Pannexin1 (Panx1) participates in several signaling events that involve ATP release, including the innate immune response, ciliary beat in airway epithelia and oxygen supply in the vasculature. The view that Panx1 forms a large ATP-release channel has been challenged by the association of a low conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K + ), Panx1 formed a high conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K + . The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single particle electron microscopic analysis revealed that K + stimulated the formation of channels with a larger pore diameter than those formed in the absence of K + . These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties.

The food dye FD&C Blue No. 1 (Brilliant Blue FCF [BB FCF]) is structurally similar to the purinergic receptor antagonist Brilliant Blue G (BBG), which is a well-known inhibitor of the ionotropic P2X7 receptor (P2X7R). The P2X7R functionally interacts with the membrane channel protein pannexin 1 (Panx1) in inflammasome signaling. Intriguingly, ligands to the P2X7R, regardless of whether they are acting as agonists or antagonists at the receptor, inhibit Panx1 channels. Thus, because both P2X7R and Panx1 are inhibited by BBG, the diagnostic value of the drug is limited. Here, we show that the food dye BB FCF is a selective inhibitor of Panx1 channels, with an IC50 of 0.27 µM. No significant effect was observed with concentrations as high as 100 µM of BB FCF on P2X7R. Differing by just one hydroxyl group from BB FCF, the food dye FD&C Green No. 3 exhibited similar selective inhibition of Panx1 channels. A reverse selectivity was observed for the P2X7R antagonist, oxidized ATP, which in contrast to other P2X7R antagonists had no significant inhibitory effect on Panx1 channels.Based on its selective action, BB FCF can be added to the repertoire of drugs to study the physiology of Panx1 channels. Furthermore, because Panx1 channels appear to be involved directly or indirectly through P2X7Rs in several disorders, BB FCF and derivatives of this “safe” food dye should be given serious consideration for pharmacological intervention of conditions such as acute Crohn’s disease, stroke, and injuries to the central nervous system.

The ATP release channel Pannexin1 (Panx1) is self-regulated, i.e. the permeant ATP inhibits the channel from the extracellular space. The affinity of the ATP binding site is lower than that of the purinergic P2X7 receptor allowing a transient activation of Panx1 by ATP through P2X7R. Here we show that the inhibition of Panx1 by ATP is abrogated by increased extracellular potassium ion concentration ([K+]o) in a dose-dependent manner. Since increased [K+]o is also a stimulus for Panx1 channels, it can be expected that a combination of ATP and increased [K+]o would be deadly for cells. Indeed, astrocytes did not survive exposure to these combined stimuli. The death mechanism, although involving P2X7R, does not appear to strictly follow a pyroptotic pathway. Instead, caspase-3 was activated, a process inhibited by Panx1 inhibitors. These data suggest that Panx1 plays an early role in the cell death signaling pathway involving ATP and K+ ions. Additionally, Panx1 may play a second role once cells are committed to apoptosis, since Panx1 is also a substrate of caspase-3.

The sequence and predicted membrane topology of pannexin1 (Panx1) places it in the family of gap junction proteins. However, rather than forming gap junction channels, Panx1 forms channels in the nonjunctional membrane. Panx1 operates in two distinct open states, depending on the mode of stimulation. The exclusively voltage-gated channel has a small conductance (<100 pS) and is highly selective for the flux of chloride ions. The Panx1 channel activated by various physiological stimuli or by increased concentrations of extracellular potassium ions has a large conductance (~500 pS, however, with multiple, long-lasting subconductance states) and is nonselectively permeable to small molecules, including ATP. To test whether the two open conformations also differ pharmacologically, the effects of di-and trivalent cations on the two Panx1 channel conformations were investigated. The rationale for this venture was that, under certain experimental conditions, ATP release from cells can be inhibited by multivalent cations, yet the literature indicates that the ATP release channel Panx1 is not affected by these ions. Consistent with previous reports, the Panx1 channel was not activated by removal of extracellular Ca and the currents through the voltage-activated channel were not altered by Ca, Zn, Ba, or Gd. In contrast, the Panx1 channel activated to the large channel conformation by extracellular K, osmotic stress, or low oxygen was inhibited by the multivalent cations in a dose-dependent way. Thus, monovalent cations activated the Panx1 channel from the closed state to the "large" conformation, while di- and trivalent cations exclusively inhibited this large channel conformation.

No abstract