The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2001
A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and desig... more A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of th...
A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid r... more A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid residues. Based on the predicted molecular mass of 57.115 kDa, it has been designated connexin-57. Similar to most other mouse connexin genes, the coding region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this new gene shows highest sequence identity to porcine connexin-60 in the alpha group of connexins. The connexin-57 gene was mapped to a position on mouse chromosome 4, 30 centimorgans proximal to a cluster of previously mapped connexin genes. Low levels of connexin-57 mRNA were detected in skin, heart, kidney, testis, ovary, intestine, and in the mouse embryo after 8 days post coitum, but expression was not detected in brain, sciatic nerve or liver. In order to analyze gene function, the connexin-57 coding region was expressed by transfection in human HeLa cells, where it restored homotypic intercellula...
Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal st... more Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP 3 , and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system.
Intracellular pH (pH i ) can change during physiological and pathological conditions causing sign... more Intracellular pH (pH i ) can change during physiological and pathological conditions causing significant changes of electrical and metabolic cell-cell communication through gap junction (GJ) channels. In HeLa cells expressing wild-type connexin45 (Cx45) as well as Cx45 and Cx43 tagged with EGFP, we examined how pH i affects junctional conductance (g j ) and g j dependence on transjunctional voltage (V j ). To characterize V j gating, we fit the g j -V j relation using a stochastic four-state model containing one V j -sensitive gate in each apposed hemichannel (aHC); aHC open probability was a Boltzmann function of the fraction of V j across it. Using the model, we estimated gating parameters characterizing sensitivity to V j and number of functional channels. In homotypic Cx45 and heterotypic Cx45/Cx43-EGFP GJs, pH i changes from 7.2 to ∼8.0 shifted g j -V j dependence of Cx45 aHCs along the V j axis resulting in increased probability of GJ channels being in the fully open state without change in the slope of g j dependence on V j . In contrast, acidification shifted g j -V j dependence in the opposite direction, reducing open probability; acidification also reduced the number of functional channels. Correlation between the number of channels in Cx45-EGFP GJs and maximal g j achieved under alkaline conditions showed that only ∼4% of channels were functional. The acid dissociation constant (pK a ) of g j -pH i dependence of Cx45/Cx45 GJs was ∼7. The pK a of heterotypic Cx45/Cx43-EGFP GJs was lower, ∼6.7, between the pK a s of Cx45 and Cx43-EGFP (∼6.5) homotypic GJs. In summary, pH i significantly modulates junctional conductance of Cx45 by affecting both V j gating and number of functional channels.
pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms propos... more pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting ( approximately 10(-7) M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.
Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauth... more Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication-chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre-and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35-and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.
It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play... more It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was approximately 220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels.
Gap junction channels and hemichannels formed of connexin subunits are found in most cell types i... more Gap junction channels and hemichannels formed of connexin subunits are found in most cell types in vertebrates. Gap junctions connect cells via channels not open to the extracellular space and permit the passage of ions and molecules of~1 kDa. Single connexin hemichannels, which are connexin hexamers, are present in the surface membrane before docking with a hemichannel in an apposed membrane. Because of their high conductance and permeability in cell-cell channels, it had been thought that connexin hemichannels remained closed until docking to form a cell-cell channel. Now it is clear that at least some hemichannels can open to allow passage of molecules between the cytoplasm and extracellular space. Here we review evidence that gap junction channels may allow intercellular diffusion of necrotic or apoptotic signals, but may also allow diffusion of ions and substances from healthy to injured cells, thereby contributing to cell survival. Moreover, opening of gap junction hemichannels may exacerbate cell injury or mediate paracrine or autocrine signaling. In addition to the cell specific features of an ischemic insult, propagation of cell damage and death within affected tissues may be affected by expression and regulation of gap junction channels and hemichannels formed by connexins. D 2004 Elsevier B.V. All rights reserved.
