SGK1.1 Reduces Kainic Acid-Induced Seizure Severity and Leads to Rapid Termination of Seizures.

Approaches to control epilepsy, one of the most important idiopathic brain disorders, are of great importance for public health. We have previously shown that in sympathetic neurons the neuronal isoform of the serum and glucocorticoid-regulated kinase (SGK1.1) increases the M-current, a well-known target for seizure control. The effect of SGK1.1 activation on kainate-induced seizures and neuronal excitability was studied in transgenic mice that express a permanently active form of the kinase, using electroencephalogram recordings and electrophysiological measurements in hippocampal brain slices. Our results demonstrate that SGK1.1 activation leads to reduced seizure severity and lower mortality rates following status epilepticus, in an M-current-dependent manner. EEG is characterized by reduced number, shorter duration, and early termination of kainate-induced seizures in the hippocampus and cortex. Hippocampal neurons show decreased excitability associated to increased M-current, without altering basal synaptic transmission or other neuronal properties. Altogether, our results reveal a novel and selective anticonvulsant pathway that promptly terminates seizures, suggesting that SGK1.1 activation can be a potent factor to secure the brain against permanent neuronal damage associated to epilepsy.

Directional coupling of oligodendrocyte connexin-47 and astrocyte connexin-43 gap junctions.

Intercellular communication via gap junction channels between oligodendrocytes and between astrocytes as well as between these cell types is essential to maintain the integrity of myelin in the central nervous system. Oligodendrocyte gap junction connexin-47 (Cx47) is a key element in this crosstalk and indeed, mutations in human Cx47 cause severe myelin disorders. However, the permeation properties of channels of Cx47 alone and in heterotypic combination with astrocyte Cx43 remain unclear. We show here that Cx47 contains three extra residues at 5' amino-terminus that play a critical role in the channel pore structure and account for relative low ionic conductivity, cationic permselectivity and voltage-gating properties of oligodendrocyte-oligodendrocyte Cx47 channels. Regarding oligodendrocyte-astrocyte coupling, heterotypic channels formed by Cx47 with Cx43 exhibit ionic and chemical rectification, which creates a directional diffusion barrier for the movement of ions and larger negatively charged molecules from cells expressing Cx47 to those with Cx43. The restrictive permeability of Cx47 channels and the diffusion barrier of Cx47-Cx43 channels was abolished by a mutation associated with leukodystrophy, the Cx47P90S, suggesting a novel pathogenic mechanism underlying myelin disorders that involves alterations in the panglial permeation.

Fast skeletal myofibers of mdx mouse, model of Duchenne muscular dystrophy, express connexin hemichannels that lead to apoptosis.

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) show numerous alterations including inflammation, apoptosis, and necrosis of myofibers. However, the molecular mechanism that explains these changes remains largely unknown. Here, the involvement of hemichannels formed by connexins (Cx HCs) was evaluated in skeletal muscle of mdx mouse model of DMD. Fast myofibers of mdx mice were found to express three connexins (39, 43 and 45) and high sarcolemma permeability, which was absent in myofibers of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice (deficient in skeletal muscle Cx43/Cx45 expression). These myofibers did not show elevated basal intracellular free Ca(2+) levels, immunoreactivity to phosphorylated p65 (active NF-κB), eNOS and annexin V/active Caspase 3 (marker of apoptosis) but presented dystrophin immunoreactivity. Moreover, muscles of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice exhibited partial decrease of necrotic features (big cells and high creatine kinase levels). Accordingly, these muscles showed similar macrophage infiltration as control mdx muscles. Nonetheless, the hanging test performance of mdx Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice was significantly better than that of control mdx Cx43(fl/fl)Cx45(fl/fl) mice. All three Cxs found in skeletal muscles of mdx mice were also detected in fast myofibers of biopsy specimens from patients with muscular dystrophy. Thus, reduction of Cx expression and/or function of Cx HCs may be potential therapeutic approaches to abrogate myofiber apoptosis in DMD.

First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit ß (ß-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and ß-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality.

Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells.

Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21(cip1.) and p16(INK4a), and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors.

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Role of connexin 32 hemichannels in the release of ATP from peripheral nerves.

