David C. Spray

Pannexin 1: The Molecular Substrate of Astrocyte “Hemichannels”

Purinergic signaling plays distinct and important roles in the CNS, including the transmission of calcium signals between astrocytes. Gap junction hemichannels are among the mechanisms proposed by which astrocytes might release ATP; however, whether the gap junction protein connexin43 (Cx43) forms these “hemichannels” remains controversial. Recently, a new group of proteins, the pannexins, have been shown to form nonselective, high-conductance plasmalemmal channels permeable to ATP, thereby offering an alternative for the hemichannel protein. Here, we provide strong evidence that, in cultured astrocytes, pannexin1 (Panx1) but not Cx43 forms hemichannels. Electrophysiological and fluorescence microscope recordings performed in wild-type and Cx43-null astrocytes did not reveal any differences in hemichannel activity, which was mostly eliminated by treating Cx43-null astrocytes with Panx1-short interfering RNA [Panx1-knockdown (Panx1-KD)]. Moreover, quantification of the amount of ATP released from wild-type, Cx43-null, and Panx1-KD astrocytes indicates that downregulation of Panx1, but not of Cx43, prevented ATP release from these cells.

P2X7 receptor-Pannexin1 complex: pharmacology and signaling

Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X(7) receptor (P2X(7)R) activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X(7)R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X(7)R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X(7)R stimulation or by membrane depolarization was blocked by Panx1 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X(7)R antagonist, prevented current activation by 2(3)-O-(4-benzoylbenzoyl)adenosine 5-triphosphate (BzATP). Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X(7)) peptides, provided evidence that the Src homology 3 death domain of the COOH-terminus of the P2X(7)R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 microM TAT-P2X(7) peptide caused 50% reduction in Src binding to the P2X(7)R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X(7) peptide, and by the Src tyrosine inhibitor PP2 and these compounds prevented both large-conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X(7)R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X(7)R.

Cytokine regulation of neuronal differentiation of hippocampal progenitor cells

THE signalling mechanisms governing haematolymphopoiesis and those regulating neural development may be closely related, as indicated by similarities of higher-order structure and function of the cytokines involved1, of the regional and temporal regulation of their transcription and translation2–6, and of their bioactivity7–10. Here we investigate this possible evolutionary connection using retroviral transduction of a temperature-sensitive mutant form of the SV40 large T antigen to develop conditionally immortalized murine embryonic hippocampal progenitor cell lines11–14. Treatment of these cells with cytokines that are thought to participate in progressive lymphoid maturation, immunoglobulin synthesis15–18 and erythropoiesis19,20 causes progressive neuronal differentiation, as defined by morphological criteria, successive expression of increasingly mature neurofilament proteins21–23, and the generation of inward currents and action potentials. The cytokine interleukin(IL)-11 induces expression of action potentials that are insensitive to tetrodotoxin, which is indicative of develop-mentally immature sodium channels24. By contrast, for expression of more mature action potentials24 (tetrodotoxin-sensitive) one of the interleukins IL-5, IL-7 or IL-9 must be applied in association with transforming growth factor-α after pretreatment with basic fibroblast growth factor. Our results suggest that the mechanisms regulating lineage commitment and cellular differentiation in the neural and haematopoietic systems are similar. Further, they define an in vitro model system that may facilitate molecular analysis of graded stages of mammalian neuronal differentiation.

Cytokine-induced programmed death of cultured sympathetic neurons

Programmed cell death (PCD) of sympathetic neurons is inhibited by nerve growth factor. However, factors that induce PCD of these cells are unknown. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, neuropoietic cytokines known to regulate sympathetic neuron gene expression, were examined for effects on survival of cultured sympathetic neurons. Treatment with LIF or ciliary neurotrophic factor caused neuronal death in a dose-dependent fashion. Inhibition of RNA or protein synthesis, or treatment with potassium, all of which prevent PCD after nerve growth factor deprivation, prevented LIF-induced death. The morphologic and ultrastructural characteristics of the neuronal death induced by LIF and by nerve growth factor deprivation were similar. Furthermore, LIF treatment resulted in DNA fragmentation with a characteristic ladder on Southern blot analysis. These observations suggest that neuron numbers may be regulated by factors which initiate PCD, as well as by factors which prevent it.

