Abstract

Pannexins constitute a family of proteins exhibiting predominantly hemichannel activity. Pannexin channels have been suggested to participate in a wide spectrum of biological functions such as propagation of calcium waves, release of IL-1β, and responses to ischemic conditions. At present, the molecular mechanisms regulating pannexin hemichannel activity are essentially unknown. Because cysteines have been shown to constitute key elements in regulating hemichannel properties of the connexin-type we performed site-directed mutagenesis of intracellular cysteine residues of Panx1. Cysteine to serine exchange (Cys → Ser) at the C-terminal position amino acid 346 led to a constitutively leaky hemichannel and subsequently to cell death. Increased channel activity was demonstrated by dye uptake and electrophysiological profiling in injected Xenopus laevis oocytes and transfected N2A cells. Mutations of the remaining intracellular cysteines did not result in major changes of Panx1 channel properties. From these data we conclude that the Cys-346 residue is important for proper functioning of the Panx1 channel.

Highlights

  • (14 –16), neuronal cell death after ischemia [17, 18], and in activation of inflammasomes [19]

  • To uncover putative mechanisms of hemichannel regulation of Panx1, we investigated the role of single intracellular cysteine residues

  • Incubation of oocytes expressing Panx1C346S in Cbx prolonged survival time, suggesting that initial cell death was due to hemichannel leakiness

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Summary

Introduction

(14 –16), neuronal cell death after ischemia [17, 18], and in activation of inflammasomes [19]. Western Blot Analysis—Total protein lysates of transfected N2A cells or injected oocytes were prepared at the time points indicated using the lysis buffer described above.

Results
Conclusion
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