Search Results (12)

Pregabalin (PGB) exerts its therapeutic effects by binding to the α₂δ auxiliary subunits of voltage-gated calcium channels and modulates synaptic transmission in the brain. However, its influence on cerebellar parallel fiber–Purkinje cell (PF–PC) synaptic transmission remains unclear. In the present study, we investigated the effects of PGB on PF–PC synaptic transmission using whole-cell patch-clamp recording, glutamate fluorescence imaging, immunohistochemistry, co-immunoprecipitation, Western blotting, and pharmacological approaches. Micro-application of PGB to the cerebellar molecular layer induced a concentration-dependent inhibition of PF–PC excitatory postsynaptic currents (EPSCs), accompanied by an increased paired-pulse ratio. The inhibitory effect of PGB on PF–PC EPSCs was abolished by extracellular blockade of N-methyl-D-aspartate receptors (NMDAR) or their GluN2A subtype, as well as by disruption of α₂δ-1–NMDAR complexes, but not by intracellular NMDAR inhibition. Glutamate sensor imaging further showed that PGB markedly reduced the fluorescence intensity of glutamate release evoked by PF stimulation. In the presence of tetrodotoxin (TTX) and a gamma-aminobutyric acid type A (GABAA) receptor antagonist, PGB reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude. The PGB-induced reduction in mEPSC frequency was fully abolished by extracellular blockade of GluN2A-containing NMDARs or disruption of α₂δ-1–NMDAR complexes. Similarly, the inhibitory effects of PGB on PF–PC EPSCs and mEPSCs were eliminated by extracellular PKA inhibition, but not by intracellular protein kinase A (PKA) inhibition. Western blot analysis showed that PGB significantly increased PKA phosphorylation in the molecular layer of the cerebellar cortex. Immunoreactivity for GluN2A and α₂δ-1 subunits was colocalized within the molecular layer and abundantly distributed around the dendrites and somata of PCs. Co-immunoprecipitation further verified that α₂δ-1 was co-precipitated with GluN1 in cerebellar molecular layer tissue samples. The results indicate that PGB depresses glutamate release from parallel-fiber terminals in the mouse cerebellar cortex through the presynaptic α₂δ-1-coupled GluN2A-containing NMDAR/PKA signaling pathway, thereby attenuating PF–PC synaptic transmission. Full article

Tetrodotoxin (TTX), a potent Site-1 sodium channel blocker (S1SCB), offers highly effective local anesthetic properties with minimal addiction potential. To fully leverage TTX’s capabilities as a local anesthetic, it is crucial to develop a drug delivery system that balances its systemic toxicity with its therapeutic efficacy. Recent studies have shown that peptide mixtures, derived from fragments of Site-1 sodium channel proteins and enhanced with hydrophobic tails (designated MP1 and MP2), can self-assemble into nanostructures that exhibit remarkable sustained-release capabilities for TTX. Despite the profound impact that the addition of a hydrophobic tail has on altering the release behavior of the original peptides, the atomic-level interactions and mechanisms underlying this phenomenon remain poorly understood. In this study, a combination of ColabFold and molecular dynamics (MD) simulations were used to investigate the binding interactions between TTX and the nanostructures formed by MP1 and MP2 at an atomic level. Our findings agree with experimental observations and indicate that the MP1/MP2 nanostructure demonstrates greater stability and higher binding affinity for TTX compared to their non-modified counterparts, P1 and P2. The analysis of the simulations revealed that charged amino acids, specifically aspartic acid (ASP) and glutamic acid (GLU), on the peptides are crucial for strong TTX binding and serve as the primary functional sites. Additionally, the stability of the nanostructure significantly affects TTX binding affinity, elucidating why P1, P2, MP1, and MP2 exhibit different binding capabilities despite containing identical charged residues. The results reported here may provide fundamental information to drive future research and enhance the development of TTX-based drug delivery systems. Full article

Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients’ induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development. Full article

