imaging-intracellular-ca2-signals-striatal-astrocytes-from-adult-mice

Imaging Intracellular Ca<sup>2+</sup> Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

University of California Los Angeles

ATP-gated P2X receptors (P2XRs) are ligand-gated ion channels (LGICs) presumably trimeric. To date, no experimental high-resolution structures are available. Recent X-ray structure of the acid-sensing ion channel 1 (ASIC1) revealed an unexpected trimeric ion channel. Beside their quaternary structure, P2XR and ASIC1 share common membrane topologies, but no significant sequence similarity. In order to overcome this low sequence resemblance, we have developed comparative models of P2X(2)R based on secondary structure predictions using the crystal structure of ASIC1 as template. These models were constrained to be consistent with known arrangement of disulfide bridges. They agreed with cross-linking experiments and supported inter-subunit ATP-binding sites. One of our models reconciled most existing data and provides new structural insights for a plausible mechanism of gating, thus encouraging new experiments.

Comparative models of P2X2 receptor support inter-subunit ATP-binding sites.

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 redox-dependent 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.

A putative extracellular salt bridge at the subunit interface contributes to the ion channel function of the ATP-gated P2X2 receptor.

Flavonoids, important plant pigments, have been shown to allosterically modulate brain GABA(A) receptors (GABA(A)Rs). We previously reported that trans-6,4'-dimethoxyretrochalcone (Rc-OMe), a hydrolytic derivative of the corresponding flavylium salt, displayed nanomolar affinity for the benzodiazepine binding site of GABA(A)Rs. Here, we evaluate the functional modulations of Rc-OMe, along with two other synthetic derivatives trans-6-bromo-4'-methoxyretrochalcone (Rc-Br) and 4,3'-dimethoxychalcone (Ch-OMe) on GABA(A)Rs.

Retrochalcone derivatives are positive allosteric modulators at synaptic and extrasynaptic GABA(A) receptors in vitro.

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 ATP-derived 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 ATP-binding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.

Agonist trapped in ATP-binding sites of the P2X2 receptor.

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.

Tightening of the ATP-binding sites induces the opening of P2X receptor channels.

Ligand-gated ion channels: new insights into neurological disorders and ligand recognition.

The molecular mechanism underlying channel opening in response to agonist binding remains a challenging issue in neuroscience. In this regard, many efforts have been recently undertaken in ATP-gated P2X receptors. Among those efforts, we have provided evidence in the P2X2 receptor that tightening of ATP sites upon agonist binding induces opening of the ion channel. Here we extend our analysis to show that the sulfhydryl-reactive ATP analog 8-thiocyano-ATP (NCS-ATP), a potent P2X2 agonist, when covalently labeled in the ATP-binding site at position Leu186 likely favors the tightening mechanism, but not the channel opening mechanism. Our data predict the existence of intermediate or preactivation state(s) trapped by NCS-ATP, in which tightening of the binding site is favored while the channel is still closed. We propose that this (these) intermediate ATP-bound state(s) prime(s) channel gating in the P2X2 receptor.

Intermediate closed channel state(s) precede(s) activation in the ATP-gated P2X2 receptor.

P2X receptors are nonselective cation channels gated by extracellular ATP. They represent new therapeutic targets, and they form channels with a unique trimeric architecture. In 2009, the first crystal structure of a P2X receptor was reported, in which the receptor was in an ATP-free, closed channel state. However, our view recently changed when a second crystal structure was reported, in which a P2X receptor was bound to ATP and resolved in an open channel conformation. This remarkable structure not only confirms many key experimental data, including the recent mechanisms of ATP binding and ion permeation, but also reveals unanticipated mechanisms. Certainly, this new information will accelerate our understanding of P2X receptor function and pharmacology at the atomic level.

Ruotian Jiang

Department of Physiology

University of California Los Angeles

Imaging Intracellular Ca²⁺ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Ruotian Jiang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology

University of California Los Angeles

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Department of Physiology

Imaging Intracellular Ca²⁺ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators