Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca²⁺-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP₃ and Ca²⁺ that initiate the propagation of the Ca²⁺-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca²⁺-wave propagation are provided by gap junction channels through the direct transfer of IP₃ and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca²⁺-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca²⁺-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca²⁺-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.     

Alpha-Helical Destabilization of the Bcl-2-BH4-Domain Peptide Abolishes Its Ability to Inhibit the IP3 Receptor

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP₃R), the primary Ca²⁺-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP₃R-mediated Ca²⁺ release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP₃R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP ₃R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine “hinges” replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP ₃R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP₃R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP₃R with significant pharmacological implications.     

Bax Inhibitor-1-mediated Ca leak is decreased by cytosolic acidosis

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca²⁺ levels. Recently, we elucidated BI-1's Ca²⁺-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca²⁺-channel pore-dead mutant BI-1 (BI-1^D213R) was developed. We determined whether BI-1 behaves as a bona fide H⁺/Ca²⁺ antiporter or as an ER Ca²⁺-leak channel by investigating the effect of pH on unidirectional Ca²⁺-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1^−/− cells increased the ER Ca²⁺-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1^D231R expression in BI-1^−/−, despite its ER localization, did not increase the ER Ca²⁺-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca²⁺ leak was blocked. Finally, a peptide representing the Ca²⁺-channel pore of BI-1 promoting Ca²⁺ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca²⁺ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca²⁺-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.     

Non-channel functions of connexins in cell growth and cell death

Cellular communication mediated by gap junction channels and hemichannels, both composed of connexin proteins, constitutes two acknowledged regulatory platforms in the accomplishment of tissue homeostasis. In recent years, an abundance of reports has been published indicating functions for connexin proteins in the control of the cellular life cycle that occur independently of their channel activities. This has yet been most exemplified in the context of cell growth and cell death, and is therefore as such addressed in the current paper. Specific attention is hereby paid to the molecular mechanisms that underpin the cellular non-channel roles of connexin proteins, namely the alteration of the expression of tissue homeostasis determinants and the physical interaction with cell growth and cell death regulators. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Connexin 43 hemichannels contribute to cytoplasmic Ca2+ oscillations by providing a bimodal Ca2+-dependent Ca2+ entry pathway

Many cellular functions are driven by changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that are highly organized in time and space. Ca(2+) oscillations are particularly important in this respect and are based on positive and negative [Ca(2+)](i) feedback on inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). Connexin hemichannels are Ca(2+)-permeable plasma membrane channels that are also controlled by [Ca(2+)](i). We aimed to investigate how hemichannels may contribute to Ca(2+) oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca(2+) oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides (32)Gap27/(43)Gap26, whereas ATP-induced oscillations were unaffected. Furthermore, these peptides inhibited the BK-triggered release of calcein, a hemichannel-permeable dye. BK-induced oscillations, but not those induced by ATP, were dependent on extracellular Ca(2+). Alleviating the negative feedback of [Ca(2+)](i) on InsP(3)Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca(2+)](i) but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca(2+)](i) inhibition. Unlike interfering with the bell-shaped dependence of InsP(3)Rs to [Ca(2+)](i), CT9 peptide prevented BK-induced oscillations but not those triggered by ATP. Collectively, these data indicate that connexin hemichannels contribute to BK-induced oscillations by allowing Ca(2+) entry during the rising phase of the Ca(2+) spikes and by providing an OFF mechanism during the falling phase of the spikes. Hemichannels were not sufficient to ignite oscillations by themselves; however, their contribution was crucial as hemichannel inhibition stopped the oscillations.     