Tetrodotoxin (TTX)-binding proteins are present in toxic TTX-bearing animals, such as pufferfish and gastropods. These may prevent autotoxicity. However, TTX-binding proteins are also found in the nontoxic marine shore crab, Hemigrapsus sanguineus. Here, we isolated the TTX-binding protein, HSTBP (Hemigrapsus sanguineus TTX-binding protein), from the hemolymph of H. sanguineus and elucidated its primary structure using cDNA cloning. HSTBP, a 400 kDa acidic glycoprotein by gel filtration high-performance liquid chromatography, comprises 3 subunits, 88 kDa (subunit-1), 65 kDa (subunit-2), and 26 kDa (subunit-3) via sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced conditions. The open reading frame of the cDNA comprises 5049 base pairs encoding 1683 amino acid residues, and the mature protein contains 1650 amino acid residues from Arg³⁴ to Ser¹⁶⁸³. The three subunits are arranged in tandem in the following order: subunit-3 (Arg³⁴-Gln²⁶¹), subunit-1 (Asp²⁶²-Phe¹¹³⁸), and subunit-2 (Val¹¹³⁹-Ser¹⁶⁸³). A BLAST homology search showed weak similarity of HSTBP to clotting proteins of crustaceans (29–40%). SMART analysis revealed a von Willebrand factor (vWF)-type (⇒delete hyphen) D domain at Phe¹³⁸⁷-Gly¹⁵⁴⁴. We confirmed that the recombinant protein of HSTBP subunit-2 containing the vWF-type (⇒delete hyphen) D domain bound to TTX at a molecular ratio of 1:1. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have triggered a recent pandemic of respiratory disease and affected almost every country all over the world. A large amount of natural bioactive compounds are under clinical investigation for various diseases. In particular, marine natural compounds are gaining more attention in the new drug development process. The present study aimed to identify potential marine-derived inhibitors against the target proteins of COVID-19 using a computational approach. Currently, 16 marine clinical-level compounds were selected for computational screening against the 4 SARS-CoV-2 main proteases. Computational screening resulted from the best drug candidates for each target based on the binding affinity scores and amino acid interactions. Among these, five marine-derived compounds, namely, chrysophaentin A (−6.6 kcal/mol), geodisterol sulfates (−6.6 kcal/mol), hymenidin (−6.4 kcal/mol), plinabulin (−6.4 kcal/mol), and tetrodotoxin (−6.3 kcal/mol) expressed minimized binding energy and molecular interactions, such as covalent and hydrophobic interactions, with the SARS CoV-2 main protease. Using molecular dynamic studies, the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (ROG), and hydrogen bond (H-Bond) values were calculated for the SARS-CoV-2 main protease with a hymenidin docked complex. Additionally, in silico drug-likeness and pharmacokinetic property assessments of the compounds demonstrated favorable druggability. These results suggest that marine natural compounds are capable of fighting SARS-CoV-2. Further in vitro and in vivo studies need to be carried out to confirm their inhibitory potential. Full article

Multiple animal species have evolved resistance to the neurotoxin tetrodotoxin (TTX) through changes in voltage-gated sodium ion channels (VGSCs). Amino acid substitutions in TTX-resistant lineages appear to be positionally convergent with changes in homologous residues associated with reductions in TTX block. We used homology modeling coupled with docking simulations to test whether positionally convergent substitutions generate functional convergence at the level of TTX–channel interactions. We found little evidence that amino acids at convergent positions generated similar patterns among TTX-resistant animal lineages across several metrics, including number of polar contacts, polar contact position, and estimates of binding energy. Though binding energy values calculated for TTX docking were reduced for some TTX-resistant channels, not all TTX-resistant channels and not all of our analyses returned reduced binding energy values for TTX-resistant channels. Our results do not support a simple model of toxin resistance where a reduced number of bonds between TTX and the channel protein prevents blocking. Rather models that incorporate flexibility and movement of the protein overall may better describe how homologous substitutions in the channel cause changes in TTX block. Full article

Pufferfish are considered a culinary delicacy but require careful preparation to avoid ingestion of the highly toxic tetrodotoxin (TTX), which accumulates in certain tissues. In this study, the tissue distribution of peroxiredoxin-1 from Takifugu bimaculatus was investigated. The peroxiredoxin-1 protein was obtained by in vitro recombinant expression and purification. The recombinant protein had a strong ability to scavenge hydroxyl radicals, protect superhelical DNA plasmids from oxidative damage, and protect L929 cells from H₂O₂ toxicity through in vitro antioxidant activity. In addition, we verified its ability to bind to tetrodotoxin using surface plasmon resonance techniques. Further, recombinant proteins were found to facilitate the entry of tetrodotoxin into cells. Through these analyses, we identified, for the first time, peroxiredoxin-1 protein from Takifugu bimaculatus as a potential novel tetrodotoxin-binding protein. Our findings provide a basis for further exploration of the application of peroxiredoxin-1 protein and the molecular mechanisms of tetrodotoxin enrichment in pufferfish. Full article

