No abstract

Summary Astrocytes are the most abundant glial cells in the central nervous system (CNS) and participate in synaptic, circuit, and behavioral functions. The well‐developed protoplasmic astrocytes contain numerous processes forming well‐delineated bushy territories that overlap by as little as 5% at their boundaries. This highly complex morphology, with up to approximately 80% of the cell's membrane constituted by fine processes with dimensions on the tens of nanometer scale and high surface area to volume ratios, comes in contact with synapses, blood vessels, and other glial cells. Recent progress is challenging the conventional view that astrocytes are morphologically homogeneous throughout the brain; instead, they display circuit‐ and region‐specific morphological diversity that may contribute to the heterogeneous astrocyte‐neuron spatiotemporal interplay in different brain areas. Further, the fine structure of astrocytes is found to be highly plastic and activity‐dependent. We are beginning to understand how astrocyte structural plasticity contributes to brain functions. The change/loss of astrocyte morphology, traditionally known as a hallmark for reactive astrogliosis, is a common pathological feature in many neurological disorders. However, recent data suggest the fine structural deficits preceding reactive astrogliosis may drive disease progression. This review summarizes recent advances in astrocyte morphological diversity, plasticity, and disease‐related deficits.

No abstract

Astrocytes are complex bushy cells that serve important functions through close contacts between their processes and synapses. However, the spatial interactions and dynamics of astrocyte processes relative to synapses have proven problematic to study in adult living brain tissue. Here, we report a genetically targeted neuron-astrocyte proximity assay (NAPA) to measure astrocyte-synapse spatial interactions within intact brain preparations and at synaptic distance scales. The method exploits resonance energy transfer between extracellularly displayed fluorescent proteins targeted to synapses and astrocyte processes. We validated the method in the striatal microcircuitry following in vivo expression. We determined the proximity of striatal astrocyte processes to distinct neuronal input pathways, to D1 and D2 medium spiny neuron synapses, and we evaluated how astrocyte-to-excitatory synapse proximity changed following cortical afferent stimulation, during ischemia and in a model of Huntington's disease. NAPA provides a simple approach to measure astrocyte-synapse spatial interactions in a variety of experimental scenarios. VIDEO ABSTRACT.

The opening of ligand-gated ion channels in response to agonist binding is a fundamental process in biology. In ATP-gated P2X receptors, little is known about the molecular events that couple ATP binding to channel opening. In this paper, we identify structural changes of the ATP site accompanying the P2X2 receptor activation by engineering extracellular zinc bridges at putative mobile regions as revealed by normal mode analysis. We provide evidence that tightening of the ATP sites shaped like open 'jaws' induces opening of the P2X ion channel. We show that ATP binding favours jaw tightening, whereas binding of a competitive antagonist prevents gating induced by this movement. Our data reveal the inherent dynamic of the binding jaw, and provide new structural insights into the mechanism of P2X receptor activation.

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATPderived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATPbinding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.affinity labeling | chemical modification | purinergic receptor P 2X receptors are oligomeric ATP-gated ion channels selective to cations (1) and are involved in physiological processes as diverse as synaptic transmission, the response to inflammation, and pain perception (2). Upon ATP binding, structural rearrangements of the subunit interface (3-5) lead to the opening of the ion channel (6-8), but the entire molecular sequence of events that couple ATP binding to channel opening remains unknown. The recent X-ray structure of the P2X4 receptor in a closed resting state represents in this regard a decisive step (9). It confirms the trimeric stoichiometry of the ion channel, in agreement with the fact that there are three activatable ATP-binding sites (10), and provides a structural context to interpret functional data (11).Early studies based on mutagenesis data have identified highly conserved extracellular residues important for ATP function and have proposed that ATP binding occurs through the extracellular domain (12-19), presumably at the subunit interface (15,17). When mapped on the crystal structure, most of these residues are observed to line a large and deep intersubunit cavity shaped like an open "jaw" and located approximately 45 Å away from the ion channel domain (9). This observation thus suggests that these residues participate in ATP binding; however, the crystal structure was solved in the absence of ATP, and therefore no direct evidence support this hypothesis to date.We used the proximity-dependent "tethering" approach (20) to localiz...

No abstract

The recent crystal structure of the ATP-gated P2X4 receptor revealed a static view of its architecture, but the molecular mechanisms underlying the P2X channels activation are still unknown. By using a P2X2 model based on the x-ray structure, we sought salt bridges formed between charged residues located in a region that directly connects putative ATP-binding sites to the ion channel. To reveal their significance for ion channel activation, we made systematic charge exchanges and measured the effects on ATP sensitivity. We found that charge reversals at the interfacial residues Glu 63 and Arg 274 produced gain-of-function phenotypes that were cancelled upon paired charge swapping. These results suggest that a putative intersubunit salt bridge formed between Glu 63 and Arg 274 contributes to the ion channel function. Engineered cysteines E63C and R274C formed redoxdependent cross-links in the absence of ATP. By contrast, the presence of ATP reduced the rate of disulfide bond formation, indicating that ATP binding might trigger relative movement of adjacent subunits at the level of Glu 63 and Arg 274 , allowing the transmembrane helices to open the channel.P2X receptors (P2XRs) 5 are membrane cation channels gated by extracellular ATP. They are widely distributed in excitable and nonexcitable cells of vertebrates (1) and play key roles in synaptic transmission (2), presynaptic modulation (3), taste sensation (4, 5), pain signaling (6, 7), and intestinal motility (8).P2XRs are allosteric trimeric ion channels formed by the oligomerization of three identical or homologous subunits (9, 10). Each subunit (there are seven identified so far in mammals, termed P2X1 through P2X7) possesses intracellular N and C termini and two transmembrane segments, termed TM1 and TM2, joined by an extracellular ectodomain. The binding of ATP to the ectodomain promotes the rapid opening of the ion channel, referred to as gating. Once the channel is opened, cations transit through the pore down their electrochemical gradients, leading to the transient influx of sodium and calcium into the cell. This in turn leads to depolarization of the cell and downstream calcium signaling. It is thought that gating involves long range conformational changes that are transduced from the ATP-binding sites to the ion channel and even to the cytosolic domain (11). However, the molecular mechanisms underlying the gating process in P2XR are still largely unknown.Very recently, the crystal structure of zebra fish P2X4R (zfP2X4R) has been solved by x-ray crystallography at a resolution of 3.1 Å (12). The structure was solved in the absence of ATP and probably represents the closed state of the ion channel. The location of the ATP-binding sites remains unknown; however, it has been suggested that the nucleotide binds to deep intersubunit grooves, located on the outside of the trimer, 45 Å from the ion channel domain, and surrounded by conserved residues previously shown to be important for ATP function (12). This structure thus represents an outstanding advance...