P2X receptors are ATP-gated nonselective cation channels. Functional receptors are assembled as homotrimers or heterotrimers of seven cloned subunits. Each subunit contains two transmembrane domains linked by a large extracellular loop that is required for agonist binding. So far, there is no direct evidence indicating whether the agonist binding site is formed within one subunit or at the interface of two neighboring subunits. Here we used a disulfide cross-linking approach to identify pairs of residues that are in close proximity within the ATP binding site of the P2X 1 homotrimer. Eight amino acid residues that have previously been shown to be essential for high ATP potency (K68, K70, F185, K190, F291, R292, R305, and K309) were substituted by cysteine residues, and the respective mutant subunits were pairwise expressed in Xenopus laevis oocytes. Nonreducing SDS-PAGE analysis of the purified receptors revealed a spontaneous and specific dimer formation between the K68C and F291C mutants. An almost complete cross-link into trimers was achieved with the K68C/F291C double mutant, consistent with the formation of intersubunit disulfide bridges. In support of this interpretation, twoelectrode voltage-clamp analysis of the K68C/F291C mutations introduced into a nondesensitizing P2X 2-1 chimera showed only small ATP-activated currents that, however, increased ϳ60-fold after extracellular application of the reducing agent dithiothreitol. In addition, we show that a K68C/K309C double mutant is nonfunctional and can be functionally rescued by coexpression with nonmutated subunits. Our data are consistent with loops from neighboring P2X subunits forming the ATP-binding site in P2X receptors.

P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodaminemaleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoylbenzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain. P 2X receptors (P2XRs) represent a family of nonselective cation channels gated by extracellular ATP. They are widely distributed in mammalian tissues and have been shown to be involved in diverse physiological functions (1). The seven known subunits all contain two transmembrane domains linked by a large extracellular loop. Functional receptors are homo-or heteromeric trimers (2, 3).Based on mutagenesis studies, it has been suggested that conserved positively charged and aromatic residues are crucial for ATP binding, presumably by interacting with the negatively charged phosphate chain of ATP (4-6) and its adenine ring (6), respectively. We have previously shown that replacement of two of these residues, K68 and F291, by cysteine residues allows disulfide cross-linking between neighboring P2X1 subunits and that this reaction is prevented in the presence of ATP. Based on these data, we concluded that the ATP binding sites are located at the subunit interfaces (7,8). This conclusion is in good agreement with the positions of the relevant amino acids in the crystal structure of the unliganded P2X4R from zebrafish (2). This zP2X4 structure revealed an ion channel architecture that resembles a dolphin, with the transmembrane helices and the extracellular region forming the fluke and the upper body, respectively. Att...

Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca2+ biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations.

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Pure Eu3+ ion doped BaF2 nanoparticles with tunable properties or property combinations are accessible via an ionic liquid-assisted solvothermal method. Structural parameters such as cell parameters, lattice strain, and especially morphology are judiciously tuned with calcination temperatures. For example, tensile strain is observed in samples calcined up to 400 °C; however, compressive strain appears in samples calcined at 600 °C and beyond. Larger surface area up to the sample calcined at 400 °C and observation of layer structure at higher calcinations temperature (650 °C and beyond) have been rationalized based on secondary nucleation. Three-dimensional island-like morphology with step-like layer structure caused by secondary nucleation and self-assembly are visualized and explained by scanning electron microscope analysis. Moreover, emission intensity, decay time, quantum yield, and Judd-Ofelt parameter of the Eu3+ ions increase systematically with calcination temperature to a maximum at 400 °C, above which they decrease and finally vanish at 800 °C. Our results suggest that smaller-sized nanoparticles with 3-dimensional island-like structures, generated due to secondary nucleation at higher calcinations temperature, may cause the increase of surface defects and subsequent luminescence quenching. To the best of our knowledge, the interplay between calcinations and secondary nucleation followed by drastic changes in the luminescence properties is new and previously unreported for the nanopowders. In addition, to improve the dispersibility, as-prepared nanoparticles are coated with silica and solubility of nanoparticles is measured in different solvents so that it can be useful for bioimaging applications also.

For the assessment of cardiac function, heartbeat represents one key parameter. Current methods of heartbeat measurements in the zebrafish larvae usually require larval immobilization, fluorescent transgenic strains and a confocal microscope, costly commercial software for analysis, or strong programming skills if the software is open-source. Here, we present a simple yet powerful method of heartbeat analysis using untethered, unlabeled zebrafish larva using ImageJ (open-source software), which does not require programming skills. We named it as ZebraPace for Zebrafish Precise Algorithm for Cardiac-rhythm Estimation. ZebraPace works directly with AVI videos and requires no image processing steps. ZebraPace uses pixel intensity change in a grayscale video to count the number of beats. We have validated the ZebraPace method by pharmacological alterations of the heartbeat in zebrafish larvae of 48 and 72 hpf stages. We have also determined beat-to-beat interval, which relates to rhythmicity of heartbeat. The results obtained by using ZebraPace corroborates well with the heartbeat values previously reported for similarly aged larvae as determined by using specialized software. We believe that the ZebraPace method is simple, cost-effective, and easy to grasp as it involves fewer steps. It not only reduces the manual workload but also eliminates sample preparation time and researcher subjectivity.