Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

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-1␤, 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 3 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.In addition to connexins, a second family of proteins, termed pannexins (Panx) 3 (1), is suggested to form hemichannels. The pannexin channel family consists of three members: Panx1, which is abundantly expressed in different organs of the vertebrate organism, Panx2, which is found mainly in the central nervous system, and Panx3, which is primarily expressed in skin and cartilage (2-8). Panx1 is capable of forming gap junctions and hemichannels in the Xenopus laevis oocytes expression system (9, 10), but it is still a matter of debate whether pannexinformed gap junctions do exist under in vivo conditions. Apparently, Panx1 primarily forms hemichannels (9,11,12). Among others, a role of Panx1 hemichannels was suggested in the initiation and propagation of calcium waves (13), the release of interleukin-1␤ due to interaction with the P2X7 receptor (14 -16), neuronal cell death after ischemia (17, 18), and in activation of inflammasomes (19). Panx1 hemichannels open in response to a rise in intracellular Ca 2ϩ , to depolarization to membrane potentials above Ϫ20 mV, and to mechanical stretch (10,11,20).Even though there is no direct sequence homology between pannexins and connexins, both protein types possess the same topology with four transmembrane domains and intracellularly located N and C termini (for review, see Ref. 21). Significant differences between the two protein families are the number of extracellular cysteines and the fact that Panx1 is glycosylated at the second extracellular loop (9, 22). Deglycosylation with N-glycosidase F enhanced gap junctional coupling, indicating that glycosylation might be a regulatory key element to distinguish between gap junctional coupling and hemichannel activity (23).Besides the importance of extracellular cysteines in gap junction channel formation of connexins (24), intracellular cysteine residues have been suggested to be responsible for sensitivity of Cx43 and Cx46 hemi...

Pannexin 1, an ATP Release Channel, Is Activated by Caspase Cleavage of Its Pore-associated C-terminal Autoinhibitory Region

“…For example, we found that a synthetic peptide containing just this sequence was not sufficient to block current from truncated hPANX1 channels, whereas larger C-terminal proteins were effective. In addition, a more proximal C-terminal residue (Cys-347) that regulates basal channel activity has been identified (16). It is also interesting that hPANX1 inhibition associated with mutations in this restricted region or obtained by cell-free reconstitution of C-terminal proteins with truncated channels was most pronounced at hyperpolarized potentials.…”

“…mSpo20 (14) and PLC␦-PH (15)). Likewise, hPANX1⌬371 currents were unaffected by a Cterminal construct that includes a point mutation (hPANX1(Ct)C347S) known to yield constitutive currents in full-length hPANX1 channels (16). Finally, a C-terminal construct truncated at Glu-391 (hPANX1(Ct)⌬391) provided a block of current that was comparable with that seen with the full-length C terminus, consistent with our observation that residues downstream of Glu-391 are not required for C-terminal inhibition of hPANX1 (cf.…”

The cellular life of pannexins

“…31,32 Moreover, there is a loss of channel function when any of the four extracellular cysteines of Panx1 are mutated. 33 The possibility of intrinsic control of pannexin channel activity by the cysteines hints at the potential regulation of the channel by such post-translational modifications as glutathionation or S-nitrosylation.…”

Function and Regulation of Pannexin 1 Channels in the Vascular Endothelium

“…Interestingly, the two sites that we identified as critical for this inhibitory modification --C40 and C346 --were previously reported to enhance activity of Panx1 channels in mutagenesis studies [259,260]. In that other work, serine substitution at either C40 [260] or C346 [259] produced "leaky" or constitutively active channels. Together with our results, these observations suggest that C40 and C346 may be localized to regions that are important for dynamic up-and downregulation of Panx1 channel activity.…”

“…Also, a previous study identified a loss in the Gly2 species in functional, plasma membrane-localized Panx1C346S [259].…”