-terminal adhesin domain and a C-terminal pilin domain that connects the adhesin to the minor pilus subunit LpfE. Surface topology and sequence conservation in the adhesin domain hint at a putative receptor-binding pocket as found in the Klebsiella pneumoniae MrkD and E. coli F17-G (GafD) adhesins. Immunohistostaining of murine intestinal tissue sections revealed that LpfD specifically binds to the intestinal mucosa and submucosa. LpfD binding was found to be resistant to treatment with O- or N-glycosidases, but was lost in collagenase-treated tissue sections, indicating the possible involvement of an intestinal matrix-associated protein as the LpfD receptor. LpfD strongly adhered to isolated fibronectin in an in vitro assay, and showed lower levels of binding to collagen V and laminin and no binding to collagens I, III and IV.

The SH3-binding domain of Cx43 participates in loop/tail interactions critical for Cx43-hemichannel activity

Connexin 43 (Cx43) hemichannels establish local signaling networks via the release of ATP and other molecules, but their excessive opening may result in cell death. Hence, the activity of Cx43-hemichannels ought to be critically controlled. This involves interactions between the C-terminal tail (CT) and the cytoplasmic loop (CL), more particularly the L2 domain within CL. Previous work revealed an important role for the last nine amino acids of the Cx43 CT by targeting the L2 domain, as these nine amino acids were sufficient to restore the activity of CT-truncated Cx43-hemichannels. However, we discovered that deletion of the last 19 amino acids of the CT only partially lowered the binding to the L2 domain, indicating that a second L2-binding region is present in the CT. We here provide evidence that the SH3-binding domain is another CT region that targets the L2 domain. At the functional level, the SH3-binding domain was able to restore the activity of CT-truncated Cx43-hemichannels and alleviate the inhibition of full-length Cx43-hemichannels by high intracellular Ca2+ concentration ([Ca2+]i) as demonstrated by various approaches including patch clamp studies of unitary Cx43-hemichannel activity. Finally, we show that in full-length Cx43-hemichannels, deletion of either the SH3-binding domain or the CT9 region suppresses the hemichannel activity, while deletion of both domains completely annihilates the hemichannel activity. These results demonstrate that the Cx43 SH3-binding domain, in addition to the CT9 region, critically controls hemichannel activity at high [Ca2+]i, which may be involved in pathological hemichannel opening.

Calcium Wave Propagation Triggered by Local Mechanical Stimulation as a Method for Studying Gap Junctions and Hemichannels

Intercellular communication is essential for the coordination and synchronization of cellular processes. Gap junction channels play an important role to communicate between cells and organs, including the brain, lung, liver, lens, retina, and heart. Gap junctions enable a direct route for ions like calcium and potassium, and low molecular weight compounds, such as inositol 1,4,5-trisphosphate, cyclic adenosine monophosphate, and various kinds of metabolites to pass between cells. Intercellular calcium wave propagation evoked by a local mechanical stimulus is one of the gap junction assays to study intercellular communication. In experimental settings, an intercellular calcium wave can be elicited by applying a mechanical stimulus to a single cell. Here, we describe the use of monolayers of primary bovine corneal endothelial cells as a model to study intercellular communication. Calcium wave propagation was assayed by imaging fluorescent calcium in bovine corneal endothelial cells loaded with a fluorescent calcium dye using a confocal microscope. Spatial changes in intercellular calcium concentration following mechanical stimulation were measured in the mechanical stimulated cell and in the neighboring cells. The active area (i.e., total surface area of responsive cells) of a calcium wave can be measured and used for studying the function and regulation of gap junction channels as well as hemichannels in a variety of cell systems.

Nutrient Starvation Decreases Cx43 Levels and Limits Intercellular Communication in Primary Bovine Corneal Endothelial Cells

Connexin (Cx) proteins form large conductance channels which function as regulators of communication between neighboring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signaling, survival and death processes. Connexin 43 (Cx43), a major connexin isoform in brain and heart, is rapidly turned over. Recent studies implicated that autophagy, a lysosomal degradation pathway induced upon nutrient starvation, mediates connexins, including Cx43, degradation. Here, we examined the impact of nutrient starvation on endogenous Cx43-protein levels and endogenous Cx43-driven intercellular communication in primary bovine corneal endothelial cells (BCECs). Hank’s Balanced Salt Solution (HBSS) was used as a starvation condition that induces autophagic flux without impacting the survival of the BCECs. Nutrient starvation of BCECs caused a rapid decline in Cx43-protein levels, both as gap junctions and as hemichannels. The time course of the decline in Cx43-protein levels coincided with the time course of the decline in intercellular communication, assessed as intercellular Ca2+-wave propagation in BCECs exposed to a single-cell mechanical stimulus. The decline in Cx43-protein levels, both as gap junctions and as hemichannels, could be prevented by the addition of bafilomycin A1, a lysosomal inhibitor, during the complete nutrient starvation period. Consistent with this, bafilomycin A1 significantly alleviated the decrease in intercellular Ca2+-wave propagation. This study further underpins the importance of autophagy as an important degradation pathway for Cx43 proteins during periods of nutrient deprivation, thereby impacting the ability of cells to perform intercellular communication.