The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1␤ 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 Pannexin 1 is a vertebrate ortholog of the invertebrate innexin gap junction proteins (1), but it does not appear to form functional gap junctions in vivo. Instead pannexin 1 acts as a membrane channel that carries ions and signaling molecules between the cytoplasm and the extracellular space (2, 3). As such, it is a candidate ATP release channel in various cell types, including erythrocytes, astrocytes, bronchial epithelial cells, and taste cells. Various functional roles have been ascribed to pannexin 1 including local vascular perfusion control and propagation of intercellular calcium waves (4 -6). Recently pannexin 1 was also shown to form the large pore of the P2X7 purinergic receptor (7, 8). P2X7 plays a major role in inflammation, and its activation by extracellular ATP results in release of interleukin (IL) 2 -1␤ from macrophages, probably involving pannexin 1 as a signaling molecule (7).IL-1␤ production and maturation are tightly regulated by caspase-1 incorporated into large protein complexes termed inflammasomes (9 -11). The molecular composition of the inflammasome depends on the identity of the NOD-like receptor (NLR) family member serving as a scaffold protein in the complex (12). The members of the cytosolic NLR family appear to recognize conserved microbial and viral components termed pathogen-associated molecular patterns in intracellular compartments (13). The bipartite adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) bridges the interaction between NLR proteins and inflammatory caspases and plays a central role in the assembly of inflammasomes and the activation of caspase-1 in response to a broad range of pathogen-associated molecular patterns and intracellular pathogens (14). In addition, the inflammasome can be activated by danger-associated molecular patterns, molecules endogenous to the organism that signal stress or injury, including extracellular ATP acting at ionotropic P2X7 receptors, fibronectin, or monosodium urate crystals (15,16). Moreover it has been suggested that a rapid K ϩ efflux through ATP-activated P2X7 receptors induces inflammasome assembly (17)(18)(19)(20).Despite the recent advances in the understanding of a...

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.connexin; transport; erythrocyte; ATP release PROBENECID HAS BEEN USED for decades for the treatment of gout. The mechanism of action of the drug is inhibition of a renal tubular transporter, thereby facilitating the excretion of the disease causative uric acid by blocking reuptake (5, 26, 37). Probenecid-sensitive transporters are widespread and are even found in plants (30,31,44,52,56). The inhibition of the transporter by probenecid is also exploited clinically to increase the effective concentrations of antibiotics, chemotherapeutics, and other medications.The inhibitory effect of probenecid on organic anion transporters is well established, and the effect is thought to be so specific that the drug is often used as a diagnostic tool, i.e., its effect is typically interpreted as an involvement of an anion transporter in the tested parameter. Accordingly, block of cAMP or cGMP release from erythrocytes (18, 25), ATP release from glia cells (1, 17), and block of dye loss in various cell types (20,21,23) by probenecid have been presented as evidence for a role of transporters in these phenomena. However, alternative pathways for the transit of these molecules across the plasma membrane have to be considered. Besides the well-documented vesicular release of ATP, a parallel release through membrane channels must exist, because the release is attenuated by drugs that do not interfere with vesicular release but affect gap junction proteins and because ATP release in several cell types is associated with uptake of dye from the extracellular medium (13).Special attention has to be given to pannexin 1 as an ATP release channel because of the specific properties of pannexin 1 channels and because of the expression pattern of pannexin 1 (2, 16, 28, 32-34, 55, 59). Pannexin 1 channels are highly permeable to ATP and to dyes typically used for dye flux measurements through gap junction channels. These dyes are in the same size range as the Ca 2ϩ indicator dyes whose loss is attenuated by probenecid. Pannexin 1 channels also are mechanosensitive, consistent with a role in Ca 2ϩ wave initiation. Expression of pannexin 1 is found in cells exhibiting ATP release, including er...

Here we describe the initial functional characterization of a cyclic nucleotide regulated ion channel from the bacterium Mesorhizobium loti and present two structures of its cyclic nucleotide binding domain, with and without cAMP. The domains are organized as dimers with the interface formed by the linker regions that connect the nucleotide binding pocket to the pore domain. Together, structural and functional data suggest the domains form two dimers on the cytoplasmic face of the channel. We propose a model for gating in which ligand binding alters the structural relationship within a dimer, directly affecting the position of the adjacent transmembrane helices.

A recently proposed model for voltage-dependent activation in K+ channels, largely influenced by the KvAP X-ray structure, suggests that S4 is located at the periphery of the channel and moves through the lipid bilayer upon depolarization. To investigate the physical distance between S4 and the pore domain in functional channels in a native membrane environment, we engineered pairs of cysteines, one each in S4 and the pore of Shaker channels, and identified two instances of spontaneous intersubunit disulfide bond formation, between R362C/A419C and R362C/F416C. After reduction, these cysteine pairs bound Cd2+ with high affinity, verifying that the residues are in atomic proximity. Molecular modeling based on the MthK structure revealed a single position for S4 that was consistent with our results and many other experimental constraints. The model predicts that S4 is located in the groove between pore domains from different subunits, rather than at the periphery of the protein.