Silviu Locovei

Pannexin membrane channels are mechanosensitive conduits for ATP

Intercellular calcium wave propagation initiated by mechanical stress is a phenomenon found in nearly all cell types. The waves utilize two pathways: transfer of InsP3 directly from cell to cell through gap junction channels and release of ATP onto extracellular purinergic receptors. The conduit for ATP has remained elusive and both a vesicular and a channel mediated release have been considered. Here, we describe the properties of single pannexin 1 channels. They have a wide expression spectrum, they are of large conductance and permeant for ATP, and they are mechanosensitive. Hence, pannexins are candidates for the release of ATP to the extracellular space upon mechanical stress.

Pannexin 1 in erythrocytes: Function without a gap

ATP is a widely used extracellular signaling molecule. The mechanism of ATP release from cells is presently unresolved and may be either vesicular or channel-mediated. Erythrocytes release ATP in response to low oxygen or to shear stress. In the absence of vesicles, the release has to be through channels. Erythrocytes do not form gap junctions. Yet, here we show with immunohistochemical and electrophysiological data that erythrocytes express the gap junction protein pannexin 1. This protein, in addition to forming gap junction channels in paired oocytes, can also form a mechanosensitive and ATP-permeable channel in the nonjunctional plasma membrane. Consistent with a role of pannexin 1 as an ATP release channel, ATP release by erythrocytes was attenuated by the gap junction blocker carbenoxolone. Furthermore, under conditions of ATP release, erythrocytes took up fluorescent tracer molecules permeant to gap junction channels.

Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium

The ability for long‐range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP‐induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.

Pannexin1 is part of the pore forming unit of the P2X7 receptor death complex

The purinergic receptor P2X7 is part of a complex signaling mechanism participating in a variety of physiological and pathological processes. Depending on the activation scheme, P2X7 receptors in vivo are non‐selective cation channels or form large pores that can mediate apoptotic cell death. Expression of P2X7R in Xenopus oocytes results exclusively in formation of a non‐selective cation channel. However, here we show that co‐expression of P2X7R with pannexin1 in oocytes leads to the complex response seen in many mammalian cells, including cell death with prolonged ATP application. While the cation channel activity is resistant to carbenoxolone treatment, this gap junction and hemichannel blocking drug suppressed the currents induced by ATP in pannexin1/P2X7R co‐expressing cells. Thus, pannexin1 appears to be the molecular substrate for the permeabilization pore (or death receptor channel) recruited into the P2X7R signaling complex.

The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes

The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1β and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K+ on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K+ was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K+ has been suggested to trigger inflammasome activation, in neural cells, high extracellular K+ activates caspase-1 probably through pannexin 1.

Probenecid, a gout remedy, inhibits pannexin 1 channels.

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.

Pannexin: To gap or not to gap, is that a question?

Vertebrates express two families of gap junction proteins: the well characterized connexins and the recently discovered pannexins. The latter are related to invertebrate innexins. Here we present the hypothesis that pannexins, rather than providing a redundant system to gap junctions formed by connexins, exert a physiological role as nonjunctional membrane channels. Specifically, we propose that pannexins can serve as ATP release channels. This function presumptively is also performed by innexins in invertebrates, in addition to their traditional gap junction role. iubmb Life, 58: 409‐419, 2006

Innexins form two types of channels

Injury to the central nervous system triggers glial calcium waves in both vertebrates and invertebrates. In vertebrates the pannexin1 ATP‐release channel appears to provide for calcium wave initiation and propagation. The innexins, which form invertebrate gap junctions and have sequence similarity with the pannexins, are candidates to form non‐junctional membrane channels. Two leech innexins previously demonstrated in glia were expressed in frog oocytes. In addition to making gap junctions, innexins also formed non‐junctional membrane channels with properties similar to those of pannexons. In addition, carbenoxolone reversibly blocked the loss of carboxyfluorescein dye into the bath from the giant glial cells in the connectives of the leech nerve cord, which are known to express the innexins we assayed.

The potassium channel subunit Kvβ3 interacts with pannexin 1 and attenuates its sensitivity to changes in redox potentials

Pannexin 1 (Panx1), a member of the second gap junction protein family identified in vertebrates, appears to preferentially form non‐junctional membrane channels. A candidate regulatory protein of Panx1 is the potassium channel subunit Kvβ3, previously identified by bacterial two‐hybrid strategies. Here, we report on the physical association of Panx1 with Kvβ3 by immunoprecipitation when co‐expressed in a neuroblastoma cell line (Neuro2A). Furthermore, in vivo co‐expression of Panx1 and Kvβ3 was shown to occur in murine hippocampus and cerebellum. Kvβ3 is known to accelerate inactivation of otherwise slowly inactivating potassium channels under reducing conditions. We subsequently found that Panx1 channel currents exhibit a significant reduction when exposed to reducing agents, and that this effect is attenuated in the presence of Kvβ3. Apparently, Kvβ3 is involved in regulating the susceptibility of Panx1 channels to redox potential. Furthermore, the Panx1 channel blockers carbenoxolone and Probenecid were less effective in inhibiting Panx1 currents when Kvβ3 was co‐expressed. The influence of Kvβ3 on Panx1 is the first example of modulation of Panx1 channel function(s) by interacting proteins, and suggests the physiological importance of sensing changes in redox potentials.