Single channel properties of pannexin-1 and connexin-43 hemichannels and P2X7 receptors in astrocytes cultured from rodent spinal cords.

Astrocytes express surface channels involved in purinergic signaling. Among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) release ATP that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, that is, open and close under physiological and pathological conditions, single channel properties of Px1 HCs in astrocytes have not been defined. Here, we developed a dual voltage clamp technique in HeLa cells expressing human Px1-YFP, and then applied this system to rodent spinal astrocytes to compare their single channel properties with other surface channels, that is, Cx43 HCs and P2X7 receptors (P2X7Rs). Channels were recorded in cell attached patches and evoked with ramp cycles applied through another pipette in whole cell voltage clamp. The mean unitary conductances of Px1 HCs were comparable in HeLa Px1-YFP cells and spinal astrocytes, ~42 and ~48 pS, respectively. Based on their unitary conductance, voltage-dependence, and unitary activity after pharmacological and gene silencing, Px1 HCs in astrocytes could be distinguished from Cx43 HCs and P2X7Rs. Channel activity of Px1 HCs and P2X7Rs was greater than that of Cx43 HCs in control astrocytes during ramps. Unitary activity of Px1 HCs was decreased and that of Cx43 HCs and P2X7Rs increased in astrocytes treated with fibroblast growth factor 1 (FGF-1). In summary, we resolved single channel properties of three different surface channels involved in purinergic signaling in spinal astrocytes, which were differentially modulated by FGF-1, a growth factor involved in neurodevelopment, inflammation and repair.

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions.

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pHi) and cytosolic magnesium ion concentration ([Mg2+]i), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pHi and [Mg2+]i affect junctional conductance (gj) in an interdependent manner; in other words, intracellular acidification cause increase or decay in gj depending on whether [Mg2+]i is high or low, respectively, and intracellular alkalization cause reduction in gj independently of [Mg2+]i. Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pHi and [Mg2+]i. Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pHi and [Mg2+]i. Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg2+]i, while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36*E8Q lost the initial increase of gj at low [Mg2+]i and double mutation lost the sensitivity to high [Mg2+]i. These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg2+ and H+ ions.

An Acute Mouse Spinal Cord Slice Preparation for Studying Glial Activation ex vivo.

Pathological conditions such as amyotrophic lateral sclerosis, spinal cord injury and chronic pain are characterized by activation of astrocytes and microglia in spinal cord and have been modeled in rodents. In vivo imaging at cellular level in these animal models is limited due to the spinal cord's highly myelinated funiculi. The preparation of acute slices may offer an alternative and valuable strategy to collect structural and functional information in vitro from dorsal, lateral and ventral regions of spinal cord. Here, we describe a procedure for preparing acute slices from mouse spinal cord (Garré et al., 2016). This preparation should allow further understanding of how glial cells in spinal cord respond acutely to various inflammatory challenges.

Functional asymmetry and plasticity of electrical synapses interconnecting neurons through a 36-state model of gap junction channel gating.

We combined the Hodgkin-Huxley equations and a 36-state model of gap junction channel gating to simulate electrical signal transfer through electrical synapses. Differently from most previous studies, our model can account for dynamic modulation of junctional conductance during the spread of electrical signal between coupled neurons. The model of electrical synapse is based on electrical properties of the gap junction channel encompassing two fast and two slow gates triggered by the transjunctional voltage. We quantified the influence of a difference in input resistances of electrically coupled neurons and instantaneous conductance-voltage rectification of gap junctions on an asymmetry of cell-to-cell signaling. We demonstrated that such asymmetry strongly depends on junctional conductance and can lead to the unidirectional transfer of action potentials. The simulation results also revealed that voltage spikes, which develop between neighboring cells during the spread of action potentials, can induce a rapid decay of junctional conductance, thus demonstrating spiking activity-dependent short-term plasticity of electrical synapses. This conclusion was supported by experimental data obtained in HeLa cells transfected with connexin45, which is among connexin isoforms expressed in neurons. Moreover, the model allowed us to replicate the kinetics of junctional conductance under different levels of intracellular concentration of free magnesium ([Mg2+]i), which was experimentally recorded in cells expressing connexin36, a major neuronal connexin. We demonstrated that such [Mg2+]i-dependent long-term plasticity of the electrical synapse can be adequately reproduced through the changes of slow gate parameters of the 36-state model. This suggests that some types of chemical modulation of gap junctions can be executed through the underlying mechanisms of voltage gating. Overall, the developed model accounts for direction-dependent asymmetry, as well as for short- and long-term plasticity of electrical synapses. Our modeling results demonstrate that such complex behavior of the electrical synapse is important in shaping the response of coupled neurons.

