1803867760     

[Erscheinungsort nicht ermittelbar] : Ghent University. Faculty of Medicine and Health Sciences, 2013

Englisch

Dissertation, Ghent University. Faculty of Medicine and Health Sciences, 2013

Elektronische Ressource: Zugang beim Produzenten (Lizenzangabe: Kostenfrei zugänglich ohne Registrierung)

Sachgruppe(n) DB (ab 2004) 572

In vertebrates, connexin hemichannels in the plasma membrane dock head-to-head with their counterparts in adjacent cells to form gap junction channels, allowing direct cell-cell transfer of electric and chemical/metabolic signals. Each hemichannel comprises a hexamer of connexin proteins which consist of four membrane-spanning domains, two extracellular loops, a cytoplasmic loop (CL) and flanking N- (NT) and C-termini (CT) in the cytoplasmic side of the cell. Accumulating evidence has suggested novel functions of connexin hemichannels beyond gap junction communication. These unapposed hemichannels can open and form a conduit between the intracellular compartment and the extracellular milieu, allowing Na+ and Ca2+ to enter the cell and K+ and paracrine messengers like ATP, glutamate and others to leave the cell. A frequently applied pharmacological tool to explore new functions of the connexin hemichannel signaling pathway consists of connexin targeting peptides like Gap26 and Gap27, which are identical to sequences located respectively on the first and second extracellular loop regions of Cx43. Intriguingly, despite the growing interest and wide use of connexin mimetic peptides in hemichannel studies, no conclusive data and arguments are available to support a direct action of these substances on hemichannels. In chapter III, we aimed to investigate the effect of Gap26/27 on Cx43 hemichannels at the single channel level. Such an approach allows unequivocal identification of hemichannel 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 hemichannels unitary currents over minutes at concentrations in the 100-200 μM range and increased the voltage threshold for hemichannel opening. By contrast, an elevation of intracellular calcium ([Ca2+]i) to 200-500 nM potentiated the unitary hemichannel current activity and lowered the voltage threshold for hemichannel activation. Interestingly, Gap26/27 inhibited the Ca2+ potentiated hemichannel currents and prevented lowering of the voltage threshold for hemichannel activation. Experiments on isolated pig ventricular cardiomyocytes, which display strong endogenous Cx43 expression, demonstrated voltage-activated unitary currents with biophysical properties of Cx43 hemichannels that were inhibited by small interfering RNA targeting Cx43. As observed in HeLa-Cx43 cells, hemichannel current activity in ventricular cardiomyocytes was potentiated by [Ca2+]i elevation to 500 nM and was inhibited by Gap26/27. Our results indicate that under pathological conditions, when [Ca2+]i is elevated, Cx43 hemichannel opening is promoted in cardiomyocytes and Gap26/27 counteracts this effect. In the mammalian heart, Cx43 is the primary connexin expressed in the working ventricular myocardium. Emerging evidence has suggested novel roles of Cx43 hemichannels in cardiac homeostasis. Residing in the periphery of gap junction plaques termed 'perinexus', these unapposed hemichannels are typically closed under control conditions, but may open in response to ischemic insults. Uncontrolled activation of hemichannels provides a nonselective conduit between the cytoplasm and extracellular fluid, introducing a current leakage pathway and liberating essential metabolites from the cell. Thus, excessive openings of Cx43 hemichannels may be deleterious for the myocardium. Currently, there is no pharmacological tool available that allows selective targeting of hemichannels without inhibiting junctional coupling. Thus, in chapter IV, we aimed to characterize a nonapeptide derived from the CL of Cx43 (further called 'Gap19'), which inhibits Cx43 hemichannels without blocking gap junctions or Cx40/pannexin-1 hemichannels. As exemplified by surface plasmon resonance, the selective effect on hemichannels is due to direct binding of Gap19 to the end of CT thereby preventing intramolecular CT-CL interactions which are essential for Cx43 hemichannel activation. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 counteracted the effect of metabolic inhibition on hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and reduced the infarct size after myocardial ischemia/reperfusion in mice in vivo. Collectively, preventing Cx43 hemichannel activation by Gap19 confers protective effects against myocardial ischemia/reperfusion injury. In summary, the present doctoral work addresses the effect of several Cx43 targeting peptides and intracellular [Ca2+]i on Cx43 hemichannels at the single-channel level. The understanding towards the mechanistic basis of these hemichannel modulators provides novel insights into the functional regulation of connexin hemichannels. Additionally, Gap19 which selectively targets connexin hemichannels without inhibiting gap junctions emerges as a valuable tool for further studies aiming at investigating the role of Cx43 hemichannels in cardiac injury.