We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluo... more We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gap junction channel gating. Previously it was suggested that each hemichannel in a cell-cell channel possesses two gates, a fast gate that closes channels to a nonzero conductance or residual state via fast (< approximately 2 ms) transitions and a slow gate that fully closes channels via slow transitions (> approximately 10 ms). Here we demonstrate that transjunctional voltage (V(j)) regulates both gates and that they are operating in series and in a contingent manner in which the state of one gate affects gating of the other. Cx43-EGFP channels lack fast V(j) gating to a residual state but show slow V(j) gating. Both Cx43 and Cx43-EGFP channels exhibit slow gating by chemical uncouplers such as CO(2) and alkanols. Chemical uncouplers do not induce obvious changes in Cx43-EGFP junctional plaques, indicating that uncoupling is not caused by dispersion or internalization of junctional plaques. Similarity of gating transitions during chemical gating and slow V(j) gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical V(j) gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast V(j) gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2005
Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is compose... more Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is composed of two hemichannels, also termed connexons, one from each of the coupled cells. Hemichannels are hexamers assembled in the ER, the Golgi, or a post Golgi compartment. They are transported to the cell surface in vesicles and inserted by vesicle fusion, and then dock with a hemichannel in an apposed membrane to form a cell-cell channel. It was thought that hemichannels should remain closed until docking with another hemichannel because of the leak they would provide if their permeability and conductance were like those of their corresponding cell-cell channels. Now it is clear that hemichannels formed by a number of different connexins can open in at least some cells with a finite if low probability, and that their opening can be modulated under various physiological and pathological conditions. Hemichannels open in different kinds of cells in culture with conductance and permeability properties predictable from those of the corresponding gap junction channels. Cx43 hemichannels are preferentially closed in cultured cells under resting conditions, but their open probability can be increased by the application of positive voltages and by changes in protein phosphorylation and/or redox state. In addition, increased activity can result from the recruitment of hemichannels to the plasma membrane as seen in metabolically inhibited astrocytes. Mutations of connexins that increase hemichannel open probability may explain cellular degeneration in several hereditary diseases. Taken together, the data indicate that hemichannels are gated by multiple mechanisms that independently or cooperatively affect their open probability under physiological as well as pathological conditions. D
Connexins are protein subunits that oligomerize into hexamers called connexons, gap junction hemi... more Connexins are protein subunits that oligomerize into hexamers called connexons, gap junction hemichannels or just hemichannels. Because some gap junction channels are permeable to negatively and/or positively charged molecules up to 1kDa in size, it was thought that hemichannels should not open to the extracellular space. A growing amount of evidence indicates that opening of hemichannels does occur under both physiological and pathological conditions in astrocytes and other cell types. Electrophysiological studies indicate that hemichannels have a low open probability under physiological conditions but may have a much higher open probability under certain pathological conditions. Some of the physiological behaviours of astrocytes that have been attributed to gap junctions may, in fact, be mediated by hemichannels. Hemichannels constituted of Cx43, the main connexin expressed by astrocytes, are permeable to small physiologically significant molecules, such as ATP, NAD + and glutamate, and may mediate paracrine as well as autocrine signalling. Hemichannels tend to be closed by negative membrane potentials, high concentrations of extracellular Ca 2+ and intracellular H + ions, gap junction blockers and protein phosphorylation. Hemichannels tend to be opened by positive membrane potentials and low extracellular Ca 2+ , and possibly by as yet unidentified cytoplasmic signalling molecules. Exacerbated hemichannel opening occurs in metabolically inhibited cells, including cortical astrocytes, which contributes to the loss of chemical gradients across the plasma membrane and speeds cell death.
Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and pur... more Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and purinergic receptors. Fibroblast growth factor 1 (FGF-1), which is released in spinal cord injury, activated spinal astrocytes in culture, induced secretion of ATP, and permeabilized them to relatively large fluorescent tracers [ethidium (Etd) and lucifer yellow (LY)] through "hemichannels" (HCs). HCs can be formed by connexins or pannexins; they can open to extracellular space or can form gap junction (GJ) channels, one HC from each cell. (Pannexins may not form gap junctions in mammalian tissues, but they do in invertebrates). HC types were differentiated pharmacologically and by Px1 knockdown with siRNA and by use of astrocytes from Cx43 knockout mice. Permeabilization was reduced by apyrase (APY), an ATPase, and by P2X 7 receptor antagonists, implicating secretion of ATP and autocrine and/or paracrine action. Increased permeability of cells exposed to FGF-1 or ATP for 2 h was mediated largely by Px1 HCs activated by P2X 7 receptors. After a 7-h treatment, the permeability was mediated by both Cx43 and Px1 HCs. FGF-1 also caused reduction in gap junctional communication. Botulinum neurotoxin A, a blocker of vesicular release, reduced permeabilization when given 30 min before FGF-1 application, but not when given 1 h after FGF-1. We infer that ATP is initially released from vesicles and then it mediates continued release by action on P2X 7 receptors and opening of HCs. These changes in HCs and gap junction channels may promote inflammation and deprive neurons of astrocyte-mediated protection in spinal cord trauma and neurodegenerative disease. astroglia | growth factor | connexon | pannexon | neurodegeneration
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2001
A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and desig... more A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of th...