Polyamines preserve connexin 43-mediated gap junctional communication during intracellular hypercalcemia and acidosis.

Changes in the regulation, formation, and gating of connexin-based gap junction channels occur in various disorders. It has been shown that H and Ca are involved in the regulation of gap junctional communication. Ischemia-induced intracellular acidification and Ca overload lead to closure of gap junctions and inhibit an exchange by ions and small molecules throughout the network of cells in the heart, brain, and other tissues. In this study, we examined the role of the polyamines in the regulation of connexin 43 (Cx43)-based gap junction channels under elevated intracellular concentrations of hydrogen ([H]i) and calcium ([Ca]i) ions. Experiments, conducted in Novikoff and A172 human glioblastoma cells, which endogenously express Cx43, showed that polyamines prevent downregulation of Cx43-mediated gap junctional communication caused by elevated [Ca]i and [H]i, accompanying ischemic and other pathological conditions. siRNA knockdown of Cx43 significantly reduces gap junctional communication, indicating that Cx43 gap junctions are the targets for spermine regulation.

FGF-1 Triggers Pannexin-1 Hemichannel Opening in Spinal Astrocytes of Rodents and Promotes Inflammatory Responses in Acute Spinal Cord Slices.

UNLABELLED: We show here that the growth factor FGF-1 is proinflammatory in the spinal cord and explore the inflammatory mechanisms. FGF-1 applied to rat spinal astrocytes in culture initiates calcium signaling and induces secretion of ATP that within minutes increases membrane permeability to ethidium (Etd(+)) and Ca(2+) by activating P2X7 receptors (P2X7Rs) that open pannexin hemichannels (Px1 HCs) that release further ATP; by 7 h treatment, connexin 43 hemichannels (Cx43 HCs) are also opened. In acute mouse spinal cord slices ex vivo, we found that FGF-1 treatment for 1 h increases the percentage of GFAP-positive astrocytes that show enhanced Px1 HC-mediated Etd(+) uptake. This response to FGF-1 was not observed in astrocytes in slices of cerebral cortex. FGF-1-induced dye uptake by astrocytes is prevented by BAPTA-AM or a phospholipase C (PLC) inhibitor. Furthermore, in spinal cord slices, P2X7R antagonists (BBG and A740003) and Px1 HC blockers ((10)Panx1 and carbenoxolone) prevent the increase in Etd(+) uptake by astrocytes, whereas Gap19, a selective Cx43 HC blocker, has no effect on dye uptake at this time. Microglia are not required for the increase in Etd(+) uptake by astrocytes induced by FGF-1, although they are activated by FGF-1 treatment. The morphological signs of microglia activation are inhibited by P2X7R antagonists and (10)Panx1 and are associated with elevated levels of proinflammatory cytokines in cord slices treated with FGF-1. The FGF-1 initiated cascade may play an important role in spinal cord inflammation in vivo SIGNIFICANCE STATEMENT: We find that FGF-1 elevates [Ca(2+)]i in spinal astrocytes, which causes vesicular release of ATP and activation of P2X7Rs to trigger opening of Px1 HCs, which release further ATP. This regenerative response occurs in astrocyte cultures and in acute spinal cord slices. In the latter, FGF-1 application promotes the activation of microglia and increases the production of proinflammatory cytokines through mechanisms depending on P2X7 receptors and Px1 HCs. This proinflammatory microenvironment may favor recruitment of leukocytes into the spinal cord and impacts negatively on neuronal structure and function in vivo Any step in these processes provides a potential therapeutic target for treatment of secondary damage in various spinal cord pathologies.

Stochastic Model of Gap Junctions Exhibiting Rectification and Multiple Closed States of Slow Gates.