Extracellular purines elicit strong signals in the nervous system. Adenosine-5'-triphosphate (ATP) does not spontaneously cross the plasma membrane, and nervous cells secrete ATP by exocytosis or through plasma membrane proteins such as connexin hemichannels. Using a combination of imaging, luminescence and electrophysiological techniques, we explored the possibility that Connexin 32 (Cx32), expressed in Schwann cells (SCs) myelinating the peripheral nervous system could be an important source of ATP in peripheral nerves. We triggered the release of ATP in vivo from mice sciatic nerves by electrical stimulation and from cultured SCs by high extracellular potassium concentration-evoked depolarization. No ATP was detected in the extracellular media after treatment of the sciatic nerve with Octanol or Carbenoxolone, and ATP release was significantly inhibited after silencing Cx32 from SCs cultures. We investigated the permeability of Cx32 to ATP by expressing Cx32 hemichannels in Xenopus laevis oocytes. We found that ATP release is coupled to the inward tail current generated after the activation of Cx32 hemichannels by depolarization pulses, and it is sensitive to low extracellular calcium concentrations. Moreover, we found altered ATP release in mutated Cx32 hemichannels related to the X-linked form of Charcot-Marie-Tooth disease, suggesting that purinergic-mediated signaling in peripheral nerves could underlie the physiopathology of this neuropathy.

Neural bases for the genesis and CO2 therapy of periodic Cheyne-Stokes breathing in neonatal male connexin-36 knockout mice.

Periodic Cheyne-Stokes breathing (CSB) oscillating between apnea and crescendo-decrescendo hyperpnea is the most common central apnea. Currently, there is no proven therapy for CSB, probably because the fundamental pathophysiological question of how the respiratory center generates this form of breathing instability is still unresolved. Therefore, we aimed to determine the respiratory motor pattern of CSB resulting from the interaction of inspiratory and expiratory oscillators and identify the neural mechanism responsible for breathing regularization induced by the supplemental CO2 administration. Analysis of the inspiratory and expiratory motor pattern in a transgenic mouse model lacking connexin-36 electrical synapses, the neonatal (P14) Cx36 knockout male mouse, with a persistent CSB, revealed that the reconfigurations recurrent between apnea and hyperpnea and vice versa result from cyclical turn on/off of active expiration driven by the expiratory oscillator, which acts as a master pacemaker of respiration and entrains the inspiratory oscillator to restore ventilation. The results also showed that the suppression of CSB by supplemental 12% CO2 in inhaled air is due to the stabilization of coupling between expiratory and inspiratory oscillators, which causes the regularization of respiration. CSB rebooted after washout of CO2 excess when the inspiratory activity depressed again profoundly, indicating that the disability of the inspiratory oscillator to sustain ventilation is the triggering factor of CSB. Under these circumstances, the expiratory oscillator activated by the cyclic increase of CO2 behaves as an "anti-apnea" center generating the crescendo-decrescendo hyperpnea and periodic breathing. The neurogenic mechanism of CSB identified highlights the plasticity of the two-oscillator system in the neural control of respiration and provides a rationale base for CO2 therapy.

Contribution of non-selective membrane channels and receptors in epilepsy.

Overcoming refractory epilepsy's resistance to the combination of antiepileptic drugs (AED), mitigating side effects, and preventing sudden unexpected death in epilepsy are critical goals for therapy of this disorder. Current therapeutic strategies are based primarily on neurocentric mechanisms, overlooking the participation of astrocytes and microglia in the pathophysiology of epilepsy. This review is focused on a set of non-selective membrane channels (permeable to ions and small molecules), including channels and ionotropic receptors of neurons, astrocytes, and microglia, such as: the hemichannels formed by Cx43 and Panx1; the purinergic P2X7 receptors; the transient receptor potential vanilloid (TRPV1 and TRPV4) channels; calcium homeostasis modulators (CALHMs); transient receptor potential canonical (TRPC) channels; transient receptor potential melastatin (TRPM) channels; voltage-dependent anion channels (VDACs) and volume-regulated anion channels (VRACs), which all have in common being activated by epileptic activity and the capacity to exacerbate seizure intensity. Specifically, we highlight evidence for the activation of these channels/receptors during epilepsy including neuroinflammation and oxidative stress, discuss signaling pathways and feedback mechanisms, and propose the functions of each of them in acute and chronic epilepsy. Studying the role of these non-selective membrane channels in epilepsy and identifying appropriate blockers for one or more of them could provide complementary therapies to better alleviate the disease.

Regulation of neuronal connexin-36 channels by pH.

