Author Response: Structure of the connexin-43 gap junction channel in a putative closed state

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 Ca²⁺ exerts a bell-shaped-dependent effect on Cx43-hemichannel opening. While low-intracellular [Ca²⁺] (<500 nM) provokes opening of the channel, high-intracellular [Ca²⁺] (> 500 nM) favours closing of the channel. The mechanism underlying this negative regulation of Cx43-hemichannel activity by high-intracellular [Ca²⁺] 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 [Ca²⁺] rise or external [Ca²⁺] 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.

Three-Dimensional Structure of Innexin GAP Junction Channels Studied by Electron Crystallography

Hearing loss, caused by gene mutations and environmental factors, is a common sensory disorder in the human population. In the developed countries, the incidence of congenital hearing loss is estimated at 1 in 1000 births, of which approximately 60% cases are attributed to genetic factors. To date, 59 auditory genes have been identified. Connexin (Cx) belongs to a large gene family, and the products of Cx gene family constitute a gap junction channel responsible for regulation of the physiological and developmental process, involving the exchange of ions and small molecules. Every six Cxs compose a hemichannel (so called as connexon), and then two hemichannel to form an integral gap junction. This study applied cell model to determine the Cx29 and Cx43 gene mutations in the pathogenic mechanisms causing hearing loss. To confirm the localization patterns seen in the immunolabeling assay, HeLa cells were transfected with Cx29 constructs that were directly ‘tagged’ with GFP or DsRed at the C-terminal end of the protein. The results reveal the Cx29WT-EGFP was expressed along apposed cell membranes in the fluorescent localization assay with a continuous staining. In contrast, the p.E269D- EGFP (missense mutation) resulted in the accumulation of the Cx29 mutant protein in the endoplasmic reticulum (ER) rather than in the plasma membrane. Co-expression of Cx29WT and Cx29E269D proteins by a bi-directional tet-on expression system demonstrated that the heteromeric connexon accumulated in the cytoplasm, thereby impairing the formation of the gap junction. Assuming these findings, we suggest that Cx29E269D has a dominant negative effect on the formation of the gap junction. To determine the crucial relationship between wild type Cx43 and three of mutant Cx43 genes( S69P、T326Iand 932 del C) were cloned and their functions were analysed in HeLa cells. Localization assay of WT Cx43 reveals a typical punctuate fluorescence pattern of a gap junction channel between neighboring expression cells. Additionally, immunoblotting analysis of the transfectants confirms the production of mutant proteins, in which their distributions along appositional membranes are determined using immunofluorescent staining procedures. Furthermore, dye transfer assay results demonstrate that gap junctional intercellular communication (GJIC) is less in HeLa cells carrying mutant GJA1 orψGJA1 gene than in WT-expressing cell. In summary, we use molecular biology methods to create the normal and mutant constructs containing Cx29 or Cx43 gene and determine the pathogenic effects of normal and mutant Cx29or Cx43 gene in transfected HeLa cells. Our data reveal the Cx29 and Cx43 gene mutations play causal roles in the pathogenesis of hearing loss.

Under physiological conditions, microglia adopt a resting phenotype associated with the production of anti-inflammatory and neurotrophic factors. In response to a wide variety of insults, these cells shift to an activated phenotype that is necessary for the proper restoration of brain homeostasis. However, when the intensity of a threat is relatively high, microglial activation worsens the progression of damage rather than providing protection, with potentially significant consequences for neuronal survival. Coordinated interactions among microglia and other brain cells, including astrocytes and neurons, are critical for the development of timely and optimal inflammatory responses in the brain parenchyma. Tissue synchronization is in part mediated by connexins and pannexins, which are protein families that form different plasma membrane channels to communicate with neighboring cells. Gap junction channels (which are exclusively formed by connexins in vertebrates) connect the cytoplasm of contacting cells to coordinate electrical and metabolic coupling. Hemichannels (HCs) and pannexons (which are formed by connexins and pannexins, respectively) communicate the intra- and extracellular compartments and serve as diffusion pathways for the exchange of ions and small molecules. In this review article, we discuss the available evidence concerning the functional expression and regulation of connexin- and pannexin-based channels in microglia and their contributions to microglial function and dysfunction. Specifically, we focus on the possible implications of these channels in microglia-to-microglia, microglia-to-astrocyte and neuron-to-microglia interactions in the inflamed brain.

Chapter 20 - Gap junction channels

Connexin43 Modulation of Osteoblast/Osteocyte Apoptosis: A Potential Therapeutic Target?

We have measured the permeability of rhodamine-6G across Cx43 hemichannels reconstituted on a pipette tip. Cx43 hemichannels were overexpressed in Sf9 cells, and affinity-purified. The hemichannels were reconstituted in a lipid bilayer on a pipette tip by the tip-dip method. R6G in the pipette permeated across the channels into the bath. The permeability of R6G was quantified by measuring R6G concentration in the bath after several hours by surface enhanced Raman spectroscopy (SERS) with 100 nm silver colloid particles. The ratio of the permeability of dye to salt, as extracted by this combined electrical-SERS technique, is compatible with similar ratios for other dyes across whole gap junction channels. The results for the permeability ratio were further compared to fluorescence measurements. The novel combination of patch and SERS techniques can be extended to quantifying the transport of biologically significant non-fluorescent molecules, such as cAMP and IP3, across 1 nm sized pores, such as the gap junction channel.