Gap-junction (GJ) channels formed from connexin (Cx) proteins provide direct pathways for electrical and metabolic cell-cell communication. Earlier, we developed a stochastic 16-state model (S16SM) of voltage gating of the GJ channel containing two pairs of fast and slow gates, each operating between open (o) and closed (c) states. However, experimental data suggest that gates may in fact contain two or more closed states. We developed a model in which the slow gate operates according to a linear reaction scheme, o↔c1↔c2, where c1 and c2 are initial-closed and deep-closed states that both close the channel fully, whereas the fast gate operates between the open state and the closed state and exhibits a residual conductance. Thus, we developed a stochastic 36-state model (S36SM) of GJ channel gating that is sensitive to transjunctional voltage (Vj). To accelerate simulation and eliminate noise in simulated junctional conductance (gj) records, we transformed an S36SM into a Markov chain 36-state model (MC36SM) of GJ channel gating. This model provides an explanation for well-established experimental data, such as delayed gj recovery after Vj gating, hysteresis of gj-Vj dependence, and the low ratio of functional channels to the total number of GJ channels clustered in junctional plaques, and it has the potential to describe chemically mediated gating, which cannot be reflected using an S16SM. The MC36SM, when combined with global optimization algorithms, can be used for automated estimation of gating parameters including probabilities of c1↔c2 transitions from experimental gj-time and gj-Vj dependencies.

Reverberation of excitation in neuronal networks interconnected through voltage-gated gap junction channels.

We combined Hodgkin-Huxley equations and gating models of gap junction (GJ) channels to simulate the spread of excitation in two-dimensional networks composed of neurons interconnected by voltage-gated GJs. Each GJ channel contains two fast and slow gates, each exhibiting current-voltage (I-V) rectification and gating properties that depend on transjunctional voltage (Vj). The data obtained show how junctional conductance (gj), which is necessary for synchronization of the neuronal network, depends on its size and the intrinsic firing rate of neurons. A phase shift between action potentials (APs) of neighboring neurons creates bipolar, short-lasting Vj spikes of approximately ± 100 mV that induce Vj gating, leading to a small decay of gj, which can accumulate into larger decays during bursting activity of neurons. We show that I-V rectification of GJs in local regions of the two-dimensional network of neurons can lead to unidirectional AP transfer and consequently to reverberation of excitation. This reverberation can be initiated by a single electrical pulse and terminated by a low-amplitude pulse applied in a specific window of reverberation cycle. Thus, the model accounts for the influence of dynamically modulatable electrical synapses in shaping the function of a neuronal network and the formation of reverberation, which, as proposed earlier, may be important for the development of short-term memory and its consolidation into long-term memory.

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors.

Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca(2+)-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.

Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels.

Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell-cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions.

Application of stochastic automata networks for creation of continuous time Markov chain models of voltage gating of gap junction channels.

The primary goal of this work was to study advantages of numerical methods used for the creation of continuous time Markov chain models (CTMC) of voltage gating of gap junction (GJ) channels composed of connexin protein. This task was accomplished by describing gating of GJs using the formalism of the stochastic automata networks (SANs), which allowed for very efficient building and storing of infinitesimal generator of the CTMC that allowed to produce matrices of the models containing a distinct block structure. All of that allowed us to develop efficient numerical methods for a steady-state solution of CTMC models. This allowed us to accelerate CPU time, which is necessary to solve CTMC models, ~20 times.

Molecular determinants of magnesium-dependent synaptic plasticity at electrical synapses formed by connexin36.

Neuronal gap junction (GJ) channels composed of connexin36 (Cx36) play an important role in neuronal synchronization and network dynamics. Here we show that Cx36-containing electrical synapses between inhibitory neurons of the thalamic reticular nucleus are bidirectionally modulated by changes in intracellular free magnesium concentration ([Mg(2+)]i). Chimeragenesis demonstrates that the first extracellular loop of Cx36 contains a Mg(2+)-sensitive domain, and site-directed mutagenesis shows that the pore-lining residue D47 is critical in determining high Mg(2+)-sensitivity. Single-channel analysis of Mg(2+)-sensitive chimeras and mutants reveals that [Mg(2+)]i controls the strength of electrical coupling mostly via gating mechanisms. In addition, asymmetric transjunctional [Mg(2+)]i induces strong instantaneous rectification, providing a novel mechanism for electrical rectification in homotypic Cx36 GJs. We suggest that Mg(2+)-dependent synaptic plasticity of Cx36-containing electrical synapses could underlie neuronal circuit reconfiguration via changes in brain energy metabolism that affects neuronal levels of intracellular ATP and [Mg(2+)]i.