Neurotransmission through electrical synapses plays an important role in the spike synchrony among neurons and oscillation of neuronal networks. Indeed, electrical transmission has been implicated in the hypersynchronous electrical activity of epilepsy. We have investigated the influence of intracellular pH on the strength of electrical coupling mediated by connexin36 (Cx36), the principal gap junction protein in the electrical synapses of vertebrates. In striking contrast to other connexin isoforms, the activity of Cx36 channels decreases following alkalosis rather than acidosis when it is expressed in Xenopus oocytes and N2A cells. This uncoupling of Cx36 channels upon alkalinization occurred in the vertebrate orthologues analyzed (human, mouse, chicken, perch, and skate). While intracellular acidification caused a mild or moderate increase in the junctional conductance of virtually all these channels, the coupling of the skate Cx35 channel was partially blocked by acidosis. The mutational analysis suggests that the Cx36 channels may contain two gating mechanisms operating with opposing sensitivity to pH. One gate, the dominant mechanism, closes for alkalosis and it probably involves an interaction between the C- and N-terminal domains, while a secondary acid sensing gate only causes minor, albeit saturating, changes in coupling following acidosis and alkalosis. Thus, we conclude that neuronal Cx36 channels undergo unique regulation by pH(i) since their activity is inhibited by alkalosis rather than acidosis. These data provide a novel basis to define the relevance and consequences of the pH-dependent modulation of Cx36 synapses under physiological and pathological conditions.

Respiratory disturbances and high risk of sudden death in the neonatal connexin-36 knockout mouse.

Neural circuits at the brainstem involved in the central generation of the motor patterns of respiration and cardiorespiratory chemoreflexes organize as cell assemblies connected by chemical and electrical synapses. However, the role played by the electrical connectivity mainly mediated by connexin36 (Cx36), which expression reaches peak value during the postnatal period, is still unknown. To address this issue, we analyzed here the respiratory phenotype of a mouse strain devoid constitutively of Cx36 at P14. Male Cx36-knockout mice at rest showed respiratory instability of variable degree, including a periodic Cheyne-Stokes breathing. Moreover, mice lacking Cx36 exhibited exacerbated chemoreflexes to normoxic and hypoxic hypercapnia characterized by a stronger inspiratory/expiratory coupling due to an increased sensitivity to CO2 . Deletion of Cx36 also impaired the generation of the recurrent episodes of transient bradycardia (ETBs) evoked during hypercapnic chemoreflexes; these EBTs constituted a powerful mechanism of cardiorespiratory coupling capable of improving alveolar gaseous exchange under hypoxic hypercapnia conditions. Approximately half of the homo- and heterozygous Cx36KO, but none WT, mice succumbed by respiratory arrest when submitted to hypoxia-hypercapnia, the principal exogenous stressor causing sudden infant death syndrome (SIDS). The early suppression of EBTs, which worsened arterial O2  saturation, and the generation of a paroxysmal generalized clonic-tonic activity, which provoked the transition from eupneic to gasping respiration, were the critical events causing sudden death in the Cx36KO mice. These results indicate that Cx36 expression plays a pivotal role in respiratory control, cardiorespiratory coordination, and protection against SIDS at the postnatal period.

Molecular basis of voltage dependence of connexin channels: an integrative appraisal.

The importance of electrical and molecular signaling through connexin (Cx) channels is now widely recognized. The transfer of ions and other small molecules between adjacent cells is regulated by multiple stimuli, including voltage. Indeed, Cx channels typically exhibit complex voltage sensitivity. Most channels are sensitive to the voltage difference between the cell interiors (or transjunctional voltage, V(j)), while other channels are also sensitive to absolute inside-outside voltage (i.e., the membrane potential, V(m)). The first part of this review is focused on the description of the distinct forms of voltage sensitivity and the gating mechanisms that regulate hemichannel activity, both individually and as components of homotypic and heterotypic gap junctions. We then provide an up to date and precise picture of the molecular and structural aspects of how V(j) and V(m) are sensed, and how they, therefore, control channel opening and closing. Mutagenic strategies coupled with structural, biochemical and electrophysical studies are providing significant insights into how distinct forms of voltage dependence are brought about. The emerging picture indicates that Cx channels can undergo transitions between multiple conductance states driven by distinct voltage-gating mechanisms. Each hemichannel may contain a set of two V(j) gates, one fast and one slow, which mediate the transitions between the main open state to the residual state and to the fully closed state, respectively. Eventually, a V(m) gate regulates channel transitions between the open and closed states. Clusters of charged residues within separate domains of the Cx molecule have been identified as integral parts of the V(j) and V(m) sensors. The charges at the first positions of the amino terminal cytoplasmic domain determine the magnitude and polarity of the sensitivity to fast V(j)-gating, as well as contributing to the V(j)-rectifying properties of ion permeation. Additionally, important advances have been made in identifying the conformational rearrangements responsible for fast V(j)-gating transitions to the residual state in the Cx43 channel. These changes involve an intramolecular particle-receptor interaction between the carboxy terminal domain and the cytoplasmic loop.

