Fluorescent Labeling of Connexin with As Complex and X-Y Coordinate Registration of Target Single Cells Based on a Triangle Standard Chip for the Image Analysis of Gap Junctional Communication.

Gap junction (GJ) research has entered a new stage focusing the concerted dynamic behavior of multiple isoforms of connexin (Cx) in the cell membrane, cytosolic vesicles, and space between them. To proceed with this research, imaging technologies are important. Here we describe two novel protocols for this purpose. At first, the adoption of a small motif of Cys-Cys-X-X-Cys-Cys as a visualization tag is described. An As complex, FlAsH, can bind to this tetra-Cys (TC) tag to form a fluorescent conjugate. Its introduction into the C-terminal of Cx43 is demonstrated. Next, a novel triangle chip for the accurate x-y registration is described. Target single cells of HeLa marked with a fluorescent dye can be easily recognized by electron microscopy based on this chip.

Gap junctions amplify spatial variations in cell volume in proliferating tumor spheroids.

Sustained proliferation is a significant driver of cancer progression. Cell-cycle advancement is coupled with cell size, but it remains unclear how multiple cells interact to control their volume in 3D clusters. In this study, we propose a mechano-osmotic model to investigate the evolution of volume dynamics within multicellular systems. Volume control depends on an interplay between multiple cellular constituents, including gap junctions, mechanosensitive ion channels, energy-consuming ion pumps, and the actomyosin cortex, that coordinate to manipulate cellular osmolarity. In connected cells, we show that mechanical loading leads to the emergence of osmotic pressure gradients between cells with consequent increases in cellular ion concentrations driving swelling. We identify how gap junctions can amplify spatial variations in cell volume within multicellular spheroids and, further, describe how the process depends on proliferation-induced solid stress. Our model may provide new insight into the role of gap junctions in breast cancer progression.

Antagonistic Functions of Connexin 43 during the Development of Primary or Secondary Bone Tumors.

Despite research and clinical advances during recent decades, bone cancers remain a leading cause of death worldwide. There is a low survival rate for patients with primary bone tumors such as osteosarcoma and Ewing's sarcoma or secondary bone tumors such as bone metastases from prostate carcinoma. Gap junctions are specialized plasma membrane structures consisting of transmembrane channels that directly link the cytoplasm of adjacent cells, thereby enabling the direct exchange of small signaling molecules between cells. Discoveries of human genetic disorders due to genetic mutations in gap junction proteins (connexins) and experimental data using connexin knockout mice have provided significant evidence that gap-junctional intercellular communication (Gj) is crucial for tissue function. Thus, the dysfunction of Gj may be responsible for the development of some diseases. Gj is thus a main mechanism for tumor cells to communicate with other tumor cells and their surrounding microenvironment to survive and proliferate. If it is well accepted that a low level of connexin expression favors cancer cell proliferation and therefore primary tumor development, more evidence is suggesting that a high level of connexin expression stimulates various cellular process such as intravasation, extravasation, or migration of metastatic cells. If so, connexin expression would facilitate secondary tumor dissemination. This paper discusses evidence that suggests that connexin 43 plays an antagonistic role in the development of primary bone tumors as a tumor suppressor and secondary bone tumors as a tumor promoter.

Visualization of Annular Gap Junction Vesicle Processing: The Interplay Between Annular Gap Junctions and Mitochondria.

It is becoming clear that in addition to gap junctions playing a role in cell⁻cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures-annular gap junctions-were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.

Blocking connexin channels during vitrification of immature cat oocytes improves maturation capacity after warming.

In the domestic cat, nuclear maturation and embryo development after vitrification of immature oocytes have been obtained but developmental competence after warming remains low. It has been reported that during folliculogenesis, the association and communication between the oocyte and the surrounding cumulus cells through connexin-based gap junctions is essential for normal oocyte and follicular development. Gap junctions result from the head-to-head interaction of two hemichannels; however, there is always a population of hemichannels not incorporated into gap junctions. These unopposed hemichannels are normally closed but may open under certain stress conditions, potentially also during vitrification and warming, turning them into toxic pores inducing cell injury and cell death. The aim of our study was to test whether inhibiting connexin 37 (Cx37) and connexin 43 (Cx43) channels with the connexin-targeting peptide Gap26 during vitrification and warming of cat immature cumulus-oocyte-complexes (COCs) could improve oocyte maturation and competence of resultant blastocysts derived by parthenogenetic activation. In the first experiment, our immunostainings confirmed the presence of Cx43 protein in the cytoplasm of immature cat oocytes and in the plasma membranes of cumulus cells. In the second experiment, COCs were randomly divided in three different groups: a control group (control), a group vitrified without Gap26 (vitrified) and a group vitrified with Gap26 (vitrified-peptide). The maturation rate was checked and oocytes from all three different experimental groups were parthenogenetically activated and cultured in vitro until day 8. After vitrification and warming, 49% of the oocytes in the control group matured, while this was 8% and 19% in the vitrified and vitrified-peptide groups, respectively. Compared to the vitrified group, oocytes in the vitrified-peptide group had significantly larger maturation rates. No blastocysts were detected at day 8 in the vitrified group, while 2% and 13% of the oocytes further developed to blastocyst at day 8 in the vitrified-peptide and control non-vitrified group, respectively. We conclude that the use of Gap26 in vitrification and warming media to vitrify immature cat oocytes improves maturation success and allows such oocytes to reach the blastocyst stage (2%) at day 8 after parthenogenetic activation.

Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics.

In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.

Communication shapes sensory response in multicellular networks.

Collective sensing by interacting cells is observed in a variety of biological systems, and yet, a quantitative understanding of how sensory information is collectively encoded is lacking. Here, we investigate the ATP-induced calcium dynamics of monolayers of fibroblast cells that communicate via gap junctions. Combining experiments and stochastic modeling, we find that increasing the ATP stimulus increases the propensity for calcium oscillations, despite large cell-to-cell variability. The model further predicts that the oscillation propensity increases with not only the stimulus, but also the cell density due to increased communication. Experiments confirm this prediction, showing that cell density modulates the collective sensory response. We further implicate cell-cell communication by coculturing the fibroblasts with cancer cells, which we show act as "defects" in the communication network, thereby reducing the oscillation propensity. These results suggest that multicellular networks sit at a point in parameter space where cell-cell communication has a significant effect on the sensory response, allowing cells to simultaneously respond to a sensory input and the presence of neighbors.

Functional formation of heterotypic gap junction channels by connexins-40 and -43.

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (Vj) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less Vj gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance - voltage (Gj-Vj) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the Vj gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic Gj-Vj curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.

Simvastatin-induced up-regulation of gap junctions composed of connexin 43 sensitize Leydig tumor cells to etoposide: an involvement of PKC pathway.

Some of lipophilic statins have been reported to enhance toxicities induced by antineoplastic agents but the underling mechanism is unclear. The authors investigated the involvement of Cx43-mediated gap junction intercellular communication (GJIC) in the effect of simvastatin on the cellular toxicity induced by etoposide in this study. The results showed that a major component of the cytotoxicity of therapeutic levels of etoposide is mediated by gap junctions composed of connexin 43(Cx43) and simvastatin at the dosage which does not induce cytotoxicity enhances etoposide toxicity by increasing gap junction coupling. The augmentative effect of simvastatin on GJIC was related to the inhibition of PKC-mediated Cx43 phosphorylation at ser368 and subsequent enhancement of Cx43 membrane location induced by the agent. The present study suggests the possibility that upregulation of gap junctions may be utilized to increase the efficacy of anticancer chemotherapies.

Super-resolution fluorescence microscopy of the cardiac connexome reveals plakophilin-2 inside the connexin43 plaque.

AIMS: Cell function requires formation of molecular clusters localized to discrete subdomains. The composition of these interactomes, and their spatial organization, cannot be discerned by conventional microscopy given the resolution constraints imposed by the diffraction limit of light (∼200-300 nm). Our aims were (i) Implement single-molecule imaging and analysis tools to resolve the nano-scale architecture of cardiac myocytes. (ii) Using these tools, to map two molecules classically defined as components 'of the desmosome' and 'of the gap junction', and defined their spatial organization. METHODS AND RESULTS: We built a set-up on a conventional inverted microscope using commercially available optics. Laser illumination, reducing, and oxygen scavenging conditions were used to manipulate the blinking behaviour of individual fluorescent reporters. Movies of blinking fluorophores were reconstructed to generate subdiffraction images at ∼20 nm resolution. With this method, we characterized clusters of connexin43 (Cx43) and of 'the desmosomal protein' plakophilin-2 (PKP2). In about half of Cx43 clusters, we observed overlay of Cx43 and PKP2 at the Cx43 plaque edge. SiRNA-mediated loss of Ankyrin-G expression yielded larger Cx43 clusters, of less regular shape, and larger Cx43-PKP2 subdomains. The Cx43-PKP2 subdomain was validated by a proximity ligation assay (PLA) and by Monte-Carlo simulations indicating an attraction between PKP2 and Cx43. CONCLUSIONS: (i) Super-resolution fluorescence microscopy, complemented with Monte-Carlo simulations and PLAs, allows the study of the nanoscale organization of an interactome in cardiomyocytes. (ii) PKP2 and Cx43 share a common hub that permits direct physical interaction. Its relevance to excitability, electrical coupling, and arrhythmogenic right ventricular cardiomyopathy, is discussed.

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 Ca(2+)-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 Ca(2+) that initiate the propagation of the Ca(2+)-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca(2+)-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 Ca(2+)-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca(2+)-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 Ca(2+)-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.

Gender-specific remodeling in atrial fibrillation?