Gap junctions are important plasma membrane organelles through which most cells in normal tissues communicate with each other. They exist in two neighboring cells and each cell contributes half of the structure. One gap junction consists of two hexameric connexons that dock with each other to create a channel. Six of the basic subunits referred to as connexins form a connexon. Less than one hundred to several thousand gap junction channels cluster together in the plane of the membrane. The gap junction channels serve as a regulated conduit for the intercellular exchange of small molecules. Maintenance of the integrity of gap junctional intercellular communication (GJIC) is important and required for normal electrical coupling, homeostasis, and embryogenesis. Aberrations of gap junctions have been related to human diseases such as cancer, cardiac arrhythmia, Charcot-Marie-tooth disease, and visceroatrial heterotaxia syndrome. This unit describes methods for measuring gap junctional intercellular communication using primary mouse hepatocytes as a model. Focus is only on functional evaluation based on dye coupling. Other methods, such as intracellular calcium waves and dual patch clamp, have been used to measure gap junctional communication, but these are not described in this unit.

Asparagine175 of Cx32 is a Critical Residue for Docking and Forming Functional Heterotypic Gap Junction Channels with Cx26

In the central nervous system (CNS), macroglial cells (astrocytes and oligodendrocytes) represent the main cell population coupled by gap junctions. Indeed, a much higher level of gap junctional communication is observed in macroglial cells than in neurons and this communication process persists up to the adult stage in these cells (1). Gap junctions are characterized by clusters of intercellular channels connecting the cytoplasm of adjacent cells. These channels are constituted by two hemichannels, the connexons, each of them being formed by six structural subunit proteins, the connexins (Cxs). Cxs are organized around a relatively large hydrated pore which allows the diffusion of ions and small molecules up to 1–1.2 kDa, providing thus the morphological basis for electrical and metabolic coupling (2). Although Cxs are expressed in neurons as well as in glial and ependymal cells (3), glial cells and astrocytes, particularly, contain the highest level of Cxs from the late embryo to the adult (4). Cx43, which is prevalent in astrocytes, is detected early during development, peaks at birth, and remains constant throughout the adult life (4). Cx30 is also found in mature astrocytes and its distribution resembles that of Cx43 (5). Experiments performed with low molecular weight tracers (Lucifer yellow, biocytin, neurobiotin) have indicated that populations of 10 to 100 astrocytes can communicate through gap junction channels in either cortical, hippocampal, or cerebellar slices (6–8). However, although Cx43 was also detected in striatal astrocytes, as reported in a previous study, the intercellular diffusion of the low molecular weight fluorescent dye Lucifer yellow was not detected in astrocytes injected with this dye in striatal slices (9,10).This is surprising because functional studies performed on astrocytic primary cultures from different brain structures, including the striatum, have demonstrated that these cells are highly coupled by gap junction channels (3,11).A marked regional heterogeneity was found in these cell culture studies (12,13) but astrocytes from either the cerebral cortex, the hippocampus, the brain stem, the cerebellum or the striatum were all shown to express Cx43 and to exchange fluorescent dyes, calcium signaling molecules, glucose, and glutamate (14).

Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure

Sequence and structure comparisons between alpha and beta connexins, Cx43 and Cx26, revealed that Cx43 has a motif, the carbamylation motif, that confers CO2-sensitivity on a subset of beta connexins. By using a fluorescent dye loading assay, whole cell patch clamp recordings and real time measurement of ATP release via GRABATP we have demonstrated that Cx43 hemichannels open in a highly CO2 sensitive manner over the range 20 to 70 mmHg. Mutational analysis confirms that the equivalent residues to those in Cx26 that have been shown to be involved in mediating the effects of CO2 on gating of hemichannels and gap junction channels, also mediate Cx43 hemichannel gating. Our data predicts that Cx43 will be partially open and able to release ATP at resting physiological levels of PCO2. We have tested this prediction in acute hippocampal slices, by showing that CO2-dependent enhancement of synaptic transmission can be blocked by the Cx43-selective mimetic peptide Gap26. Our data resolves an inconsistency in the literature between in vivo studies suggesting that Cx43 hemichannels are at least partially open at rest, and in vitro studies, performed in the absence of HCO3-/CO2 buffering that show Cx43 hemichannels are shut. Our evidence suggests that the ancestral gene that duplicated to give the alpha and beta connexin clades must have possessed the carbamylation motif. CO2 sensitivity is thus a fundamental ancient characteristic of several connexins that has been lost in more recently derived members of this gene family.

Gap junctions facilitate direct cytoplasmic communication between neighboring cells, facilitating the transfer of small molecular weight molecules involved in cell signaling and metabolism. Gap junction channels are formed by the joining of two hemichannels from adjacent cells, each composed of six oligomeric protein subunits called connexins. Of paramount importance to CNS homeostasis are astrocyte networks formed by gap junctions, which play a critical role in maintaining the homeostatic regulation of extracellular pH, K+, and glutamate levels. Inflammation is a hallmark of several diseases afflicting the CNS. Within the past several years, the number of publications reporting effects of cytokines and pathogenic stimuli on glial gap junction communication has increased dramatically. The purpose of this review is to discuss recent observations characterizing the consequences of inflammatory stimuli on homocellular gap junction coupling in astrocytes and microglia as well as changes in connexin expression during various CNS inflammatory conditions.

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 Role of Amino Terminus of Mouse Cx50 in Determining Transjunctional Voltage-Dependent Gating and Unitary Conductance