Manipulating Connexin Communication Channels: Use of Peptidomimetics and the Translational Outputs

Gap junctions are key components underpinning multicellularity. They provide cell to cell channel pathways that enable direct intercellular communication and cellular coordination in tissues and organs. The channels are constructed of a family of connexin (Cx) membrane proteins. They oligomerize inside the cell, generating hemichannels (connexons) composed of six subunits arranged around a central channel. After transfer to the plasma membrane, arrays of Cx hemichannels (CxHcs) interact and couple with partners in neighboring attached cells to generate gap junctions. Cx channels have been studied using a range of technical approaches. Short peptides corresponding to sequences in the extra- and intracellular regions of Cxs were used first to generate epitope-specific antibodies that helped studies on the organization and functions of gap junctions. Subsequently, the peptides themselves, especially Gap26 and -27, mimetic peptides derived from each of the two extracellular loops of connexin43 (Cx43), a widely distributed Cx, have been extensively applied to block Cx channels and probe the biology of cell communication. The development of a further series of short peptides mimicking sequences in the intracellular loop, especially the extremity of the intracellular carboxyl tail of Cx43, followed. The primary inhibitory action of the peptidomimetics occurs at CxHcs located at unapposed regions of the cell’s plasma membrane, followed by inhibition of cell coupling occurring across gap junctions. CxHcs respond to a range of environmental conditions by increasing their open probability. Peptidomimetics provide a way to block the actions of CxHcs with some selectivity. Furthermore, they are increasingly applied to address the pathological consequences of a range of environmental stresses that are thought to influence Cx channel operation. Cx peptidomimetics show promise as candidates in developing new therapeutic approaches for containing and reversing damage inflicted on CxHcs, especially in hypoxia and ischemia in the heart and in brain functions.     

The contractile system as a negative regulator of the connexin 43 hemichannel

The molecular mechanisms underlying the regulation of gap junction (GJ) channels based on the 43-kDa connexin isoform (Cx43) have been studied extensively. GJ channels are formed by the docking of opposed hemichannels in adjacent cells. Mounting data indicate that unopposed Cx43 hemichannels are also functional in the plasma membrane. However, our understanding of how Cx43-hemichannel opening and closing is regulated at the molecular level is only poorly understood. Recent work elucidated that actomyosin contractility inhibits potently Cx43 hemichannels. It is known that intracellular Ca(2+) exerts a bell-shaped-dependent effect on Cx43-hemichannel opening. While low-intracellular [Ca(2+) ] (<500 nM) provokes opening of the channel, high-intracellular [Ca(2+) ] (> 500 nM) favours closing of the channel. The mechanism underlying this negative regulation of Cx43-hemichannel activity by high-intracellular [Ca(2+) ] seems to be dependent on the activation of the actomyosin contractile system. The activity of Cx43 hemichannels is critically controlled by molecular interactions between the intracellular loop and the C-terminal tail. These interactions are essential for Cx43-hemichannel opening in response to triggers such as cytosolic [Ca(2+) ] rise or external [Ca(2+) ] lowering. In this review, we present the hypothesis that the actomyosin contractile system can function as an important brake mechanism on Cx43-hemichannel opening. By controlling loop-tail interactions, the contractile system would prevent aberrant or excessive opening of Cx43 hemichannels.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling.

BACKGROUND INFORMATION: The IP(3)R (inositol 1,4,5-trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca(2+) release in virtually all cell types. We have previously demonstrated that caspase-3-mediated cleavage of IP(3)R1 during cell death generates a C-terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP(3)R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. RESULTS: In the present study, we demonstrate that expression of the caspase-3-cleaved C-terminus of IP(3)R1 increased the rate of thapsigargin-mediated Ca(2+) leak and decreased the rate of Ca(2+) uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca(2+) stores. We detected the truncated IP(3)R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP(3)R1 blocked sperm factor-induced Ca(2+) oscillations and induced an apoptotic phenotype. CONCLUSIONS: In the present study, we show that caspase-3-mediated truncation of IP(3)R1 enhanced the Ca(2+) leak from the ER. We suggest a model in which, in normal conditions, the increased Ca(2+) leak is largely compensated by enhanced Ca(2+)-uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca(2+) leak acts as a feed-forward mechanism to divert the cell into apoptosis.