Synaptic transmission from horizontal cells to cones is impaired by loss of connexin hemichannels

In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a long-standing debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Disruption in Connexin-Based Communication Is Associated with Intracellular Ca2+ Signal Alterations in Astrocytes from Niemann-Pick Type C Mice

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1^−/−) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1^−/− hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1^−/− astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1^−/− astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.     

Connexin hemichannel and pannexin channel electrophysiology: How do they differ?

Connexin hemichannels are postulated to form a cell permeabilization pore for the uptake of fluorescent dyes and release of cellular ATP. Connexin hemichannel activity is enhanced by low external [Ca²⁺]_o, membrane depolarization, metabolic inhibition, and some disease-causing gain-of-function connexin mutations. This paper briefly reviews the electrophysiological channel conductance, permeability, and pharmacology properties of connexin hemichannels, pannexin 1 channels, and purinergic P2X₇ receptor channels as studied in exogenous expression systems including Xenopus oocytes and mammalian cell lines such as HEK293 cells. Overlapping pharmacological inhibitory and channel conductance and permeability profiles makes distinguishing between these channel types sometimes difficult. Selective pharmacology for Cx43 hemichannels (Gap19 peptide), probenecid or FD&C Blue #1 (Brilliant Blue FCF, BB FCF) for Panx1, and A740003, A438079, or oxidized ATP (oATP) for P2X7 channels may be the best way to distinguish between these three cell permeabilizing channel types. Endogenous connexin, pannexin, and P2X₇ expression should be considered when performing exogenous cellular expression channel studies. Cell pair electrophysiological assays permit the relative assessment of the connexin hemichannel/gap junction channel ratio not often considered when performing isolated cell hemichannel studies.     

Connexin channels, connexin mimetic peptides and ATP release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP(3) elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Connexin Channels, Connexin Mimetic Peptides and ATP Release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP₃ elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.     

Pathological hemichannels associated with human Cx26 mutations causing Keratitis-Ichthyosis-Deafness syndrome

Connexin (Cx) proteins form intercellular gap junction channels by first assembling into single membrane hemichannels that then dock to connect the cytoplasm of two adjacent cells. Gap junctions are highly specialized structures that allow the direct passage of small molecules between cells to maintain tissue homeostasis. Functional activity of nonjunctional hemichannels has now been shown in several experimental systems. Hemichannels may constitute an important diffusional exchange pathway with the extracellular space, but the extent of their normal physiological role is currently unknown. Aberrant hemichannel activity has been linked to mutations of connexin proteins involved in genetic diseases. Here, we review a proposed role for hemichannels in the pathogenesis of Keratitis-Ichthyosis-Deafness (KID) syndrome associated with connexin26 (Cx26) mutations. Continued functional evaluation of mutated hemichannels linked to human hereditary disorders may provide additional insights into the mechanisms governing their regulation in normal physiology and dysregulation in disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Properties of Connexin26 Hemichannels Expressed in Xenopus Oocytes

1. Hemichannels formed by connexin26 (Cx26) on the horizontal cell dendrites that invaginate cone terminals in the vertebrate retina have been implicated in the feedback mechanism by which horizontal cells regulate transmitter release from cone photoreceptors. However, their membrane properties had not been studied previously, and it was unclear whether they could subserve their purported function at the membrane potentials over which horizontal cells operate. 2. We used the two-electrode voltage clamp technique to record the membrane currents and pharmacological properties of Cx26 hemichannels formed in the Xenopus oocyte expression system. 3. Oocytes expressing Cx26 exhibited large membrane conductances over a broad range of hyperpolarizing and depolarizing membrane potentials, and displayed little evidence of voltage-dependent gating, indicating that the hemichannels are constitutively open. The Cx26-mediated nonjunctional currents were relatively insensitive to quinine, a cinchona alkaloid that opens hemichannels formed by several other connexins. However, the hemichannel currents were blocked by carbenoxolone, a rise in extracellular calcium, or lowering intracellular pH. The currents could also be suppressed by reducing extracellular pH, and by the chloride channel blocker NPPB through its direct interaction with Cx26 hemichannels. 4. These findings provide a basis with which to evaluate the in situ pharmacological studies that attempt to assess the putative role of Cx26 hemichannels in the feedback pathway in the distal retina.     

