96 research outputs found
A fast neutron spectrometer of advanced design Final report, 1 May 1966 - 30 Jun. 1967
Helium 3 sandwich type fast neutron spectrometer desig
Fast-neutron spectrometer developments
Li6 sandwich-type neutron spectrometer is equipped with proportional counter for particle identification. System uses current-sensitive preamplifiers to minimize pile-up of gamma-ray and particle pulses
Identifying Regulators for EAG1 Channels with a Novel Electrophysiology and Tryptophan Fluorescence Based Screen
Ether-à-go-go (EAG) channels are expressed throughout the central nervous system and are also crucial regulators of cell cycle and tumor progression. The large intracellular amino- and carboxy- terminal domains of EAG1 each share similarity with known ligand binding motifs in other proteins, yet EAG1 channels have no known regulatory ligands.Here we screened a library of small biologically relevant molecules against EAG1 channels with a novel two-pronged screen to identify channel regulators. In one arm of the screen we used electrophysiology to assess the functional effects of the library compounds on full-length EAG1 channels. In an orthogonal arm, we used tryptophan fluorescence to screen for binding of the library compounds to the isolated C-terminal region.Several compounds from the flavonoid, indole and benzofuran chemical families emerged as binding partners and/or regulators of EAG1 channels. The two-prong screen can aid ligand and drug discovery for ligand-binding domains of other ion channels
Oocyte expression with injection of purified T7 RNA polymerase.
International audienceThe Xenopus oocyte is a widely used system for protein expression. Investigators have had the choice between two different techniques: injection into the cytoplasm of in vitro transcribed complementary RNA (cRNA) or injection into the nucleus of complementary DNA (cDNA). We report on a third expression technique that is based on the combined injection of cDNA and purified T7 RNA polymerase directly into the cytoplasm of oocytes
Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels
Molecular determinants of ion channel tetramerization are well characterized, but those involved in heteromeric channel assembly are less clearly understood. The heteromeric composition of native channels is often precisely controlled. Cyclic nucleotide-gated (CNG) channels from rod photoreceptors exhibit a 3:1 stoichiometry of CNGA1 and CNGB1 subunits that tunes the channels for their specialized role in phototransduction. Here we show, using electrophysiology, fluorescence, biochemistry, and X-ray crystallography, that the mechanism for this controlled assembly is the formation of a parallel 3-helix coiled-coil domain of the carboxy-terminal leucine zipper region of CNGA1 subunits, constraining the channel to contain three CNGA1 subunits, followed by preferential incorporation of a single CNGB1 subunit. Deletion of the carboxy-terminal leucine zipper domain relaxed the constraint and permitted multiple CNGB1 subunits in the channel. The X-ray crystal structures of the parallel 3-helix coiled-coil domains of CNGA1 and CNGA3 subunits were similar, suggesting that a similar mechanism controls the stoichiometry of cone CNG channels
Co-Crystal Structures of PKG Iβ (92–227) with cGMP and cAMP Reveal the Molecular Details of Cyclic-Nucleotide Binding
Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity
Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel
An unusual mechanism of ion channel regulation generates voltage-dependent gating in the absence of a canonical voltage-sensing domain
Lipid-dependent gating of a voltage-gated potassium channel
Recent studies hypothesized that phospholipids stabilize two voltage-sensing arginine residues of certain voltage-gated potassium channels in activated conformations. It remains unclear how lipids directly affect these channels. Here, by examining the conformations of the KvAP in different lipids, we showed that without voltage change, the voltage-sensor domains switched from the activated to the resting state when their surrounding lipids were changed from phospholipids to nonphospholipids. Such lipid-determined conformational change was coupled to the ion-conducting pore, suggesting that parallel to voltage gating, the channel is gated by its annular lipids. Our measurements recognized that the energetic cost of lipid-dependent gating approaches that of voltage gating, but kinetically it appears much slower. Our data support that a channel and its surrounding lipids together constitute a functional unit, and natural nonphospholipids such as cholesterol should exert strong effects on voltage-gated channels. Our first observation of lipid-dependent gating may have general implications to other membrane proteins
Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels
Combinations of bio-active dietary constituents affect human white adipocyte function in-vitro
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