ATP signaling is deficient in cultured pannexin1‐null mouse astrocytes

Abstract

Pannexins (Panx1, 2 and 3) comprise a group of proteins expressed in vertebrates that share weak yet significant sequence homology with the invertebrate gap junction proteins, the innexins. In contrast to the other vertebrate gap junction protein family (connexin), pannexins do not form intercellular channels, but at least Panx1 forms non-junctional plasma membrane channels. Panx1 is ubiquitously expressed and has been shown to form large conductance (500pS) channels that are voltage-dependent, mechanosensitive and permeable to relatively large molecules, such as ATP. Pharmacological and knockdown approaches have indicated that Panx1 is the molecular substrate for the so-called “hemichannel” originally attributed to connexin43 (Cx43) and that Panx1 is the pore forming unit of the P2X7 receptor. Here, we describe, for the first time, conductance and permeability properties of Panx1-null astrocytes. The electrophysiological and fluorescence imaging analysis performed on these cells fully support our previous pharmacological and Panx1 knockdown studies that showed profoundly lower dye uptake and ATP release than wild-type untreated astrocytes. As a consequence of decreased ATP paracrine signaling, intercellular calcium wave spread is altered in Panx1-null astrocytes. Moreover, we found that in astrocytes as in Panx1 expressing oocytes, elevated extracellular K+ activates Panx1 channels independently of membrane potential. Thus, based on the present findings and our previous report, we propose that Panx1 channels serve as K+ sensors for changes in the extracellular milieu such as those occurring under pathological conditions.

Intrarenal localization of the plasma membrane ATP channel pannexin1

“…These transcripts are currently being evaluated (personal communication from Prof. Eliana Scemes). Nevertheless, recent studies indicate that tissues and cells derived from the same Panx1 knockout mouse model function as total Panx1 knockouts (29,39). Importantly, urinary [ATP] was ϳ30% lower in Panx1 Ϫ/Ϫ mice compared with wild-type mice ( Ϫ/Ϫ mice (20) suggests the presence of alternative ATP release pathways in renal tubules that are not Panx1 channels or Cx hemichannels.…”

“…Immunoblotting and ATP measurements were performed using samples from Panx1 Ϫ/Ϫ mice and their wild-type littermates. The generation of Panx1 Ϫ/Ϫ mice used for these studies was described previously (29,39). All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Southern California.…”

Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations

“…Given the widespread distribution of connexin and pannexin-1 (Panx1) hemichannels on neurones and astrocytes (Cisneros-Mejorado et al, 2014;De Bock et al, 2014) and the strong evidence for a contribution of connexin and pannexin-1 hemichannel-mediated ATP release during neuronal and astrocytic activity (Garre et al, 2010;Santiago et al, 2011;Stout et al, 2002;Suadicani et al, 2012), we decided to test the hypothesis that GI mGluR-evoked ATP release occurs via hemichannels.…”

“…Whereas the P2X 7 receptor antagonist A438079 had no effect on DHPG-induced bursting, the fact that probenecid, as well as a low concentration of carbenoxolone, strongly suppressed both DHPG-induced neuronal activity and the release of ATP is suggestive of the requirement of Pannexin-1 hemichannels for the induction of this activity. Pannexin-1 hemichannels, unlike connexins, which form junctions between two adjacent cells, preferentially form a channel with the extracellular environment permeable to ATP (Iglesias et al, 2009;Leybaert and Sanderson, 2012;Locovei et al, 2006;Suadicani et al, 2012). Furthermore, Panx1 is activated by elevated extracellular K + Silverman et al, 2009) which would be expected during the synchronised neuronal bursting induced by DHPG.…”

Functional outcome of pannexin-deficient mice after cerebral ischemia

“…subunit of astrocyte channels and both Px1 and Px2 play no role has been challenged by a recent study claiming that Px1 is the key channel subunit of astrocytes. 3,[16][17][18] Possibly, there is a partial compensation between pannexins and Cx43 in astrocytes. Irrespective of channel function in astrocytes, there is good evidence that pannexin channels are a useful target for stroke therapy, as interference with pannexin function robustly improves the functional outcome after stroke.…”