The Connexin40A96S mutation from a patient with atrial fibrillation causes decreased atrial conduction velocities and sustained episodes of induced atrial fibrillation in mice.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and a major cause of stroke. In the mammalian heart the gap junction proteins connexin40 (Cx40) and connexin43 (Cx43) are strongly expressed in the atrial myocardium mediating effective propagation of electrical impulses. Different heterozygous mutations in the coding region for Cx40 were identified in patients with AF. We have generated transgenic Cx40A96S mice harboring one of these mutations, the loss-of-function Cx40A96S mutation, as a model for atrial fibrillation. Cx40A96S mice were characterized by immunochemical and electrophysiological analyses. Significantly reduced atrial conduction velocities and strongly prolonged episodes of atrial fibrillation were found after induction in Cx40A96S mice. Analyses of the gating properties of Cx40A96S channels in cultured HeLa cells also revealed significantly lower junctional conductance and enhanced sensitivity voltage gating of Cx40A96S in comparison to Cx40 wild-type gap junctions. This is caused by reduced open probabilities of Cx40A96S gap junction channels, while single channel conductance remained the same. Similar to the corresponding patient, heterozygous Cx40A96S mice revealed normal expression levels and localization of the Cx40 protein. We conclude that heterozygous Cx40A96S mice exhibit prolonged episodes of induced atrial fibrillation and severely reduced atrial conduction velocities similar to the corresponding human patient.

Molecular and functional asymmetry at a vertebrate electrical synapse.

Electrical synapses are abundant in the vertebrate brain, but their functional and molecular complexities are still poorly understood. We report here that electrical synapses between auditory afferents and goldfish Mauthner cells are constructed by apposition of hemichannels formed by two homologs of mammalian connexin 36 (Cx36) and that, while Cx35 is restricted to presynaptic hemiplaques, Cx34.7 is restricted to postsynaptic hemiplaques, forming heterotypic junctions. This molecular asymmetry is associated with rectification of electrical transmission that may act to promote cooperativity between auditory afferents. Our data suggest that, in similarity to pre- and postsynaptic sites at chemical synapses, one side in electrical synapses should not necessarily be considered the mirror image of the other. While asymmetry based on the presence of two Cx36 homologs is restricted to teleost fish, it might also be based on differences in posttranslational modifications of individual connexins or in the complement of gap junction-associated proteins.

Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels.

The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function.

Intracellular magnesium-dependent modulation of gap junction channels formed by neuronal connexin36.

Gap junction (GJ) channels composed of Connexin36 (Cx36) are widely expressed in the mammalian CNS and form electrical synapses between neurons. Here we describe a novel modulatory mechanism of Cx36 GJ channels dependent on intracellular free magnesium ([Mg(2+)]i). We examined junctional conductance (gj) and its dependence on transjunctional voltage (Vj) at different [Mg(2+)]i in cultures of HeLa or N2A cells expressing Cx36. We found that Cx36 GJs are partially inhibited at resting [Mg(2+)]i. Thus, gj can be augmented or reduced by lowering or increasing [Mg(2+)]i, respectively. Similar changes in gj and Vj-gating were observed using MgATP or K2ATP in pipette solutions, which increases or decreases [Mg(2+)]i, respectively. Changes in phosphorylation of Cx36 or in intracellular free calcium concentration were not involved in the observed Mg(2+)-dependent modulation of gj. Magnesium ions permeate the channel and transjunctional asymmetry in [Mg(2+)]i resulted in asymmetric Vj-gating. The gj of GJs formed of Cx26, Cx32, Cx43, Cx45, and Cx47 was also reduced by increasing [Mg(2+)]i, but was not increased by lowering [Mg(2+)]i; single-channel conductance did not change. We showed that [Mg(2+)]i affects both open probability and the number of functional channels, likely through binding in the channel lumen. Finally, we showed that Cx36-containing electrical synapses between neurons of the trigeminal mesencephalic nucleus in rat brain slices are similarly affected by changes in [Mg(2+)]i. Thus, this novel modulatory mechanism could underlie changes in neuronal synchronization under conditions in which ATP levels, and consequently [Mg(2+)]i, are modified.

Regulation of connexin36 gap junction channels by n-alkanols and arachidonic acid.