Characterization of gap junctions between cultured leptomeningeal cells

Leptomeningeal cells in intact meninges or dissociated and cultured for 2 h to several weeks were dye-coupled (Lucifer yellow), and voltage-clamped pairs of freshly dissociated leptomeningeal cells were well coupled electrically. Unitary conductances of junctional channels were predominantly 40-90 pS. Junctional conductance was reversibly reduced by 2 mM halothane, 1 mM heptanol and 100% CO2 and was increased by 1 mM 8 Br-cAMP. Two gap junction proteins, connexin 26 and connexin 43, were identified between leptomeningeal cells using immunocytochemical methods; Northern blot analyses of RNA isolated from cultured leptomeningeal cells showed specific hybridization to cDNAs encoding connexins 26 and 43, but not to a cDNA encoding connexin 32. These studies demonstrate co-expression of two connexins in a single cell type in the nervous system; biophysical properties do not differ significantly from those of astrocytes and cardiac myocytes, which express only connexin 43.

TNFα inhibits Schwann cell proliferation, connexin46 expression, and gap junctional communication

Abstract Schwann cell responses to nerve injury are stimulated, in part, by inflammatory cytokines. This study compares changes in the phenotype of cultured Schwann cells after exposure to the cytokine tumor necrosis factor (TNF)-α or the mitogen neu differentiation factor (NDF)-β. TNFα inhibited proliferation in a dose-dependent manner without altering Schwann cell survival. TNFα also reduced both gap junctional conductance and Lucifer yellow dye coupling between Schwann cells. Moreover, both P0and glial fibrillary acidic protein (GFAP) immunoreactivity were reduced. By contrast, NDFβ initially had little effect on cell division although it reduced junctional coupling within 8xa0h. However, by 48 h, NDFβ stimulated proliferation with a concomitant increase in coupling. Dividing Schwann cells (BrdU+) were preferentially dye coupled compared to nondividing cells, indicating an association between proliferation and coupling. Moreover, cultured Schwann cells expressed connexin46 mRNA and protein, and changes in the levels of the protein correlated with the degree of proliferation and coupling. The data thus provide evidence for cytokine-induced modulation of Schwann cell antigenic phenotype, proliferation, and gap junction properties. These observations suggest that enhanced gap junctional communication among Schwann cells after nerve injury could help to coordinate cellular responses to the injury, and that TNFα may be a signal which terminates proliferation as well as junctional communication.

The Gap Junction Protein Connexin32 Interacts with the Src Homology 3/Hook Domain of Discs Large Homolog 1

Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.

Transfection of mammalian cells with connexins and measurement of voltage sensitivity of their gap junctions.

Vertebrate gap junction channels are formed by a family of more than 20 connexin proteins. These gap junction proteins are expressed with overlapping cellular and tissue specificity, and coding region mutations can cause human hereditary diseases. Here we present a summary of what has been learned from voltage clamp studies performed on cell pairs either endogenously expressing gap junctions or in which connexins are exogenously expressed. General protocols presented here are currently used to transfect mammalian cells with connexins and to study the biophysical properties of the heterologously expressed connexin channels. Transient transfection is accomplished overnight with maximal expression occurring at about 36 h; stable transfectants normally can be generated within three or four weeks through colony selection. Electrophysiological protocols are presented for analysis of voltage dependence and single-channel conductance of gap junction channels as well as for studies of chemical gating of these channels.

Molecular Organization and Regulation of the Cardiac Gap Junction Channel Connexin43

Gap junction channels provide a pathway for direct cell-to-cell communication between adjacent cells. These channels are involved in a number of biologic functions such as electrical conduction, embryogenesis, and cell growth. Mutations of their constituent proteins in humans have been associated with nonsyndromic deafness, Charcot-Marie-Tooth syndrome, oculodentodigital dysplasia, 1 and congenital cataracts among other congenital human diseases. 2 3 In the heart, gap junctions are necessary for the propagation of the action potential 4 5 as well as for normal cardiac embryogenesis. 6 In this chapter, we review the structure of the cardiac gap junctions and correlate it to our current knowledge, with the function of the channel. It is one of the expectations of structure-function analysis that this information may one day translate into, among other things, the rational design of molecules that may either enhance or interfere with the function of the channel, thus providing a new tool for the manipulation of electrical and metabolic synchrony in the heart.

Gap Junctions in the Nervous System: An Introduction

Acentury ago, it was generally believed that consciousness and movement resulted from the flow of substances freely throughout the interconnected neural network. This Reticular Theory eventually gave way to the Neuronal Doctrine in which the nervous system was envisioned as a composite of discrete cells, where direct transfer of information among neurons is a rare event, occurring only in specialized nuclei and under specific circumstances. Nevertheless, as chapters in this volume testify, gap junctions, the structural elements responsible for direct intercellular communication, are increasingly detected between cells in both the central and peripheral nervous systems of mammals, including man.