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 1beta 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.

Structural and energetic analysis of activation by a cyclic nucleotide binding domain.

MlotiK1 is a prokaryotic homolog of cyclic-nucleotide-dependent ion channels that contains an intracellular C-terminal cyclic nucleotide binding (CNB) domain. X-ray structures of the CNB domain have been solved in the absence of ligand and bound to cAMP. Both the full-length channel and CNB domain fragment are easily expressed and purified, making MlotiK1 a useful model system for dissecting activation by ligand binding. We have used X-ray crystallography to determine three new MlotiK1 CNB domain structures: a second apo configuration, a cGMP-bound structure, and a second cAMP-bound structure. In combination, the five MlotiK1 CNB domain structures provide a unique opportunity for analyzing, within a single protein, the structural differences between the apo state and the bound state, and the structural variability within each state. With this analysis as a guide, we have probed the nucleotide selectivity and importance of specific residue side chains in ligand binding and channel activation. These data help to identify ligand-protein interactions that are important for ligand dependence in MlotiK1 and, more globally, in the class of nucleotide-dependent proteins.

The MlotiK1 channel transports ions along the canonical conduction pore.

Although the cyclic nucleotide-modulated potassium channel from Mesorhizobium loti, MlotiK1, is easily studied using a 86Rb+ flux assay, its comparatively low activity raises serious concerns about the integrity of the purified protein. We investigated the pathway of uptake using a multi-pronged approach. First, we probed the conduction pathway using quaternary ammonium compounds known to block conduction in eukaryotic K+ channels. Second, we examined the effect of chemical modification of putative pore-lining residues. Our results are consistent with ions traversing MlotiK1 along a conduction pathway like that of the eukaryotic channels, but at a much slower rate.

Mechanism of increased open probability by a mutation of the BK channel.

A missense mutation (D434G) has recently been identified in the alpha subunit of the human large-conductance calcium-activated potassium (BK) channel. Interestingly, although the mutation causes an increase in open probability, individuals that carry the mutation have epilepsy and/or paroxysmal dyskinesia, disorders of increased brain excitability. To define the mechanisms of the mutation, we have used recordings from single channels and measurement of macroscopic conductances to examine the gating of the alpha subunit, modulation by the regulatory beta4 subunit, and the effect of Mg2+ on channel properties. Although there was relatively little difference in open dwell times for the mutant and wild-type alpha subunits, the mutant channel spent less time in a long-lived closed state. Co-expression of the beta4 subunit caused the wild-type channel to be less sensitive to calcium at low Ca2+ concentrations but had little effect on the mutant channel, further accentuating the difference between the wild-type and the mutant channels. In the absence of Ca2+, there was no difference in Mg2+ or voltage sensitivity of the mutant and wild-type channels, whereas in 2 mM Ca2+, the mutant channel had greater open probability at every Mg2+ concentration tested. We conclude that the D434G mutation modifies Ca2+ -dependent activation, but we find no evidence of a direct effect on activation by Mg2+ or voltage. The resulting enhancement of BK channel function leads to an increase in brain excitability, possibly due to more rapid repolarization of action potentials.

Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel.

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.

Binding site in eag voltage sensor accommodates a variety of ions and is accessible in closed channel.

In ether-a-go-go K+ channels, voltage-dependent activation is modulated by ion binding to a site located in an extracellular-facing crevice between transmembrane segments S2 and S3 in the voltage sensor. We find that acidic residues D278 in S2 and D327 in S3 are able to coordinate a variety of divalent cations, including Mg2+, Mn2+, and Ni2+, which have qualitatively similar functional effects, but different half-maximal effective concentrations. Our data indicate that ions binding to individual voltage sensors in the tetrameric channel act without cooperativity to modulate activation gating. We have taken advantage of the unique phenotype of Ni2+ in the D274A channel, which contains a mutation of a nonbinding site residue, to demonstrate that ions can access the binding site from the extracellular solution when the voltage sensor is in the resting conformation. Our results are difficult to reconcile with the x-ray structure of the KvAP K+ channel, in which the binding site residues are widely separated, and with the hydrophobic paddle model for voltage-dependent activation, in which the voltage sensor domain, including the S3-S4 loop, is near the cytoplasmic side of the membrane in the closed channel.

Atomic proximity between S4 segment and pore domain in Shaker potassium channels.

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.

Structural basis of two-stage voltage-dependent activation in K+ channels.

The structure of the voltage sensor and the detailed physical basis of voltage-dependent activation in ion channels have not been determined. We now have identified conserved molecular rearrangements underlying two major voltage-dependent conformational changes during activation of divergent K(+) channels, ether-à-go-go (eag) and Shaker. Two conserved arginines of the S4 voltage sensor move sequentially into an extracellular gating pocket, where they interact with an acidic residue in S2. In eag, these transitions are modulated by a divalent ion that binds in the gating pocket. Conservation of key molecular details in the activation mechanism confirms that voltage sensors in divergent K(+) channels share a common structure. Molecular modeling reveals that structural constraints derived from eag and Shaker specify the unique packing arrangement of transmembrane segments S2, S3, and S4 within the voltage sensor.

Structural organization of the voltage sensor in voltage-dependent potassium channels.

The structural organization of the voltage sensor in K+ channels has been investigated by second site suppressor analysis in Shaker and by identification of a metal ion binding site in ether-à-go-go (eag). In Shaker, two groups of interacting charged residues have been identified. K374 in the S4 segment interacts with E293 in S2 and D316 in S3, whereas E283 in S2 interacts with R368 and R371, two voltage-sensing residues in S4. Interactions of E283 with R368 and R371 are voltage dependent. The results suggest that E283 is located in a water-filled pocket near the extracellular surface of the protein. During voltage-dependent activation of Shaker channels, R368 and R371 move into this pocket and come into proximity with E283. In eag channels, extracellular Mg2+ directly modulates the activation process by binding to two acidic residues that are located in an analogous pocket. These acidic residues are found only in eag family members, accounting for the specificity of Mg2+ modulation to that family. These compatible results from Shaker and eag suggest a model for the packing and conformational changes of transmembrane segments in the voltage sensor of K+ channels.