We examined junctional conductance (gj) and its dependence on transjunctional voltage in gap junction (GJ) channels formed of wild-type connexin36 (Cx36) or its fusion form with green fluorescent protein (Cx36-EGFP) transfected in HeLa cells or endogenously expressed in primary culture of pancreatic ß-cells. Only a very small fraction (∼0.8%) of Cx36-EGFP channels assembled into junctional plaques of GJs were open under control conditions. We found that short carbon chain n-alkanols (SCCAs) increased gj, while long carbon chain n-alkanols resulted in full uncoupling; cutoff is between heptanol and octanol. The fraction of functional channels and gj increased several fold under an exposure to SCCAs, or during reduction of endogenous levels of arachidonic acid (AA) by exposure to fatty acid-free BSA or cytosolic phospholipase A2 inhibitors. Moreover, uncoupling caused by exogenously applied AA can be rescued by BSA, which binds AA and other polyunsaturated fatty acids (PUFAs), but not by BSA modified with 1,2-cyclohexanedione, which does not bind AA and other PUFAs. We propose that under control conditions, Cx36 GJ channels in HeLa transfectants and ß-cells are inhibited by endogenous AA, which stabilizes a closed conformational state of the channel that leads to extremely low fraction of functional channels. In addition, SCCAs increase gj by interfering with endogenous AA-dependent inhibition, increasing open probability and the fraction of functional channels.

Paracrine signaling through plasma membrane hemichannels.

Plasma membrane hemichannels composed of connexin (Cx) proteins are essential components of gap junction channels but accumulating evidence suggests functions of hemichannels beyond the communication provided by junctional channels. Hemichannels not incorporated into gap junctions, called unapposed hemichannels, can open in response to a variety of signals, electrical and chemical, thereby forming a conduit between the cell's interior and the extracellular milieu. Open hemichannels allow the bidirectional passage of ions and small metabolic or signaling molecules of below 1-2kDa molecular weight. In addition to connexins, hemichannels can also be formed by pannexin (Panx) proteins and current evidence suggests that Cx26, Cx32, Cx36, Cx43 and Panx1, form hemichannels that allow the diffusive release of paracrine messengers. In particular, the case is strong for ATP but substantial evidence is also available for other messengers like glutamate and prostaglandins or metabolic substances like NAD(+) or glutathione. While this field is clearly in expansion, evidence is still lacking at essential points of the paracrine signaling cascade that includes not only messenger release, but also downstream receptor signaling and consequent functional effects. The data available at this moment largely derives from in vitro experiments and still suffers from the difficulty of separating the functions of connexin-based hemichannels from gap junctions and from pannexin hemichannels. However, messengers like ATP or glutamate have universal roles in the body and further defining the contribution of hemichannels as a possible release pathway is expected to open novel avenues for better understanding their contribution to a variety of physiological and pathological processes. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury.

Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell-cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT-CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.

Connexin mimetic peptides inhibit Cx43 hemichannel opening triggered by voltage and intracellular Ca2+ elevation.

Connexin mimetic peptides (CxMPs), such as Gap26 and Gap27, are known as inhibitors of gap junction channels but evidence is accruing that these peptides also inhibit unapposed/non-junctional hemichannels (HCs) residing in the plasma membrane. We used voltage clamp studies to investigate the effect of Gap26/27 at the single channel level. Such an approach allows unequivocal identification of HC currents by their single channel conductance that is typically ~220 pS for Cx43. In HeLa cells stably transfected with Cx43 (HeLa-Cx43), Gap26/27 peptides inhibited Cx43 HC unitary currents over minutes and increased the voltage threshold for HC opening. By contrast, an elevation of intracellular calcium ([Ca(2+)](i)) to 200-500 nM potentiated the unitary HC current activity and lowered the voltage threshold for HC opening. Interestingly, Gap26/27 inhibited the Ca(2+)-potentiated HC currents and prevented lowering of the voltage threshold for HC opening. Experiments on isolated pig ventricular cardiomyocytes, which display strong endogenous Cx43 expression, demonstrated voltage-activated unitary currents with biophysical properties of Cx43 HCs that were inhibited by small interfering RNA targeting Cx43. As observed in HeLa-Cx43 cells, HC current activity in ventricular cardiomyocytes was potentiated by [Ca(2+)](i) elevation to 500 nM and was inhibited by Gap26/27. Our results indicate that under pathological conditions, when [Ca(2+)](i) is elevated, Cx43 HC opening is promoted in cardiomyocytes and CxMPs counteract this effect.