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

Errors in the measurement of voltage-activated ion channels in cell-attached patch-clamp recordings

Patch-clamp recording techniques have revolutionized understanding of the function and sub-cellular location of ion channels in excitable cells. The cell-attached patch-clamp configuration represents the method of choice to describe the endogenous properties of voltage-activated ion channels in the axonal, somatic and dendritic membrane of neurons, without disturbance of the intracellular milieu. Here, we directly examine the errors associated with cell-attached patch-clamp measurement of ensemble ion channel activity. We find for a number of classes of voltage-activated channels, recorded from the soma and dendrites of neurons in acute brain-slices and isolated cells, that the amplitude and kinetics of ensemble ion channel activity recorded in cell-attached patches is significantly distorted by transmembrane voltage changes generated by the flow of current through the activated ion channels. We outline simple error–correction procedures that allow a more accurate description of the density and properties of voltage-activated channels to be incorporated into computational models of neurons

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