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

Fundamentals of Cellular Calcium Signaling: A Primer

Ionized calcium (Ca2+) is the most versatile cellular messenger. All cells use Ca2+ signals to regulate their activities in response to extrinsic and intrinsic stimuli. Alterations in cellular Ca2+ signaling and/or Ca2+ homeostasis can subvert physiological processes into driving pathological outcomes. Imaging of living cells over the past decades has demonstrated that Ca2+ signals encode information in their frequency, kinetics, amplitude, and spatial extent. These parameters alter depending on the type and intensity of stimulation, and cellular context. Moreover, it is evident that different cell types produce widely varying Ca2+ signals, with properties that suit their physiological functions. This primer discusses basic principles and mechanisms underlying cellular Ca2+ signaling and Ca2+ homeostasis. Consequently, we have cited some historical articles in addition to more recent findings. A brief summary of the core features of cellular Ca2+ signaling is provided, with particular focus on Ca2+ stores and Ca2+ transport across cellular membranes, as well as mechanisms by which Ca2+ signals activate downstream effector systems

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

OrganiZational communication and organiSational communication: Binaries and the fragments of a field

In this paper, I employ personal narrative to help cast light on connections and tensions between organiZational communication research, as produced in the United States, and organiSational communication research, as produced in Aotearoa New Zealand. I address the issue by highlighting three sets of differences between these bodies of research: canonical, institutional and theoretical. I then unpack how these differences are apparent in my own university before sketching out three ways in which we might productively use such tensions to achieve radical engagement, and critique disciplinary others, identities, and locations

Deleterious effects of calcium indicators within cells; an inconvenient truth

The study of cellular Ca⁠2+ signalling is indebted to Roger Tsien for the invention of fluorescent indicators that can be readily loaded into living cells and provide the means to measure cellular Ca⁠⁠2+ changes over long periods of time with sub-second resolution and microscopic precision. However, a recent study [1] reminds us that as useful as these tools are they need to be employed with caution as there can be off-target effects. This article summarises these recent findings within the wider context of confounding issues that can be encountered when using chemical and genetically-encoded Ca⁠⁠2+ indicators, and briefly discusses some approaches that may mitigate against misleading outcomes

Alzheimer’s disease-associated peptide Aβ₄₂ mobilizes ER Ca²⁺ via InsP₃R-dependent and -independent mechanisms

Dysregulation of Ca2+ homeostasis is considered to contribute to the toxic action of the Alzheimer’s Disease (AD) associated Amyloid β-peptide (Aβ). Ca2+ fluxes across the plasma membrane and release from intracellular stores have both been reported to underlie the Ca2+ fluxes induced by Aβ42. Here, we investigated the contribution of Ca2+ release from the endoplasmic reticulum (ER) to the effects of Aβ42 upon Ca2+ homeostasis and the mechanism by which Aβ42 elicited these effects. Consistent with previous reports, application of soluble oligomeric forms of Aβ42 exhibited Ca2+ mobilizing properties. The Aβ42-stimulated Ca2+ signals persisted in the absence of extracellular Ca2+ indicating a significant contribution of Ca2+ release from the ER Ca2+ store to the generation of these signals. Moreover, inositol 1,4,5-trisphosphate (InsP3) signaling contributed to Aβ42-stimulated Ca2+ release. The Ca2+ mobilizing effect of Aβ42 was also observed when applied to permeabilized cells deficient in InsP3 receptors revealing an additional direct effect of internalized Aβ42 upon the ER, and a mechanism for induction of toxicity by intracellular Aβ42

The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca2+-release channel

AbstractThe inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) type 2 (IP3R2) is an intracellular Ca2+-release channel located on the endoplasmic reticulum (ER). IP3R2 is characterized by a high sensitivity to both IP3 and ATP and is biphasically regulated by Ca2+. Furthermore, IP3R2 is modulated by various protein kinases. In addition to its regulation by protein kinase A, IP3R2 forms a complex with adenylate cyclase 6 and is directly regulated by cAMP. Finally, in the ER, IP3R2 is less mobile than the other IP3R isoforms, while its functional properties appear dominant in heterotetramers. These properties make the IP3R2 a Ca2+ channel with exquisite properties for setting up intracellular Ca2+ signals with unique characteristics. IP3R2 plays a crucial role in the function of secretory cell types (e.g. pancreatic acinar cells, hepatocytes, salivary gland, eccrine sweat gland). In cardiac myocytes, the role of IP3R2 appears more complex, because, together with IP3R1, it is needed for normal cardiogenesis, while its aberrant activity is implicated in cardiac hypertrophy and arrhythmias. Most importantly, its high sensitivity to IP3 makes IP3R2 a target for anti-apoptotic proteins (e.g. Bcl-2) in B-cell cancers. Disrupting IP3R/Bcl-2 interaction therefore leads in those cells to increased Ca2+ release and apoptosis. Intriguingly, IP3R2 is not only implicated in apoptosis but also in the induction of senescence, another tumour-suppressive mechanism. These results were the first to unravel the physiological and pathophysiological role of IP3R2 and we anticipate that further progress will soon be made in understanding the function of IP3R2 in various tissues and organs

RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho) physiological conditions of stress

The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes

In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap 19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap 19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by reversible phosphorylation and dephosphorylation

AbstractThe inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is a universal intracellular Ca2+-release channel. It is activated after cell stimulation and plays a crucial role in the initiation and propagation of the complex spatio-temporal Ca2+ signals that control cellular processes as different as fertilization, cell division, cell migration, differentiation, metabolism, muscle contraction, secretion, neuronal processing, and ultimately cell death. To achieve these various functions, often in a single cell, exquisite control of the Ca2+ release is needed. This review aims to highlight how protein kinases and protein phosphatases can interact with the IP3R or with associated proteins and so provide a large potential for fine tuning the Ca2+-release activity and for creating efficient Ca2+ signals in subcellular microdomains