A four-state Markov model of sleep-wakefulness dynamics along light/dark cycle in mice.

Behavioral states alternate between wakefulness (wk), rapid eye movement (rem) and non-rem (nrem) sleep at time scale of hours i.e., light and dark cycle rhythms and from several tens of minutes to seconds (i.e., brief awakenings during sleep). Using statistical analysis of bout duration, Markov chains of sleep-wk dynamics and quantitative EEG analysis, we evaluated the influence of light/dark (ld) changes on brain function along the sleep-wk cycle. Bout duration (bd) histograms and Kaplan-Meier (km) survival curves of wk showed a bimodal statistical distribution, suggesting that two types of wk do exist: brief-wk (wkb) and long-wk (wkl). Light changes modulated specifically wkl bouts, increasing its duration during active/dark period. In contrast, wkb, nrem and rem bd histograms and km curves did not change significantly along ld cycle. Hippocampal eeg of both types of wk were different: in comparison wkb showed a lower spectral power in fast gamma and fast theta bands and less emg tone. After fitting a four-states Markov chain to mice hypnograms, moreover in states transition probabilities matrix was found that: in dark/active period, state-maintenance probability of wkl increased, and probability of wkl to nrem transition decreased; the opposite was found in light period, favoring the hypothesis of the participation of brief wk into nrem-rem intrinsic sleep cycle, and the role of wkl in SWS homeostasis. In conclusion, we propose an extended Markov model of sleep using four stages (wkl, nrem, rem, wkb) as a fully adequate model accounting for both modulation of sleep-wake dynamics based on the differential regulation of long-wk (high gamma/theta) epochs during dark and light phases.

Species specificity of mammalian connexin-26 to form open voltage-gated hemichannels.

Mutations of connexin-26 (Cx26) cause nonsyndromic hearing loss and other syndromes affecting ectoderm-derived tissues. While the exact mechanisms underlying these diseases remain elusive, Cx's are generally considered to mediate cell-to-cell communication by forming gap junction channels. We show here that unlike rat Cx26, human and sheep Cx26 form voltage-gated hemichannels when expressed in oocytes and Neuro2A cells. A single evolutionary amino acidic change at position 159 of the rodent protein, the replacement of aspartic acid with asparagine in the human and sheep proteins, accounts for this species specificity. At the resting potential and in normal millimolar extracellular calcium, open human Cx26 hemichannels can be detected both electrophysiologically and by dye uptake, although they did not affect cell viability. These hemichannels opened at approximately -50 mV and their activation increased by depolarization until they inactivate at positive membrane potentials. Single-channel analysis revealed that activation and inactivation involved two distinct voltage gating mechanisms and that the fully open hemichannel displays a conductance twice that of the intercellular channel. The existence of a hemichannel that opens under physiological control of the membrane potential may have important implications for the normal and pathological activity of Cx26 in humans, particularly with respect to hearing and the epidermis.

Long-term dynamics of somatosensory activity in a stroke model of distal middle cerebral artery oclussion.

A constant challenge in experimental stroke is the use of appropriate tests to identify signs of recovery and adverse effects linked to a particular therapy. In this study, we used a long-term longitudinal approach to examine the functional brain changes associated with cortical infarction in a mouse model induced by permanent ligation of the middle cerebral artery (MCA). Sensorimotor function and somatosensory cortical activity were evaluated with fault-foot and forelimb asymmetry tests in combination with somatosensory evoked potentials. The stroke mice exhibited both long-term deficits in the functional tests and impaired responses in the infarcted and intact hemispheres after contralateral and ipsilateral forepaw stimulation. In the infarcted hemisphere, reductions in the amplitudes of evoked responses were detected after contralateral and ipsilateral stimulation. In the intact hemisphere, and similar to cortical stroke patients, a gradual hyperexcitability was observed after contralateral stimulation but no parallel evidence of a response was detected after ipsilateral stimulation. Our results suggest the existence of profound and persistent changes in the somatosensory cortex in this specific mouse cortical stroke model. The study of evoked potentials constitutes a feasible and excellent tool for evaluating the fitness of the somatosensory cortex in relation to functional recovery after preclinical therapeutic intervention.

Connexins: Intercellular Signal Transmitters in Lymphohematopoietic Tissues.

Life-long hematopoietic demands are met by a pool of hematopoietic stem cells (HSC) with self-renewal and multipotential differentiation ability. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment control HSC activity. Cell-to-cell communication through connexin (Cx) containing gap junctions (GJs) allows pluricellular coordination and synchronization through transfer of small molecules with messenger activity. Hematopoietic and surrounding nonhematopoietic cells communicate each other through GJs, which regulate fetal and postnatal HSC content and function in hematopoietic tissues. Traffic of HSC between peripheral blood and BM is also dependent on Cx proteins. Cx mutations are associated with human disease and hematopoietic dysfunction and Cx signaling may represent a target for therapeutic intervention. In this review, we illustrate and highlight the importance of Cxs in the regulation of hematopoietic homeostasis under normal and pathological conditions.