BACKGROUND: The authors wanted to investigate whether the remodeling process in AF regarding gap junction proteins, collagen I, and amyloid may be gender dependent in humans. METHODS: In total, 123 patients with sinus rhythm (SR, n = 41) or atrial fibrillation (AF, n = 82) suffering from mitral valve disease undergoing cardiac surgery were included. Of the 123 patients, 66 patients (SR: n = 17, AF: n = 49) were investigated biochemically for the expression of the atrial gap junction proteins connexin40 (Cx40), connexin43 (Cx43) and collagen I and 57 patients (SR: n = 24; AF: n = 33) using histochemical methods for possible amyloid depositions. RESULTS: AF led to increased levels of Cx40, Cx43, and collagen I protein. Regarding Cx40 this upregulation was significantly higher in female than in male patients. For AF-induced changes in collagen or Cx43, there were no significant gender-dependent differences. Amyloid depositions were found with increasing age, but were not significantly related to AF or gender. CONCLUSIONS: Remodeling in AF seems to be similar in men and women, with a tendency for women exhibiting somewhat stronger AF-induced changes in Cx40, which is probably a secondary effect because there is nothing known about hormone sensitivity of the Cx40 promoter, and a not significant tendency for higher Cx43 and collagen I.

Characterization of the structure and intermolecular interactions between the connexin 32 carboxyl-terminal domain and the protein partners synapse-associated protein 97 and calmodulin.

In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca(2+), pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis.

Prostaglandin-induced cervical remodelling in humans in the first trimester is associated with increased expression of specific tight junction, but not gap junction proteins.

BACKGROUND: Prostaglandins (PG) are widely employed to induce cervical remodelling (CR) in pregnancy. However, the underlying molecular mechanisms are not fully elucidated. Tight junctions (TJ) and gap junctions (GJ) regulate paracellular and intercellular solute transport respectively but their role in the process of CR remains unexplored. We hypothesized that the synthetic prostaglandin E1 analogue Misoprostol (M), widely used in clinical practice to induce CR, may alter TJ and GJ expression as part of the changes in the extracellular matrix (ECM) associated with remodelling. We investigated the effects of Misoprostol exposure on the expression of cervical TJ (claudins 1, 2, 4, 5, 7 and occludin) and GJ (connexins 43, 30 and 26) in the 1st trimester. METHODS: Cervical biopsies were obtained from pregnant women and comparisons of TJ and GJ protein expression (by western blotting) and immunolocalisation (laser scanning confocal microscopy) made between those who were administered vaginal Misoprostol (n=10) and those who were not (n=5). RESULTS: We found that Misoprostol-treated tissue (M+) had higher expression of Claudins 1,2,4,7 and occludin (p<0.05) than untreated (M-) tissue. Expression levels of Claudins 1, 2 and 4 were positively correlated to interval from Misoprostol treatment to biopsy, whilst occludin was negatively correlated. Misoprostol-treated cervical tissue demonstrated more endothelial claudin-5 and occludin, whilst expression of GJs were unchanged. CONCLUSION: Our observations suggest, for the first time, that increased expression of tight junction proteins may be one of the mechanisms by which Misoprostol induces CR in humans. Further studies are needed to explore if TJ proteins may be therapeutic targets to alter timing of CR in clinical practice.

In vitro motility of liver connexin vesicles along microtubules utilizes kinesin motors.

Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin- and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 µm/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 µM of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 µM ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4-0.5 µm/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 µM vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.

SCAM analysis of Panx1 suggests a peculiar pore structure.

Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct.

The carboxy-terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells.

Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C-tail) to demonstrate that the C-tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C-tail in mediating Cx43-dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1-242) and a construct encompassing only the C-tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C-tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C-tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time-lapse imaging to examine the behavior of migrating cells and it was apparent that the C-tail was associated with a lamellipodia-based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C-tail is sufficient to induce Cx43-dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton.

Somatic cAMP signaling regulates MSP-dependent oocyte growth and meiotic maturation in C. elegans.

Soma-germline interactions control fertility at many levels, including stem cell proliferation, meiosis and gametogenesis, yet the nature of these fundamental signaling mechanisms and their potential evolutionary conservation are incompletely understood. In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and fertilization. Sperm release the major sperm protein (MSP) signal to trigger meiotic resumption (meiotic maturation) and to promote contraction of the follicle-like gonadal sheath cells that surround oocytes. Using genetic mosaic analysis, we show that all known MSP-dependent meiotic maturation events in the germline require Galpha(s)-adenylate cyclase signaling in the gonadal sheath cells. We show that the MSP hormone promotes the sustained actomyosin-dependent cytoplasmic streaming that drives oocyte growth. Furthermore, we demonstrate that efficient oocyte production and cytoplasmic streaming require Galpha(s)-adenylate cyclase signaling in the gonadal sheath cells, thereby providing a somatic mechanism that coordinates oocyte growth and meiotic maturation with sperm availability. We present genetic evidence that MSP and Galpha(s)-adenylate cyclase signaling regulate oocyte growth and meiotic maturation in part by antagonizing gap-junctional communication between sheath cells and oocytes. In the absence of MSP or Galpha(s)-adenylate cyclase signaling, MSP binding sites are enriched and appear clustered on sheath cells. We discuss these results in the context of a model in which the sheath cells function as the major initial sensor of MSP, potentially via multiple classes of G-protein-coupled receptors. Our findings highlight a remarkable similarity between the regulation of meiotic resumption by soma-germline interactions in C. elegans and mammals.

Conformational changes in a pore-forming region underlie voltage-dependent "loop gating" of an unapposed connexin hemichannel.

The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32*Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32*Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.