A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid r... more A new mouse connexin gene has been isolated that codes for a connexin protein of 505 amino acid residues. Based on the predicted molecular mass of 57.115 kDa, it has been designated connexin-57. Similar to most other mouse connexin genes, the coding region of connexin-57 is not interrupted by introns and exists in the mouse genome as a single-copy gene. Within the connexin family, this new gene shows highest sequence identity to porcine connexin-60 in the alpha group of connexins. The connexin-57 gene was mapped to a position on mouse chromosome 4, 30 centimorgans proximal to a cluster of previously mapped connexin genes. Low levels of connexin-57 mRNA were detected in skin, heart, kidney, testis, ovary, intestine, and in the mouse embryo after 8 days post coitum, but expression was not detected in brain, sciatic nerve or liver. In order to analyze gene function, the connexin-57 coding region was expressed by transfection in human HeLa cells, where it restored homotypic intercellula...
Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal st... more Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP 3 , and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system.
Intracellular pH (pH i ) can change during physiological and pathological conditions causing sign... more Intracellular pH (pH i ) can change during physiological and pathological conditions causing significant changes of electrical and metabolic cell-cell communication through gap junction (GJ) channels. In HeLa cells expressing wild-type connexin45 (Cx45) as well as Cx45 and Cx43 tagged with EGFP, we examined how pH i affects junctional conductance (g j ) and g j dependence on transjunctional voltage (V j ). To characterize V j gating, we fit the g j -V j relation using a stochastic four-state model containing one V j -sensitive gate in each apposed hemichannel (aHC); aHC open probability was a Boltzmann function of the fraction of V j across it. Using the model, we estimated gating parameters characterizing sensitivity to V j and number of functional channels. In homotypic Cx45 and heterotypic Cx45/Cx43-EGFP GJs, pH i changes from 7.2 to ∼8.0 shifted g j -V j dependence of Cx45 aHCs along the V j axis resulting in increased probability of GJ channels being in the fully open state without change in the slope of g j dependence on V j . In contrast, acidification shifted g j -V j dependence in the opposite direction, reducing open probability; acidification also reduced the number of functional channels. Correlation between the number of channels in Cx45-EGFP GJs and maximal g j achieved under alkaline conditions showed that only ∼4% of channels were functional. The acid dissociation constant (pK a ) of g j -pH i dependence of Cx45/Cx45 GJs was ∼7. The pK a of heterotypic Cx45/Cx43-EGFP GJs was lower, ∼6.7, between the pK a s of Cx45 and Cx43-EGFP (∼6.5) homotypic GJs. In summary, pH i significantly modulates junctional conductance of Cx45 by affecting both V j gating and number of functional channels.
pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms propos... more pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting ( approximately 10(-7) M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side.
Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauth... more Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication-chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre-and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35-and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.
It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play... more It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was approximately 220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels.
Gap junction channels and hemichannels formed of connexin subunits are found in most cell types i... more Gap junction channels and hemichannels formed of connexin subunits are found in most cell types in vertebrates. Gap junctions connect cells via channels not open to the extracellular space and permit the passage of ions and molecules of~1 kDa. Single connexin hemichannels, which are connexin hexamers, are present in the surface membrane before docking with a hemichannel in an apposed membrane. Because of their high conductance and permeability in cell-cell channels, it had been thought that connexin hemichannels remained closed until docking to form a cell-cell channel. Now it is clear that at least some hemichannels can open to allow passage of molecules between the cytoplasm and extracellular space. Here we review evidence that gap junction channels may allow intercellular diffusion of necrotic or apoptotic signals, but may also allow diffusion of ions and substances from healthy to injured cells, thereby contributing to cell survival. Moreover, opening of gap junction hemichannels may exacerbate cell injury or mediate paracrine or autocrine signaling. In addition to the cell specific features of an ischemic insult, propagation of cell damage and death within affected tissues may be affected by expression and regulation of gap junction channels and hemichannels formed by connexins. D 2004 Elsevier B.V. All rights reserved.
