Connexin 43 Hemichannels Are Permeable to ATP

Abstract

Astrocytes are electrically nonexcitable cells that communicate by means of Ca 2ϩ signaling. Long-distance intercellular Ca 2ϩ waves are initiated by release of ATP and activation of purinergic receptors on nearby cells. Previous studies have implicated connexin 43 (Cx43) in ATP release, but definitive proof that ATP exits through Cx43 hemichannels does not exist. Here, through several alternative approaches, we show that ATP anions can permeate through Cx43 hemichannels. First, openings of Cx43 hemichannels were detected in both cell-attached and inside-out patch recordings in C6 cells expressing Cx43, but not in C6 cells expressing Cx43-eGFP (enhanced green fluorescent protein) or a C-terminus truncation mutant of Cx43. Second, Cx43 hemichannel openings were inhibited by three structurally different gap-junction channel blockers, but not by the P2X 7 blocker Brilliant blue G. Third, bioluminescence imaging of ATP combined with single-channel recording in the inside-out patch configuration showed that ATP efflux coincided with channel openings and was absent when the Cx43 hemichannel was closed. Fourth, ion replacement experiments confirmed that Cx43 hemichannels are permeable to ATP. In summary, these observations provide the first direct evidence for efflux of ATP through Cx43 hemichannels. Furthermore, a putative Cx43 hemichannel with characteristics identical to the Cx43 hemichannel in C6 cells was identified in the membrane of hippocampal astrocytes in acutely prepared slices.

Astrocytes in Alzheimer's Disease

“…[97][98][99][100][101][102][103] The leading mechanism for gliotransmitters release is exocytotic, [104][105][106] although diffusion though large-pore plasmalemmal channels can also be involved. [107][108][109][110] 5) Glia and information processing in the brain.…”

Targeting Astrocytes for Stroke Therapy

“…Astrocytes release ATP through hemichannels formed by Cx43. 120 The extracellular ATP released by astrocytes is converted to adenosine through the actions of ecto-nucleoside triphosphate diphosphohydrolases (NTPDase) and ecto 5=-nucleotidases (CD73). 121 One recent study illustrates the absolute requirement of astrocyte-derived ATP and adenosine for hypoxic preconditioning-mediated neuroprotection.…”

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.…”