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The maxi-anion channel plays a classically recognized role in controlling the membrane potential through the chloride conductance. It also has novel functions as a regulated pathway for the release of the anionic signaling molecules ATP and excitatory amino acids from cells subjected to osmotic perturbation, ischemia, or hypoxia. Because hemichannels formed by pannexins and connexins have been reported to mediate ATP release from a number of cell types, these hemichannels may represent the molecular correlate of the maxi-anion channel. Here, we found that L929 fibrosarcoma cells express functional maxi-anion channels which mediate a major portion of swelling-induced ATP release, and that ATP released via maxi-anion channels facilitates the regulatory volume decrease after osmotic swelling. Also, it was found that the cells express the mRNA for pannexin 1, pannexin 2, and connexin 43. Hypotonicity-induced ATP release was partially suppressed not only by known blockers of the maxi-anion channel but also by several blockers of pannexins including the pannexin 1-specific blocking peptide (10)Panx1 and small interfering (si)RNA against pannexin 1 but not pannexin 2. The inhibitory effects of maxi-anion channel blockers and pannexin 1 antagonists were additive. In contrast, maxi-anion channel activity was not affected by pannexin 1 antagonists and siRNAs against pannexins 1 and 2. Although a connexin 43-specific blocking peptide, Gap27, slightly suppressed hypotonicity-induced ATP release, maxi-anion channel activity was not affected by Gap27 or connexin 43-specific siRNA. Thus, it is concluded that the maxi-anion channel is a molecular entity distinct from pannexin 1, pannexin 2, and connexin 43, and that the maxi-anion channel and the hemichannels constitute separate pathways for swelling-induced ATP release in L929 cells.

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.

Pannexin 1 forms a large membrane channel that, based on its biophysical properties and its expression pattern, is a prime candidate to represent an ATP release channel. Pannexin 1 channel activity is potentially deleterious for cells as indicated by its involvement in the P2X7 death complex. Here we describe a negative feedback loop controlling pannexin 1 channel activity. ATP, permeant to pannexin 1 channels, was found to inhibit its permeation pathway when applied extracellularly to oocytes expressing pannexin 1 exogenously. ATP analogues, including benzoylbenzoyl-ATP, suramin, and brilliant blue G were even more effective inhibitors of pannexin 1 currents than ATP. These compounds also attenuated the uptake of dyes by erythrocytes, which express pannexin 1. The rank order of the compounds in attenuation of pannexin 1 currents was similar to their binding affinities to the P2X7 receptor, except that receptor agonists and antagonists both were inhibitory to the channel. Mutational analysis identified R75 in pannexin 1 to be critical for ATP inhibition of pannexin 1 currents.

Probenecid is a well-established drug for the treatment of gout and is thought to act on an organic anion transporter, thereby affecting uric acid excretion in the kidney by blocking urate reuptake. Probenecid also has been shown to affect ATP release, leading to the suggestion that ATP release involves an organic anion transporter. Other pharmacological evidence and the observation of dye uptake, however, suggest that the nonvesicular release of ATP is mediated by large membrane channels, with pannexin 1 being a prominent candidate. In the present study we show that probenecid inhibited currents mediated by pannexin 1 channels in the same concentration range as observed for inhibition of transport processes. Probenecid did not affect channels formed by connexins. Thus probenecid allows for discrimination between channels formed by connexins and pannexins.