Connexin Hemichannels in Astrocytes: An Assessment of Controversies Regarding Their Functional Characteristics

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a ‘docked’ counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.     

Pathogenic connexin-31 forms constitutively active hemichannels to promote necrotic cell death

Mutations in Connexin-31 (Cx31) are associated with multiple human diseases including erythrokeratodermia variabilis (EKV). The molecular action of Cx31 pathogenic mutants remains largely elusive. We report here that expression of EKV pathogenic mutant Cx31R42P induces cell death with necrotic characteristics. Inhibition of hemichannel activity by a connexin hemichannel inhibitor or high extracellular calcium suppresses Cx31R42P-induced cell death. Expression of Cx31R42P induces ER stress resulting in reactive oxygen species (ROS) production, in turn, to regulate gating of Cx31R42P hemichannels and Cx31R42P induced cell death. Moreover, Cx31R42P hemichannels play an important role in mediating ATP release from the cell. In contrast, no hemichannel activity was detected with cells expressing wildtype Cx31. Together, the results suggest that Cx31R42P forms constitutively active hemichannels to promote necrotic cell death. The Cx31R42P active hemichannels are likely resulted by an ER stress mediated ROS overproduction. The study identifies a mechanism of EKV pathogenesis induced by a Cx31 mutant and provides a new avenue for potential treatment strategy of the disease.     

Following tracks of hemichannels

It has been suggested that plasma membrane-bound hemichannels perform physiological and pathophysiological functions per se. Such functions require the presence of hemichannels on the cell surface and their accessibility to the extracellular environment for at least some limited period of time. We have previously shown that hemichannels can be labeled by means of antibodies directed to an external loop domain of connexin (Cx) 43. We now provide evidence that trafficking of hemichannel vesicles can be visualized upon binding of a labeled homophilic peptide corresponding to a region of the first extracellular loop (EL1) of Cx43. In vivo imaging was performed after labeling hemichannels from the extracellular site with a mimetic peptide tagged with a fluorochrome (Alexa-546). Using a Cx43-CFP transfected HeLa cell line for incubation with the mimetic peptide, a significant number of double-labeled vesicles were found inside the cells. This double labeling indicates that a portion of Cx43 within the cell had accessed the cell surface as hemichannels where it bound to the peptide and was subsequently endocytosed. Pulse labeling with the peptide showed a decrease in the number of dual-labeled vesicles over time, indicating degradation and/or concurrent recycling of hemichannel vesicles.     

Connexin Channels,Connexin Mimetic Peptides and ATP Release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP₃elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Bisphosphonate-Induced,Hemichannel-Mediated,Anti-Apoptosis Through the Src/ERK Pathway: A Gap Junction-Independent Action of Connexin43

Preservation of the mechanosensory function of osteocytes by inhibiting their apoptosis might contribute to the beneficial effects of bisphosphonates in bone. We report herein a mechanism by which connexin43 hemichannel opening by bisphosphonates triggers the activation of the kinases Src and ERK.S and promotes cell survival. Bisphosphonate-induced anti-apoptosis requires connexin channel integrity, but not gap junctions. Osteocytic cells express functional hemichannels that are opened by bisphosphonates, as demonstrated by dye uptake, regulation by established agonists and antagonists, and cell surface biotinylation. The anti-apoptotic effect of bisphosphonates depends on connexin43 expression in mouse embryonic fibroblasts and osteoblastic cells. Transfection of connexin43, but not other connexins, into connexin43 naive cells confers de novo responsiveness to the drugs. The signal transducing property of connexin43 requires the pore-forming, as well as the C-terminal domains of the protein, the interaction of connexin43 with Src, and the activation of both Src and ERK kinases. These studies establish a role for connexin43 hemichannels in bisphosphonate action, and a novel function of connexin43—beyond gap junction communication—in the regulation of survival signaling pathways.     

