Connexin 43 Hemichannels Contribute to Cytoplasmic Ca2+ Oscillations by Providing a Bimodal Ca2+-dependent Ca2+ Entry Pathway

Activation, Permeability, and Inhibition of Astrocytic and Neuronal Large Pore (Hemi)channels

“…It must be noted, however, that removal of divalent cations may have a variety of secondary cellular effects such as (i) membrane depolarization which may (52) or may not (30,53) promote Cx43 hemichannel opening, and (ii) changes in intracellular Ca 2ϩ concentration that may, in itself, affect hemichannel opening (54,55). Once the opening was induced in the respective hemichannels, the permeation pattern differed among the connexins: Cx30-expressing oocytes displayed Gd 3ϩ -sensitive ion conductance and permeability to both ethidium, Yo-Pro, and propidium, whereas Cx36-and Cx43-expressing oocytes displayed only ethidium permeability (in a Gd 3ϩ -sensitive fashion), and Cx26-expressing oocytes solely exhibited Gd 3ϩ -sensitive membrane conductance and no dye permeability.…”

At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries

“…Hemichannels are permeable to Ca 2þ and may thus contribute to cellular Ca 2þ entry 4 and diffusive ATP release. 5 Moreover, hemichannel opening is [Ca 2þ ] i -dependent in a bimodal manner, [6][7][8][9][10] with moderate [Ca 2þ ] i elevation favouring opening and above $500 nM [Ca 2þ ] i inhibiting opening. The bimodal Ca 2þ -dependence of hemichannels resembles the Ca 2þ -dependence of inositol-trisphosphate receptor (IP 3 R) channels, which are Ca 2þ release channels in the endoplasmic reticulum (ER) that play a central role in generating Ca 2þ oscillations in diverse cell types including SMCs.…”

“…25,26 In contrast to this, L2 inhibits Cx43-hemichannels as concluded from indirect measurements of hemichannel function. 10 CT9 corresponds to the last 9 CT amino acids of Cx43 and dye uptake studies suggested it prevents Cx43 hemichannel closure at micromolar [Ca 2þ ] i 9,10 , thereby promoting hemichannel opening. Thus, L2-inhibition of hemichannels and CT9-prevention of high [Ca 2þ ] i hemichannel closure makes these peptides interesting tools to determine the role of Cx43-hemichannels in Ca 2þ oscillations.…”

Electroporation Loading and Flash Photolysis to Investigate Intra- and Intercellular Ca2+Signaling

“…Cytochrome C (CytC) has been shown to alleviate the negative feedback of Ca 2+ on IP 3 Rs by binding to the carboxy-terminal tail of IP 3 R type 1 (amino acids 2621-2636) (Boehning et al 2003). Consequently, we have used loading of CytC into Madin Darby Canine Kidney (MDCK) cells to show that abolishing the negative feedback of Ca 2+ on the IP 3 R inhibits Ca 2+ oscillations triggered by extracellulary applied ATP or bradykinin (De Bock et al 2012b). Importantly, the low concentrations of CytC did not induce any detectable apoptosis during the 15 min time period of these experiments.…”

“…Single-cell electroporation is in principle also possible when using a locally positioned micropipette as one electrode and a bath Ag/AgCl pellet as the other electrode. Using this approach, cells can be loaded with a variety of membrane-impermeable molecules, including fluorescent dyes, caged compounds, molecules/peptides interfering with signaling pathways, and even recombinantly expressed and purified enzymes (Braet et al 2001(Braet et al , 2003(Braet et al , 2004De Vuyst et al 2008;Decrock et al 2009Decrock et al , 2012De Bock et al 2012b;Monaco et al 2012). Importantly, enzyme activity remains intact when introducing them into cells, as emphasized by the ability of a human type I IP 3 5-phosphatase to reduce IP 3 -induced Ca 2+ increases after its electroporation loading into C6 glioma cells .…”