The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K + channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltagesensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.oltage-sensitive cation channels play crucial roles in brain and cardiac excitability. These channels are endowed with two main transmembrane modules, a voltage-sensing domain (VSD) and a pore domain. Mutations of ion channel genes in humans lead to severe inherited neurological, cardiovascular, or metabolic disorders, called "channelopathies" (1). So far, the medicinal toolbox has focused on the pore domain and its gate in an attempt to cure ion channel-related dysfunctions by channel blockers or openers (2).In contrast, the VSD of voltage-gated cation channels was virtually not exploited for therapeutic purposes. VSDs are found in voltage-dependent cation channels and other voltage-regulated proteins (3). In voltage-gated cation channels, the linker S4-S5 of the VSD serves as an electromechanical coupling device, which opens the channel pore. VSDs have also been recently characterized in voltage-regulated proteins that lack associated ion channel pores (4-6). A voltage-sensitive phosphatase, Ci-VSP, has a VSD that is coupled to a phosphatase domain (4). In the human voltage-activated proton channel (Hv1), the VSD itself functions as a proton channel (5-7).Crystallographic studies of voltage-gated K + channels (Kv) have described the VSD architecture in its open-state conformation. It forms a module of four membrane-spanning segments (S1-S4) with the S3b helix and the charge-bearing S4 helix forming a helix-turn-helix structure, termed the "paddle ...

The unfolded protein response PERK branch induces recruitment of misfolded proteins and the ubiquitin ligase HRD1 to the ER-derived quality control compartment (ERQC), a staging ground for ER-associated degradation (ERAD). This is accomplished by up-regulation of homocysteine-induced ER protein (Herp), which recruits the ERAD complex at the ERQC.

We present high field DEER (double electron-electron resonance) distance measurements using Gd(3+) (S = 7/2) spin labels for probing peptides' conformations in solution. The motivation for using Gd(3+) spin labels as an alternative for the standard nitroxide spin labels is the sensitivity improvement they offer because of their very intense EPR signal at high magnetic fields. Gd(3+) was coordinated by dipicolinic acid derivative (4MMDPA) tags that were covalently attached to two cysteine thiol groups. Cysteines were introduced in positions 15 and 27 of the peptide melittin and then two types of spin labeled melittins were prepared, one labeled with two nitroxide spin labels and the other with two 4MMDPA-Gd(3+) labels. Both types were subjected to W-band (95 GHz, 3.5 T) DEER measurements. For the Gd(3+) labeled peptide we explored the effect of the solution molar ratio of Gd(3+) and the labeled peptide, the temperature, and the maximum dipolar evolution time T on the DEER modulation depth. We found that the optimization of the [Gd(3+)]/[Tag] ratio is crucial because excess Gd(3+) masked the DEER effect and too little Gd(3+) resulted in the formation of Gd(3+)-tag(2) complexes, generating peptide dimers. In addition, we observed that the DEER modulation depth is sensitive to spectral diffusion processes even at Gd(3+) concentrations as low as 0.2 mM and therefore experimental conditions should be chosen to minimize it as it decreases the DEER effect. Finally, the distance between the two Gd(3+) ions, 3.4 nm, was found to be longer by 1.2 nm than the distance between the two nitroxides. The origin and implications of this difference are discussed.

We present a new approach to obtain details on the distribution and average structure and locations of membrane-associated peptides. The approach combines (i) pulse double electron-electron resonance (DEER) to determine intramolecular distances between residues in spin labeled peptides, (ii) electron spin echo envelope modulation (ESEEM) experiments to measure water exposure and the direct interaction of spin labeled peptides with deuterium nuclei on the phospholipid molecules, and (iii) Monte Carlo (MC) simulations to derive the peptide-membrane populations, energetics, and average conformation of the native peptide and mutants mimicking the spin labeling. To demonstrate the approach, we investigated the membrane-bound and solution state of the well-known antimicrobial peptide melittin, used as a model system. A good agreement was obtained between the experimental results and the MC simulations regarding the distribution of distances between the labeled amino acids, the side chain mobility, and the peptide's orientation. A good agreement in the extent of membrane penetration of amino acids in the peptide core was obtained as well, but the EPR data reported a somewhat deeper membrane penetration of the termini compared to the simulations. Overall, melittin adsorbed on the membrane surface, in a monomeric state, as an amphipatic helix with its hydrophobic residues in the hydrocarbon region of the membrane and its charged and polar residues in the lipid headgroup region.

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