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val¹¹⁵, ASP¹¹⁶, Val¹¹⁷, and Lys¹²²) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein–toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish. Full article

Nassarius has caused serious people poisoning and death incident as a popular food due to tetrodotoxin (TTX) accumulation in their body. Understanding the genetic basis of tetrodotoxin (TTX) transformation and resistance in animals could lead to significant insights into adaptive evolution to toxins and toxin poisoning cures in medicine. Here we performed comparative transcriptome analysis for toxic and non-toxic communities in Nassarius succinctus and Nassarius variciferus to reveal their genetic expression and mutation patterns. For both species, the cellular and metabolic process, and binding and catalytic activity accounted for the top classification categories, and the toxic communities generally produced more up-regulated genes than non-toxic communities. Most unigenes and different expression genes were related to disease, e.g., heat shock protein and tissue factor pathway inhibitors, which involve detoxification and coagulation. In mutation levels, the sodium channel gene of N. succinctus had one amino acid mutation “L”, which is different from that of other animals. In conclusion, the comparative transcriptome analysis of different species and populations provided an important genetic basis for adaptive evolution to toxins, health and toxin poisoning cure research for TTX in marine gastropoda mollusk. Future studies will focus on the action mechanism of the important functional gene for TTX accumulation and resistance. Full article

Pufferfish saxitoxin and tetrodotoxin (TTX) binding protein (PSTBP) is a glycoprotein that we previously isolated from the blood plasma of the pufferfish Takifugu pardalis; this protein was also detected in seven species of the genus Takifugu. We proposed that PSTBP is a carrier protein for TTX in pufferfish; however, PSTBP had not yet been found in genera other than Takifugu. In this study, we investigated the presence of PSTBP-like proteins in the toxic pufferfish Arothron nigropunctatus, A. hispidus, A. manilensis, and Chelonodon patoca. On the basis of ultrafiltration experiments, TTX was found to be present and partially bound to proteins in the plasma of these pufferfish, and Western blot analyses with anti-PSTBP antibody revealed one or two bands per species. The observed decreases in molecular mass following deglycosylation with glycopeptidase F suggest that these positive proteins are glycoproteins. The molecular masses of the deglycosylated proteins detected in the three Arothron species were larger than that of PSTBP in the genus Takifugu, whereas the two bands detected in C. patoca had molecular masses similar to that of tributyltin-binding protein-2 (TBT-bp2). The N-terminal amino acid sequences of 23–29 residues of these detected proteins were all homologous with those of PSTBP and TBT-bp2. Full article

Tetrodotoxin (TTX) is believed to be one of the most selective inhibitors of voltage-gated fast Na⁺ channels in excitable tissues. Recently, however, TTX has been shown to block L-type Ca²⁺ current (I_Ca) in canine cardiac cells. In the present study, the TTX-sensitivity of I_Ca was studied in isolated canine ventricular myocytes as a function of (1) channel phosphorylation, (2) extracellular pH and (3) the redox potential of the bathing medium using the whole cell voltage clamp technique. Fifty-five micromoles of TTX (IC₅₀ value obtained under physiological conditions) caused 60% ± 2% inhibition of I_Ca in acidic (pH = 6.4), while only a 26% ± 2% block in alkaline (pH = 8.4) milieu. Similarly, the same concentration of TTX induced 62% ± 6% suppression of I_Ca in a reductant milieu (containing glutathione + ascorbic acid + dithiothreitol, 1 mM each), in contrast to the 31% ± 3% blockade obtained in the presence of a strong oxidant (100 μM H₂O₂). Phosphorylation of the channel protein (induced by 3 μM forskolin) failed to modify the inhibiting potency of TTX; an IC₅₀ value of 50 ± 4 μM was found in forskolin. The results are in a good accordance with the predictions of our model, indicating that TTX binds, in fact, to the selectivity filter of cardiac L-type Ca channels. Full article

Tetrodotoxin and saxitoxin are small, compact asymmetrical marine toxins that block voltage-gated Na channels with high affinity and specificity. They enter the channel pore’s outer vestibule and bind to multiple residues that control permeation. Radiolabeled toxins were key contributors to channel protein purification and subsequent cloning. They also helped identify critical structural elements called P loops. Spacial organization of their mutation-identified interaction sites in molecular models has generated a molecular image of the TTX binding site in the outer vestibule and the critical permeation and selectivity features of this region. One site in the channel’s domain I P loop determines affinity differences in mammalian isoforms. Full article