Biomedical subjects

Feliksas F Bukauskas

Publications and source records attributed to Feliksas F Bukauskas.

At least 19 recordsLinked to original sources

PubMed2006

Gating properties of heterotypic gap junction channels formed of connexins 40, 43, and 45.

Connexins (Cxs) 40, 43, and 45 are expressed in many different tissues, but most abundantly in the heart, blood vessels, and the nervous system. We examined formation and gating properties of heterotypic gap junction (GJ) channels assembled between cells expressing wild-type Cx40, Cx43, or Cx45 and their fusion forms tagged with color variants of green fluorescent protein. We show that these Cxs, with exception of Cxs 40 and 43, are compatible to form functional heterotypic GJ channels. Cx40 and Cx43 hemichannels are unable or effectively impaired in their ability to dock and/or assemble into junctional plaques. When cells expressing Cx45 contacted those expressing Cx40 or Cx43 they readily formed junctional plaques with cell-cell coupling characterized by asymmetric junctional conductance dependence on transjunctional voltage, V(j). Cx40/Cx45 heterotypic GJ channels preferentially exhibit V(j)-dependent gating transitions between open and residual states with a conductance of approximately 42 pS; transitions between fully open and closed states with conductance of approximately 52 pS in magnitude occur at substantially lower ( approximately 10-fold) frequency. Cx40/Cx45 junctions demonstrate electrical signal transfer asymmetry that can be modulated between unidirectional and bidirectional by small changes in the difference between holding potentials of the coupled cells. Furthermore, both fast and slow gating mechanisms of Cx40 exhibit a negative gating polarity.

Connexin 43↗

PubMed2006

Pathogenetic role of the deafness-related M34T mutation of Cx26.

Mutations in the GJB2 gene, which encodes the gap junction protein connexin26 (Cx26), are the major cause of genetic non-syndromic hearing loss. The role of the allelic variant M34T in causing hereditary deafness remains controversial. By combining genetic, clinical, biochemical, electrophysiological and structural modeling studies, we have re-assessed the pathogenetic role of the M34T mutation. Genetic and audiological data indicate that the majority of heterozygous carriers and all five compound heterozygotes exhibited an impaired auditory function. Functional expression in transiently transfected HeLa cells showed that, although M34T was correctly synthesized and targeted to the plasma membrane, it inefficiently formed intercellular channels that displayed an abnormal electrical behavior and retained only 11% of the unitary conductance of the wild-type protein (HCx26wt). Moreover, M34T channels failed to support the intercellular diffusion of Lucifer Yellow and the spreading of mechanically induced intercellular Ca2+ waves. When co-expressed together with HCx26wt, M34T exerted dominant-negative effects on cell-cell coupling. Our findings are consistent with a structural model, predicting that the mutation leads to a constriction of the channel pore. These data support the view that M34T is a pathological variant of Cx26 associated with hearing impairment.

Adolescent↗

PubMed2006

Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels: implications for atrioventricular conduction in the heart.

Four connexins (Cxs), mouse (m)Cx30.2, Cx40, Cx43, and Cx45, determine cell-cell electrical signaling in mouse heart, and Cx43 and Cx45 are known to form unapposed hemichannels. Here we show that mCx30.2, which is most abundantly expressed in sinoatrial and atrioventricular nodal regions of the heart, and its putative human ortholog, human (h)Cx31.9, also form functional hemichannels, which, like mCx30.2 cell-cell channels, are permeable to cationic dyes up to approximately 400 Da in size. DAPI uptake by HeLa cells expressing mCx30.2 was >10-fold faster than that by HeLa parental cells. In Ca(2+)-free medium, uptake of DAPI by HeLaCx30.2-EGFP cells was increased approximately 2-fold, but uptake by parental cells was not affected. Conversely, acidification by application of CO(2) reduced DAPI uptake by HeLaCx30.2-EGFP cells but had little effect on uptake by parental cells. Cells expressing mCx30.2 exhibited higher rates of DAPI uptake than did cells expressing any of the other cardiac Cxs. Cardiomyocytes of 2-day-old rats transfected with hCx31.9-EGFP took up DAPI and ethidium bromide 5-10 times faster than wild-type cardiomyocytes. Mefloquine, a close derivative of quinine and quinidine that exhibits antimalarial and antiarrhythmic properties, reduced conductance of cell-cell junctions and dye uptake through mCx30.2 hemichannels with approximately the same affinity (IC(50) = approximately 10 microM) and increased dependence of junctional conductance on transjunctional voltage. Unitary conductance of mCx30.2 hemichannels was approximately 20 pS, about twice the cell-cell channel conductance. Hemichannels formed of mCx30.2 and hCx31.9 may slow propagation of excitation in the sinoatrial and atrioventricular nodes by shortening the space constant and depolarizing the excitable membrane.

Animals↗

PubMed2006

Properties of human connexin 31, which is implicated in hereditary dermatological disease and deafness.

The connexins are a family of at least 20 homologous proteins in humans that form aqueous channels connecting the interiors of coupled cells and mediating electrical and chemical communication. Mutations in the gene for human connexin 31 (hCx31) are associated with disorders of the skin and auditory system. Alterations in functional properties of Cx31 junctions are likely to play a role in these diseases; nonetheless, little is known about the properties of the wild-type channels. Here we show that hCx31 channels, like other connexin channels, are gated by voltage and close at low pH and when exposed to long-chain alkanols. Single-channel conductance of the fully open channel is approximately 85 pS, and it is permeable to Lucifer yellow, Alexa Fluor(350), ethidium bromide, and DAPI, which have valences of -2, -1, +1, and +2, respectively. In contrast to what has been reported for mouse Cx31, hCx31 appears to form functional heterotypic channels with all four connexins tested, Cx26, Cx30, Cx32, and Cx45. These findings provide an important first step in evaluating the pathogenesis of inherited human diseases associated with mutations in the gene for Cx31.

Animals↗

PubMed2006

Connexin-mediated cardiac impulse propagation: connexin 30.2 slows atrioventricular conduction in mouse heart.

In mouse heart, four connexins (Cxs), Cx30.2, Cx40, Cx43, and Cx45, form gap junction (GJ) channels for electric and metabolic cell-to-cell signaling. Extent and pattern of Cx isoform expression together with cytoarchitecture and excitability of cells determine the velocity of excitation spread in different regions of the heart. In the SA node, cell-cell coupling is mediated by Cx30.2 and Cx45, which form low-conductance (approximately 9 and 32 pS, respectively) GJ channels. In contrast, the working cardiomyocytes of atria and ventricles express mainly Cx40 and Cx43, which form GJ channels of high conductance (approximately 180 and 115 pS, respectively) that facilitate the fast conduction necessary for efficient mechanical contraction. In the AV node, cell-cell coupling is mediated by abundantly expressed Cx30.2 and Cx45 and Cx40, which is expressed to a lesser extent. Cx30.2 and Cx45 may determine higher intercellular resistance and slower conduction in the SA- and AV-nodal regions than in the ventricular conduction system or the atrial and ventricular working myocardium. Cx30.2 and its putative human ortholog, Cx31.9, under physiologic conditions form unapposed hemichannels in nonjunctional plasma membrane; these hemichannels have a conductance of approximately 20 pS and are permeable to cationic dyes up to approximately 400 Da in molecular mass. Genetic ablation of Cxs confirmed that Cx40 and Cx43 are important in determining the high conduction velocities in atria and ventricles, whereas the deletion of the Cx30.2 complementary DNA led to accelerated conduction in the AV node and reduced the Wenckebach period. We suggest that these effects are caused by (1) a dominant-negative effect of Cx30.2 on junctional conductance via formation of low-conductance homotypic and heterotypic GJ channels, and (2) open Cx30.2 hemichannels in non-junctional membranes, which shorten the space constant and depolarize the excitable membrane.

Animals↗

PubMed2005

Functional properties of mouse connexin30.2 expressed in the conduction system of the heart.