Rats with minimal hepatic encephalopathy show reduced cGMP-dependent protein kinase activity in hypothalamus correlating with circadian rhythms alterations.

Patients with liver cirrhosis show disturbances in sleep and in its circadian rhythms which are an early sign of minimal hepatic encephalopathy (MHE). The mechanisms of these disturbances are poorly understood. Rats with porta-caval shunt (PCS), a model of MHE, show sleep disturbances reproducing those of cirrhotic patients. The aims of this work were to characterize the alterations in circadian rhythms in PCS rats and analyze the underlying mechanisms. To reach these aims, we analyzed in control and PCS rats: (a) daily rhythms of spontaneous and rewarding activity and of temperature, (b) timing of the onset of activity following turning-off the light, (c) synchronization to light after a phase advance and (d) the molecular mechanisms contributing to these alterations in circadian rhythms. PCS rats show altered circadian rhythms of spontaneous and rewarding activities (wheel running). PCS rats show more rest bouts during the active phase, more errors in the onset of motor activity and need less time to re-synchronize after a phase advance than control rats. Circadian rhythm of body temperature is also slightly altered in PCS rats. The internal period length (tau) of circadian rhythm of motor activity is longer in PCS rats. We analyzed some mechanisms by which hypothalamus modulate circadian rhythms. PCS rats show increased content of cGMP in hypothalamus while the activity of cGMP-dependent protein kinase was reduced by 41% compared to control rats. Altered cGMP-PKG pathway in hypothalamus would contribute to altered circadian rhythms and synchronization to light.

Regulation of connexin hemichannel activity by membrane potential and the extracellular calcium in health and disease.

Connexins are thought to solely mediate cell-to-cell communication by forming gap junction channels composed of two membrane-spanning hemichannels positioned end-to-end. However, many if not all connexin isoforms also form functional hemichannels (i.e., the precursors of complete channels) that mediate the rapid exchange of ions, second messengers and metabolites between the cell interior and the interstitial space. Electrical and molecular signaling via connexin hemichannels is now widely recognized to be important in many physiological scenarios and pathological conditions. Indeed, mutations in connexins that alter hemichannel function have been implicated in several diseases. Here, we present a comprehensive overview of how hemichannel activity is tightly regulated by membrane potential and the external calcium concentration. In addition, we discuss the genetic mutations known to alter hemichannel function and their deleterious effects, of which a better understanding is necessary to develop novel therapeutic approaches for diseases caused by hemichannel dysfunction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Effect of charge substitutions at residue his-142 on voltage gating of connexin43 channels.

Previous studies indicate that the carboxyl terminal of connexin43 (Cx43CT) is involved in fast transjunctional voltage gating. Separate studies support the notion of an intramolecular association between Cx43CT and a region of the cytoplasmic loop (amino acids 119-144; referred to as "L2"). Structural analysis of L2 shows two alpha-helical domains, each with a histidine residue in its sequence (H126 and H142). Here, we determined the effect of H142 replacement by lysine, alanine, and glutamate on the voltage gating of Cx43 channels. Mutation H142E led to a significant reduction in the frequency of occurrence of the residual state and a prolongation of dwell open time. Macroscopically, there was a large reduction in the fast component of voltage gating. These results resembled those observed for a mutant lacking the carboxyl terminal (CT) domain. NMR experiments showed that mutation H142E significantly decreased the Cx43CT-L2 interaction and disrupted the secondary structure of L2. Overall, our data support the hypothesis that fast voltage gating involves an intramolecular particle-receptor interaction between CT and L2. Some of the structural constrains of fast voltage gating may be shared with those involved in the chemical gating of Cx43.

Severe neuropathy with leaky connexin32 hemichannels.

X-linked Charcot-Marie-Tooth disease is one of a set of diseases caused by mutations in gap junction proteins called connexins. We identified a connexin32 missense mutation (F235C) in a girl with unusually severe neuropathy. The localization and trafficking of the mutant protein in cell culture was normal, but electrophysiological studies showed that the mutation caused abnormal hemichannel opening, with excessive permeability of the plasma membrane and decreased cell survival. Abnormal leakiness of connexin hemichannels is likely a mechanism of cellular toxicity in this and perhaps other diseases caused by connexin mutations.