Bisphosphonate-induced,hemichannel-mediated,anti-apoptosis through the Src/ERK pathway: a gap junction-independent action of connexin43

Preservation of the mechanosensory function of osteocytes by inhibiting their apoptosis might contribute to the beneficial effects of bisphosphonates in bone. We report herein a mechanism by which connexin43 hemichannel opening by bisphosphonates triggers the activation of the kinases Src and ERKs and promotes cell survival. Bisphosphonate-induced anti-apoptosis requires connexin channel integrity, but not gap junctions. Osteocytic cells express functional hemichannels that are opened by bisphosphonates, as demonstrated by dye uptake, regulation by established agonists and antagonists, and cell surface biotinylation. The anti-apoptotic effect of bisphosphonates depends on connexin43 expression in mouse embryonic fibroblasts and osteoblastic cells. Transfection of connexin43, but not other connexins, into connexin43 na?ve cells confers de novo responsiveness to the drugs. The signal transducing property of connexin43 requires the pore-forming, as well as the C-terminal domains of the protein, the interaction of connexin43 with Src. and the activation of both Src and ERK kinases. These studies establish a role for connexin43 hemichannels in bisphosphonate action, and a novel function of connexin43--beyond gap junction communication--in the regulation of survival signaling pathways.     

Pathological hemichannels associated with human Cx26 mutations causing Keratitis–Ichthyosis–Deafness syndrome

Connexin (Cx) proteins form intercellular gap junction channels by first assembling into single membrane hemichannels that then dock to connect the cytoplasm of two adjacent cells. Gap junctions are highly specialized structures that allow the direct passage of small molecules between cells to maintain tissue homeostasis. Functional activity of nonjunctional hemichannels has now been shown in several experimental systems. Hemichannels may constitute an important diffusional exchange pathway with the extracellular space, but the extent of their normal physiological role is currently unknown. Aberrant hemichannel activity has been linked to mutations of connexin proteins involved in genetic diseases. Here, we review a proposed role for hemichannels in the pathogenesis of Keratitis–Ichthyosis–Deafness (KID) syndrome associated with connexin26 (Cx26) mutations. Continued functional evaluation of mutated hemichannels linked to human hereditary disorders may provide additional insights into the mechanisms governing their regulation in normal physiology and dysregulation in disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Following Tracks of Hemichannels

It has been suggested that plasma membrane-bound hemichannels perform physiological and pathophysiological functions per se. Such functions require the presence of hemichannels on the cell surface and their accessibility to the extracellular environment for at least some limited period of time. We have previously shown that hemichannels can be labeled by means of antibodies directed to an external loop domain of connexin (Cx) 43 (1). We now provide evidence that trafficking of hemichannel vesicles can be visualized upon binding of a labeled homophilic peptide corresponding to a region of the first extracellular loop (EL1) of Cx43. In vivoimaging was performed after labeling hemichannels from the extracellular site with a mimetic peptide tagged with a fluorochrome (Alexa-546). Using a Cx43-CFP transfected HeLa cell line for incubation with the mimetic peptide, a significant number of double-labeled vesicles were found inside the cells. This double labeling indicates that a portion of Cx43 within the cell had accessed the cell surface as hemichannels where it bound to the peptide and was subsequently endocytosed. Pulse labeling with the peptide showed a decrease in the number of dual-labeled vesicles over time, indicating degradation and/or concurrent recycling of hemichannel vesicles.     

Functioning of cx43 hemichannels demonstrated by single channel properties

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.     

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.     

Functioning of Cx43 Hemichannels Demonstrated by Single Channel Properties

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 ～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 ～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.