Gap junction channels composed of connexin (Cx) 40, Cx43, and Cx45 proteins are known to be necessary for impulse propagation through the heart. Here, we report mouse connexin30.2 (mCx30.2) to be a new cardiac connexin that is expressed mainly in the conduction system of the heart. Antibodies raised to the cytoplasmic loop or the C-terminal regions of mCx30.2 recognized this protein in mouse heart as well as in HeLa cells transfected with wild-type mCx30.2 or mCx30.2 fused with enhanced green fluorescent protein (mCx30.2-EGFP). Immunofluorescence analyses of adult hearts yielded positive signals within the sinoatrial node, atrioventricular node, and A-V bundle of the cardiac conduction system. Dye transfer studies demonstrated that mCx30.2 and mCx30.2-EGFP channels discriminate poorly on the basis of charge, but do not allow permeation of tracers >400 Da. Both mCx30.2 and mCx30.2-EGFP gap junctional channels exhibited weak sensitivity to transjunctional voltage (Vj) and a single channel conductance of approximately 9 pS, which is the lowest among all members of the connexin family measured in HeLa cell transfectants. HeLa mCx30.2-EGFP transfectants when paired with cells expressing Cx40, Cx43, or Cx45 formed functional heterotypic gap junction channels that exhibited low unitary conductances (15 to 18 pS), rectifying open channel I-V relations and asymmetric Vj dependence. The electrical properties of homo- and hetero-typic junctions involving mCx30.2 may contribute to slow propagation velocity in nodal tissues and directional asymmetry of excitation spread in the AV nodal region.

Animals↗

PubMed2005

Connexin-based gap junction hemichannels: gating mechanisms.

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.

Animals↗

PubMed2004

Correlative studies of gating in Cx46 and Cx50 hemichannels and gap junction channels.

Transjunctional voltage (V(j)) gating of gap junction (GJ) channels formed of connexins has been proposed to occur by gating of the component hemichannels. We took advantage of the ability of Cx46 and Cx50 to function as unapposed hemichannels to identify gating properties intrinsic to hemichannels and how they contribute to gating of GJ channels. We show that Cx46 and Cx50 hemichannels contain two distinct gating mechanisms that generate reductions in conductance for both membrane polarities. At positive voltages, gating is similar in Cx46 and Cx50 hemichannels, primarily showing increased transitioning to long-lived substates. At negative voltages, Cx46 currents deactivate completely and the underlying single hemichannels exhibit transitions to a fully closed state. In contrast, Cx50 currents do not deactivate completely at negative voltages and the underlying single hemichannels predominantly exhibit transitions to various substates. Transitions to a fully closed state occur, but are infrequent. In the respective GJ channels, both forms of gating contribute to the reduction in conductance by V(j). However, examination of gating of mutant hemichannels and GJ channels in which the Asp at position 3 was replaced with Asn (D3N) showed that the positive hemichannel gate predominantly closes Cx50 GJs, whereas the negative hemichannel gate predominantly closes Cx46 GJs in response to V(j). We also report, for the first time, single Cx50 hemichannels in oocytes to be inwardly rectifying, high conductance channels (gamma = 470 pS). The antimalarial drug mefloquine, which selectively blocks Cx50 and not Cx46 GJs, shows the same selectivity in Cx50 and Cx46 hemichannels indicating that the actions of such uncoupling agents, like voltage gating, are intrinsic hemichannel properties.

Animals↗

PubMed2004

Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness.

Connexins are membrane proteins that assemble into gap-junction channels and are responsible for direct, electrical and metabolic coupling between connected cells. Here we describe an investigation of the properties of a recombinantly expressed recessive mutant of connexin 26 (Cx26), the V84L mutant, associated with deafness. Unlike other Cx26 mutations, V84L affects neither intracellular sorting nor electrical coupling, but specifically reduces permeability to the Ca(2+)-mobilizing messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)). Both the permeability to Lucifer Yellow and the unitary channel conductance of V84L-mutant channels are indistinguishable from those of the wild-type Cx26. Injection of Ins(1,4,5)P(3) into supporting cells of the rat organ of Corti, which abundantly express Cx26, ensues in a regenerative wave of Ca(2+) throughout the tissue. Blocking the gap junction communication abolishes wave propagation. We propose that the V84L mutation reduces metabolic coupling mediated by Ins(1,4,5)P(3) to an extent sufficient to impair the propagation of Ca(2+) waves and the formation of a functional syncytium. Our data provide the first demonstration of a specific defect of metabolic coupling and offer a mechanistic explanation for the pathogenesis of an inherited human disease.

