Voltage Inc Games Crack Site
Harriet Wehrenberg
The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.
Voltage-gated sodium (Nav) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Nav channel inhibitors that exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) or Nav1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1-S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the "extracellular" facing regions of the S2 and S3 transmembrane segments of Nav1.3 and Nav1.7 seem to be major determinants of Nav subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Nav1.3)/D1586 (Nav1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Nav channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.
Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.
Light-oxygen-voltage (LOV) proteins form a sensory photoreceptor class that elicit a wide palette of physiological responses to blue light across archaea, bacteria, protists, fungi, and plants1,2,3. Complementing their eminent role in nature, LOV receptors also serve as genetically encoded actuators in optogenetics4 for the spatiotemporally precise control by light of cellular state and processes5. At the heart of these responses lies the flavin-binding LOV photosensor module which belongs to the Per-ARNT-Sim superfamily6 and comprises several α-helices (denoted Cα, Dα, Eα, and Fα) arranged around a five-stranded antiparallel β-sheet (strands Aβ, Bβ, Gβ, Hβ, and Iβ)7,8 (Suppl. Fig. 1). Light absorption by the flavin triggers a well-studied photocycle2,9,10,11, as part of which an initial electronically excited singlet state (S1) decays within nanoseconds to a triplet state (T1) (Fig. 1a). Likely via a radical-pair mechanism12, T1 reacts within microseconds to the signaling state, characterized by a covalent thioadduct between a highly conserved cysteine residue in the LOV photosensor and the C4a atom of the flavin isoalloxazine ring system. Once illumination ceases, the signaling state passively reverts to the resting state in the base-catalyzed dark-recovery reaction13. Thioadduct formation entails a hybridization change of the flavin C4a atom from sp2 to sp3 and concomitant protonation of the adjacent N5 atom. The resultant conversion of the N5 position from a hydrogen bond acceptor to a donor serves as the principal trigger14 for a raft of conformational and dynamic transitions, that depending upon LOV receptor, culminate in order-disorder transitions15, oligomerization16, or other tertiary and quaternary structural changes17. A highly conserved glutamine residue in strand Iβ is situated immediately adjacent to the flavin and has been identified as instrumental in reading out the flavin N5 position and eliciting the downstream transitions. Supported by spectroscopy, structural and functional data, chemical reasoning, and molecular simulations8,18,19,20,21,22,23,24, the glutamine is widely held to rotate its amide sidechain to accommodate N5 protonation in the signaling state. As a corollary, additional hydrogen-bond rearrangements permeate the LOV photosensor and propagate towards the β-sheet scaffold. As recently proposed25, glutamine reorientation, and signal propagation may be aided by transient rearrangements of two conserved asparagine residues that coordinate the pteridin portion of the flavin.
Notwithstanding the strong conservation of the glutamine residue and its established role in LOV receptors, recent reports indicate that at least in certain proteins, productive signaling responses to blue light may occur without the glutamine26,27,28. Potentially, these responses harness steric interactions rather than hydrogen-bonding changes as a means of signal transduction26,29. By contrast, reports on other LOV receptors considered the glutamine essential for eliciting blue-light responses21,30.
To rationalize these conflicting findings and to provide further insight into signal transduction, here we systematically investigated the role of the conserved glutamine in several model LOV receptors (Fig. 1b and Suppl. Fig. 1). Unexpectedly, the glutamine residue is not essential in LOV signaling as productive blue-light responses were generally maintained even in its absence. Almost all other amino acids could functionally substitute for the conserved glutamine, with notable exceptions. High-resolution crystal structures of the paradigm Avena sativa phototropin 1 LOV2 (AsLOV2) domain revealed that after glutamine substitution by leucine, closely similar structural changes are evoked by light as in the wild type. Based on structural data, chemical reasoning, and molecular simulations, we propose that in the absence of the glutamine, water molecules relay hydrogen-bonding signals from the flavin N5 position to the LOV β-sheet. The ability to transduce light signals without the glutamine appears to be an inherent, general trait of LOV receptors and may reflect their evolutionary origin. This notion finds support in the existence in nature of numerous LOV receptors that lack the conserved glutamine and presumably serve as blue-light receptors, as we confirm for a glutamine-deficient, proteobacterial LOV-diguanylate cyclase.