We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluo... more We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gap junction channel gating. Previously it was suggested that each hemichannel in a cell-cell channel possesses two gates, a fast gate that closes channels to a nonzero conductance or residual state via fast (< approximately 2 ms) transitions and a slow gate that fully closes channels via slow transitions (> approximately 10 ms). Here we demonstrate that transjunctional voltage (V(j)) regulates both gates and that they are operating in series and in a contingent manner in which the state of one gate affects gating of the other. Cx43-EGFP channels lack fast V(j) gating to a residual state but show slow V(j) gating. Both Cx43 and Cx43-EGFP channels exhibit slow gating by chemical uncouplers such as CO(2) and alkanols. Chemical uncouplers do not induce obvious changes in Cx43-EGFP junctional plaques, indicating that uncoupling is not caused by dispersion or internalization of junctional plaques. Similarity of gating transitions during chemical gating and slow V(j) gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical V(j) gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast V(j) gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2005
Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is compose... more Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is composed of two hemichannels, also termed connexons, one from each of the coupled cells. Hemichannels are hexamers assembled in the ER, the Golgi, or a post Golgi compartment. They are transported to the cell surface in vesicles and inserted by vesicle fusion, and then dock with a hemichannel in an apposed membrane to form a cell-cell channel. It was thought that hemichannels should remain closed until docking with another hemichannel because of the leak they would provide if their permeability and conductance were like those of their corresponding cell-cell channels. Now it is clear that hemichannels formed by a number of different connexins can open in at least some cells with a finite if low probability, and that their opening can be modulated under various physiological and pathological conditions. Hemichannels open in different kinds of cells in culture with conductance and permeability properties predictable from those of the corresponding gap junction channels. Cx43 hemichannels are preferentially closed in cultured cells under resting conditions, but their open probability can be increased by the application of positive voltages and by changes in protein phosphorylation and/or redox state. In addition, increased activity can result from the recruitment of hemichannels to the plasma membrane as seen in metabolically inhibited astrocytes. Mutations of connexins that increase hemichannel open probability may explain cellular degeneration in several hereditary diseases. Taken together, the data indicate that hemichannels are gated by multiple mechanisms that independently or cooperatively affect their open probability under physiological as well as pathological conditions. D
Connexins are protein subunits that oligomerize into hexamers called connexons, gap junction hemi... more Connexins are protein subunits that oligomerize into hexamers called connexons, gap junction hemichannels or just hemichannels. Because some gap junction channels are permeable to negatively and/or positively charged molecules up to 1kDa in size, it was thought that hemichannels should not open to the extracellular space. A growing amount of evidence indicates that opening of hemichannels does occur under both physiological and pathological conditions in astrocytes and other cell types. Electrophysiological studies indicate that hemichannels have a low open probability under physiological conditions but may have a much higher open probability under certain pathological conditions. Some of the physiological behaviours of astrocytes that have been attributed to gap junctions may, in fact, be mediated by hemichannels. Hemichannels constituted of Cx43, the main connexin expressed by astrocytes, are permeable to small physiologically significant molecules, such as ATP, NAD + and glutamate, and may mediate paracrine as well as autocrine signalling. Hemichannels tend to be closed by negative membrane potentials, high concentrations of extracellular Ca 2+ and intracellular H + ions, gap junction blockers and protein phosphorylation. Hemichannels tend to be opened by positive membrane potentials and low extracellular Ca 2+ , and possibly by as yet unidentified cytoplasmic signalling molecules. Exacerbated hemichannel opening occurs in metabolically inhibited cells, including cortical astrocytes, which contributes to the loss of chemical gradients across the plasma membrane and speeds cell death.
Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and pur... more Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and purinergic receptors. Fibroblast growth factor 1 (FGF-1), which is released in spinal cord injury, activated spinal astrocytes in culture, induced secretion of ATP, and permeabilized them to relatively large fluorescent tracers [ethidium (Etd) and lucifer yellow (LY)] through "hemichannels" (HCs). HCs can be formed by connexins or pannexins; they can open to extracellular space or can form gap junction (GJ) channels, one HC from each cell. (Pannexins may not form gap junctions in mammalian tissues, but they do in invertebrates). HC types were differentiated pharmacologically and by Px1 knockdown with siRNA and by use of astrocytes from Cx43 knockout mice. Permeabilization was reduced by apyrase (APY), an ATPase, and by P2X 7 receptor antagonists, implicating secretion of ATP and autocrine and/or paracrine action. Increased permeability of cells exposed to FGF-1 or ATP for 2 h was mediated largely by Px1 HCs activated by P2X 7 receptors. After a 7-h treatment, the permeability was mediated by both Cx43 and Px1 HCs. FGF-1 also caused reduction in gap junctional communication. Botulinum neurotoxin A, a blocker of vesicular release, reduced permeabilization when given 30 min before FGF-1 application, but not when given 1 h after FGF-1. We infer that ATP is initially released from vesicles and then it mediates continued release by action on P2X 7 receptors and opening of HCs. These changes in HCs and gap junction channels may promote inflammation and deprive neurons of astrocyte-mediated protection in spinal cord trauma and neurodegenerative disease. astroglia | growth factor | connexon | pannexon | neurodegeneration
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Papers by F. Bukauskas