Animals↗

PubMed2004

Gap junction channel gating.

Over the last two decades, the view of gap junction (GJ) channel gating has changed from one with GJs having a single transjunctional voltage-sensitive (V(j)-sensitive) gating mechanism to one with each hemichannel of a formed GJ channel, as well as unapposed hemichannels, containing two, molecularly distinct gating mechanisms. These mechanisms are termed fast gating and slow or 'loop' gating. It appears that the fast gating mechanism is solely sensitive to V(j) and induces fast gating transitions between the open state and a particular substate, termed the residual conductance state. The slow gating mechanism is also sensitive to V(j), but there is evidence that this gate may mediate gating by transmembrane voltage (V(m)), intracellular Ca(2+) and pH, chemical uncouplers and GJ channel opening during de novo channel formation. A distinguishing feature of the slow gate is that the gating transitions appear to be slow, consisting of a series of transient substates en route to opening and closing. Published reports suggest that both sensorial and gating elements of the fast gating mechanism are formed by transmembrane and cytoplamic components of connexins among which the N terminus is most essential and which determines gating polarity. We propose that the gating element of the slow gating mechanism is located closer to the central region of the channel pore and serves as a 'common' gate linked to several sensing elements that are responsive to different factors and located in different regions of the channel.

Animals↗

PubMed2004

Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue.

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 approximately 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.

Animals↗

PubMed2003

Connexins and apoptotic transformation.

We examined the influence of connexin (Cx) expression on the development of apoptosis in HeLa parental cells (coupling deficient cell line) and HeLa cells expressing wild-type Cx43 and Cxs fused with enhanced green fluorescent protein (EGFP). EGFP was attached to the C-terminus of Cx32 and Cx43, Cx32-EGFP and Cx43-EGFP, respectively, and to the N-terminus of Cx32, EGFP-Cx32. All fusion proteins assembled into junctional plaques (JPs) at areas of cell-cell contact, but only the C-terminal fusion proteins formed functional gap junction (GJ) channels as well as hemichannels. In each cell line, apoptosis was induced by treatment with various agents including anisomycin, camptothecin, cis-platinum, colchicine, cycloheximide, etoposide, staurosporin and taxol. Using fluorescence microscopy, time-lapse imaging and dual whole-cell voltage clamp techniques, we correlated the changes in functional properties of GJ channels and Cx distribution with the progression of apoptosis based on cells' labeling with acridine orange and ethidium bromide (EB). The early phase of apoptosis (a viable apoptotic (VA) state) was characterized by shrinkage of the cells and by increased internalization of JPs accompanied by decreased cell-cell coupling. The apoptotic reagents had no direct effect on electrical cell-cell coupling. Transformation from a VA to a nonviable apoptotic (NVA) state was faster in HeLa cells expressing Cx43 or Cx43-EGFP than in HeLa parental cells. The potent GJ uncoupler, octanol, slowed the transition of HelaCx43-EGFP cells into a NVA state. In the absence of apoptotic reagents, the rate of EB uptake was higher in HeLaCx43-EGFP than in HeLa parental cells consistent with the presence of open Cx43-EGFP hemichannels. However, in both cell lines the rate of EB uptake decreased proportionally during the development of apoptosis suggesting that membrane permeability ascribed to Cx hemichannels is reduced. Cells expressing Cx32-EGFP and EGFP-Cx32 demonstrate the same apoptotic patterns as HeLaCx43-EGFP and HeLa parental cells, respectively. Intracellular levels of ATP in HeLaCx43-EGFP cells were substantially lower than in HeLa parental cells, and ATP added to the medium abolished the accelerated transition from a VA to a NVA state in HeLaCx43-EGFP cells. In summary, Cx32 or Cx43 accelerates transformation of cells into a NVA state or secondary necrosis and this depends on the ability of Cxs to form functional GJ channels and hemichannels.