To probe the role of the active-site glutamine (position Q123) in signal transduction, we substituted this residue for all 19 other canonical amino acids. Strikingly, most of the resultant glutamine-deficient YF1 variants prompted a blue-light-induced reduction of reporter gene fluorescence, similar to the original YF1 and almost regardless of which residue replaced the glutamine. These data clearly indicate that at least in the pDusk setup, the majority of residue substitutions, including alanine, cysteine, glutamic acid and leucine, leave light-dependent signal transduction largely unimpaired. Merely, the substitution by proline and the bulky aromatic amino acids His, Trp, and Tyr abolished responsiveness and resulted in high reporter expression independently of light. Similarly, the Q123R variant did not react to light but exhibited constitutively low reporter fluorescence. The Q123A and Q123N exchanges were previously assessed in Bacillus subtilis YtvA from which YF1 derives34,35. As probed by photocalorimetry and in vivo analysis, the Q123A substitution slightly impaired signal transduction, but Q123N completely abolished any light responsiveness. Whereas the Q123A findings are consistent with the present data, YF1 Q123N retained attenuated light responses. The divergent observations for the Q123N exchange might be tied to the different effector modules in BsYtvA and YF1. We note that asparagine in this position can principally support LOV signal transduction, as indicated by partial preservation of light responsiveness in the corresponding Q513N variant of AsLOV221.
We next recorded the dark recovery after blue-light exposure and found the return to the dark-adapted state 10-fold decelerated in Q123L relative to YF1 (Suppl. Fig. 2). The Q123P variant exhibited even slower kinetics that was not completed even after several days. Given that the Q123L variant principally retained the capability of transducing signals (see Fig. 2b), we reasoned that modification of the active-glutamine provides an additional, little-tapped means of altering recovery kinetics39 and thus modulating photosensitivity at photostationary state40. To explore this effect, we assessed the response of YF1 Q123L to pulsatile blue-light illumination41 in the pDawn system that derives from pDusk but exhibits an inverted response to blue light31. The Q123L variant was toggled by much lower light doses than YF1, fully consistent with its retarded dark recovery (Suppl. Fig. 3). Compared to the V28I substitution, which also decelerates dark recovery by around 10-fold39,41,42, the Q123L exchange was somewhat less sensitive to blue light. Combining the substitutions V28I and Q123L did not provide a further gain but slightly reduced the effective light sensitivity.
We next addressed whether the striking ability to transduce light signals without the conserved glutamine residue is specific for YF1 or more widely shared across LOV receptors. To this end, we examined light-dependent signaling responses in Nakamurella multipartita PAL46, as a naturally occurring LOV receptor, and the A. sativa phototropin 1 LOV2 domain, as the arguably best-studied and optogenetically most widely used LOV module5,15,47,48. Notably, NmPAL differs from YF1 by an unusual C-terminal arrangement of its LOV photosensor and binds a small RNA aptamer sequence-specifically and in a light-activated manner46. By embedding this aptamer directly upstream of the Shine-Dalgarno sequence in an mRNA encoding the fluorescent DsRed protein, NmPAL activity and response to light can be assessed in a bacterial reporter assay (Fig. 3a). In its dark-adapted state, wild-type NmPAL has little affinity for the aptamer, and DsRed is readily expressed. Light-induced binding by NmPAL interferes with expression, presumably at the translational level, and reporter fluorescence is diminished by 10-fold (Fig. 3b). Using this assay, we tested the effect of replacing the active-site glutamine (residue Q347 in NmPAL) with histidine, leucine, or proline. Consistent with the findings for YF1, the resultant Q347H and Q347P variants no longer exhibited light-induced changes in reporter fluorescence. As in the YF1 case, the proline variant had constitutive activity similar to the dark-adapted parental wild-type NmPAL. Conversely, for Q347H we observed constitutively low fluorescence values, indicative of RNA binding and thus corresponding to light-adapted wild-type NmPAL. This contrasts with YF1 where the corresponding histidine variant functionally corresponded to the dark-adapted state of the parental receptor. The Q347L variant exhibited a light-induced decrease of DsRed fluorescence by around 17-fold, thus even surpassing the value for wild-type NmPAL. Taken together, the results from the NmPAL reporter assay are broadly consistent with the findings for YF1 in that the leucine substitution supported light responses to a significant extent whereas the histidine and proline substitutions incurred a loss of light-dependent signal transduction.
7fc3f7cf58