Adenosine Triphosphate↗

PubMed2003

Gating and regulation of connexin 43 (Cx43) hemichannels.

Connexin 43 (Cx43) nonjunctional or "unapposed" hemichannels can open under physiological or pathological conditions. We characterize hemichannels comprised of Cx43 or Cx43-EGFP (Cx43 with enhanced GFP fused to the C terminus) expressed in HeLa cells. Channel opening was induced at potentials greater than +60 mV. Open probability appeared to be very low. No comparable opening was detected in the parental, nontransfected HeLa cells. Conductance of fully open single hemichannels was approximately 220 pS, which is approximately double that of Cx43 cell-cell channels. Cx43 hemichannels exhibited two types of gating: fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS and slow transitions (>5 ms) between either open state and the fully closed state. Cx43-EGFP hemichannels exhibited only slow transitions (>5 ms) between closed and fully open states. These properties resemble those of the corresponding Cx43 and Cx43-EGFP cell-cell channels. Cx43 with EGFP on the N terminus (EGFP-Cx43) inserted into the surface and formed plaques but did not form hemichannels or cell-cell channels. Hemichannel blockers, 18beta-glycyrrhetinic acid or La3+, blocked depolarization-induced currents. Uptake of ethidium bromide (i) was faster in Cx43 and Cx43-EGFP than parental and EGFP-Cx43 cells, (ii) was directly correlated with Cx43-EGFP expression, (iii) was reduced by hemichannel blockers, and (iv) occurred at the same low rate in EGFP-Cx43 and parental cells. Although hemichannel opening was not detected electrophysiologically at the resting potential, infrequent or brief opening could account for ethidium bromide uptake. Opening of Cx43 hemichannels may mediate normal signaling or be deleterious.

Connexin 43↗

PubMed2003

New roles for astrocytes: gap junction hemichannels have something to communicate.

Gap junctions are clusters of aqueous channels that connect the cytoplasm of adjoining cells. Each cell contributes a hemichannel, or connexon, to each cell-cell channel. The cell-cell channels are permeable to relatively large molecules, and it was thought that opening of hemichannels to the extracellular space would kill cells through loss of metabolites, collapse of ionic gradients and influx of Ca(2+). Recent findings indicate that specific non-junctional hemichannels do open under both physiological and pathological conditions, and that opening is functional or deleterious depending on the situation. Most of these studies utilized cells in tissue culture that expressed a specific gap junction protein, connexin 43. Several such examples are reviewed here, with a particular focus on astrocytes.

Animals↗

PubMed2002

Coupling asymmetry of heterotypic connexin 45/ connexin 43-EGFP gap junctions: properties of fast and slow gating mechanisms.

Although fast and slow gating mechanisms have been described in gap junctions (GJs), their relative contributions to dependence on transjunctional voltage, V(j), is still unclear. We used cell lines expressing wild-type connexin 45 (Cx45) and connexin 43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gating in homo- and heterotypic GJs formed of these connexins. Macroscopically Cx45/Cx45 channels show high sensitivity to V(j). Cx45 channels demonstrate two types of gating: fast transitions between open and residual states and slow transitions between open and completely closed states. Single-channel conductance of the Cx45 channel is approximately 32 pS for the open state and approximately 4 pS for the residual state. Cx45/Cx43-EGFP heterotypic junctions exhibit very asymmetrical V(j) gating with the maximum junctional conductance shifted to V(j) positive on the Cx45 side. Conductance of single Cx45/Cx43-EGFP channels is approximately 55 pS for the open state and approximately 4 pS for the residual state, values consistent with the simple-series connection of Cx45 and Cx43-EGFP hemichannels. At V(j) = 0, the slow gate of many Cx45 hemichannels is closed in both homotypic Cx45/Cx45 and heterotypic Cx45/Cx43-EGFP junctions. Fast and slow V(j) gates of both Cx45 and Cx43 hemichannels close for relative negativity at their cytoplasmic end. Coupling mediated by Cx45/Cx43-EGFP junctions can exhibit asymmetry that can be strongly modulated by small changes in difference of holding potentials. Cx45/Cx43 junctions are likely to be found in brain and heart and may mediate rectifying electrical transmission or modulatable chemical communication.

Connexin 43↗

PubMed2002

Conductance and permeability of the residual state of connexin43 gap junction channels.

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell-cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (V(j)) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher V(j)s that are negative on the side of gated hemichannel. The degree of rectification increases when Cl(-) is replaced by Asp(-) and decreases when K(+) is replaced by TEA(+). These data are consistent with an increased anionic selectivity of the residual state. The V(j)-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast V(j)-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.

Animals↗

PubMed2002

Connexin-GFPs shed light on regulation of cell-cell communication by gap junctions.

Important roles for connexins have emerged from studies linking connexin mutations to human disease. Use of connexins tagged with GFP have provided a clearer picture of the mechanisms that govern connexin channel function and it is now evident that functional forms of connexin channel include cell-cell channels and unapposed hemichannels. Clustering appears to be a requirement for opening of cell-cell channels and suggests that dynamic changes occur in plaques (clusters) as they form and grow that are critical for channel function. In particular, recruitment or generation of 'silent' channels has gained support as a mechanism by which coupling can be dynamically regulated within formed plaques. Two distinct voltage sensitive gating mechanisms appear to be built-into each hemichannel, one putatively located at the cytoplasmic entrance and the other at the extracellular end, each differing in sensitivity, kinetics and degree of channel/hemichannel closure. The extracellular gate may also be that which opens unapposed hemichannels in the plasma membrane and be the final target of many known chemical agents that act as uncouplers of cell-cell communication. An understanding of the structural requirements for regulation via gating and clustering represents an important preclinical step in the design of therapeutic agents to treat disorders arising from connexin channel and hemichannel dysfunction.

Animals↗

PubMed2001

Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture.

Rat cortical astrocytes in pure culture are functionally coupled to neighboring cells via connexin (Cx) 43 gap junctions under ordinary conditions. Small fluorescent molecules such as Lucifer yellow (LY) pass between cell interiors via gap junctions, but do not enter the cells when externally applied. Subjecting rat and mouse cortical astrocytes to "chemical ischemia" by inhibition of glycolytic and oxidative metabolism induced permeabilization of cells to Lucifer yellow and ethidium bromide before loss of membrane integrity determined by dextran uptake and lactate dehydrogenase release. The gap junction blockers octanol and 18alpha-glycyrrhetinic acid markedly reduced dye uptake, suggesting that uptake was mediated by opening of unapposed hemichannels. Extracellular La(3+) also reduced dye uptake and delayed cell death. The purinergic blocker, oxidized ATP, was ineffective. Astrocytes isolated from mice with targeted deletion of the Cx43 coding DNA exhibited greatly reduced dye coupling and ischemia-induced dye uptake, evidence that dye uptake is mediated by Cx43 hemichannels. Dye coupling was reduced but not blocked by metabolic inhibition. Blockade of lipoxygenases or treatment with free radical scavengers reduced dye uptake by rat astrocytes, suggesting a role for arachidonic acid byproducts in hemichannel opening. Furthermore, permeabilization was accompanied by reduction in ATP levels and dephosphorylation of Cx43. Although hemichannel opening would tend to collapse electrochemical and metabolic gradients across the plasma membrane of dying cells, healthy cells might rescue dying cells by transfer of ions and essential metabolites via Cx43 gap junctions. Alternatively, dying astrocytes might compromise the health of neighboring cells via Cx43 gap junctions, thereby promoting the propagation of cell death.

Adenosine Triphosphate↗