Search

Membrane lipids extensively modulate the activation gating of voltage-gated potassium channels (K V ), however, much less is known about the mechanisms of ceramide and glucosylceramide actions including which structural element is the main intramolecular target and whether there is any contribution of indirect, membrane biophysics-related mechanisms to their actions. We used two-electrode voltage-clamp fluorometry capable of recording currents and fluorescence signals to simultaneously monitor movements of the pore domain (PD) and the voltage sensor domain (VSD) of the K V 1.3 ion channel after attaching an MTS-TAMRA fluorophore to a cysteine introduced into the extracellular S3-S4 loop of the VSD. We observed rightward shifts in the conductance-voltage (G-V) relationship, slower current activation kinetics, and reduced current amplitudes in response to loading the membrane with C16-ceramide (Cer) or C16-glucosylceramide (GlcCer). When analyzing VSD movements, only Cer induced a rightward shift in the fluorescence signal-voltage (F-V) relationship and slowed fluorescence activation kinetics, whereas GlcCer exerted no such effects. These results point at a distinctive mechanism of action with Cer primarily targeting the VSD, while GlcCer only the PD of K V 1.3. Using environment-sensitive probes and fluorescence-based approaches, we show that Cer and GlcCer similarly increase molecular order in the inner, hydrophobic regions of bilayers, however, Cer induces a robust molecular reorganization at the membrane-water interface. We propose that this unique ordering effect in the outermost membrane layer in which the main VSD rearrangement involving an outward sliding of the top of S4 occurs can explain the VSD targeting mechanism of Cer, which is unavailable for GlcCer., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis H V 1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: The unique microenvironment in tumors inhibits the normal functioning of tumor-infiltrating lymphocytes, leading to immune evasion and cancer progression. Over-activation of KCa3.1 using positive modulators has been proposed to rescue the anti-tumor response. One of the key characteristics of the tumor microenvironment is extracellular acidity. Herein, we analyzed how intra- and extracellular pH affects K + currents through KCa3.1 and if the potency of two of its positive modulators, Riluzole and SKA-31, is pH sensitive., Methods: Whole-cell patch-clamp was used to measure KCa3.1 currents either in activated human peripheral lymphocytes or in CHO cells transiently transfected with either the H192A mutant or wild-type hKCa3.1 in combination with T79D-Calmodulin, or with KCa2.2., Results: We found that changes in the intra- and extracellular pH minimally influenced the KCa3.1-mediated K + current. Extracellular pH, in the range of 6.0-8.0, does not interfere with the capacity of Riluzole and SKA-31 to robustly activate the K + currents through KCa3.1. Contrariwise, an acidic intracellular solution causes a slow, but irreversible loss of potency of both the activators. Using different protocols of perfusion and depolarization we demonstrated that the loss of potency is strictly time and pH-dependent and that this peculiar effect can be observed with a structurally similar channel KCa2.2. While two different point mutations of both KCa3.1 (H192A) and its associated protein Calmodulin (T79D) do not limit the effect of acidity, increasing the cytosolic Ca 2+ concentration to saturating levels eliminated the loss-of-potency phenotype., Conclusion: Based on our data we conclude that KCa3.1 currents are not sensitive the either the intracellular or the extracellular pH in the physiological and pathophysiological range. However, intracellular acidosis in T cells residing in the tumor microenvironment could hinder the potentiating effect of KCa3.1 positive modulators administered to boost their activity. Further research is warranted both to clarify the molecular interactions between the modulators and KCa3.1 at different intracellular pH conditions and to define whether this loss of potency can be observed in cancer models as well., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Cozzolino and Panyi.)

Despite significant advances in the development of therapeutic interventions targeting autoimmune diseases and chronic inflammatory conditions, lack of effective treatment still poses a high unmet need. Modulating chronically activated T cells through the blockade of the Kv1.3 potassium channel is a promising therapeutic approach; however, developing selective Kv1.3 inhibitors is still an arduous task. Phage display-based high throughput peptide library screening is a rapid and robust approach to develop promising drug candidates; however, it requires solid-phase immobilization of target proteins with their binding site preserved. Historically, the KcsA bacterial channel chimera harboring only the turret region of the human Kv1.3 channel was used for screening campaigns. Nevertheless, literature data suggest that binding to this type of chimera does not correlate well with blocking potency on the native Kv1.3 channels. Therefore, we designed and successfully produced advanced KcsA-Kv1.3, KcsA-Kv1.1, and KcsA-Kv1.2 chimeric proteins in which both the turret and part of the filter regions of the human Kv1.x channels were transferred. These T+F (turret-filter) chimeras showed superior peptide ligand-binding predictivity compared to their T-only versions in novel phage ELISA assays. Phage ELISA binding and competition results supported with electrophysiological data confirmed that the filter region of KcsA-Kv1.x is essential for establishing adequate relative affinity order among selected peptide toxins (Vm24 toxin, Hongotoxin-1, Kaliotoxin-1, Maurotoxin, Stichodactyla toxin) and consequently obtaining more reliable selectivity data. These new findings provide a better screening tool for future drug development efforts and offer insight into the target-ligand interactions of these therapeutically relevant ion channels., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

CD8 + T cells play a role in the suppression of tumor growth and immunotherapy. Ion channels control the Ca 2+ -dependent function of CD8 + lymphocytes such as cytokine/granzyme production and tumor killing. Kv1.3 and KCa3.1 K + channels stabilize the negative membrane potential of T cells to maintain Ca 2+ influx through CRAC channels. We assessed the expression of Kv1.3, KCa3.1 and CRAC in CD8 + cells from ovarian cancer (OC) patients ( n = 7). We found that the expression level of Kv1.3 was higher in patients with malignant tumors than in control or benign tumor groups while the KCa3.1 activity was lower in the malignant tumor group as compared to the others. We demonstrated that the Ca 2+ response in malignant tumor patients is higher compared to control groups. We propose that altered Kv1.3 and KCa3.1 expression in CD8 + cells in OC could be a reporter and may serve as a biomarker in diagnostics and that increased Ca 2+ response through CRAC may contribute to the impaired CD8 + function.

Diverse structural scaffolds have been described in peptides from sea anemones, with the ShKT domain being a common scaffold first identified in ShK toxin from Stichodactyla helianthus. ShK is a potent blocker of voltage-gated potassium channels (K V 1.x), and an analog, ShK-186 (dalazatide), has completed Phase 1 clinical trials in plaque psoriasis. The ShKT domain has been found in numerous other species, but only a tiny fraction of ShKT domains has been characterized functionally. Despite adopting the canonical ShK fold, some ShKT peptides from sea anemones inhibit K V 1.x, while others do not. Mutagenesis studies have shown that a Lys-Tyr (KY) dyad plays a key role in K V 1.x blockade, although a cationic residue followed by a hydrophobic residue may also suffice. Nevertheless, ShKT peptides displaying an ShK-like fold and containing a KY dyad do not necessarily block potassium channels, so additional criteria are needed to determine whether new ShKT peptides might show activity against potassium channels. In this study, we used a combination of NMR and molecular dynamics (MD) simulations to assess the potential activity of a new ShKT peptide. We determined the structure of ShKT-Ts1, from the sea anemone Telmatactis stephensoni, examined its tissue localization, and investigated its activity against a range of ion channels. As ShKT-Ts1 showed no activity against K V 1.x channels, we used MD simulations to investigate whether solvent exposure of the dyad residues may be informative in rationalizing and potentially predicting the ability of ShKT peptides to block K V 1.x channels. We show that either a buried dyad that does not become exposed during MD simulations, or a partially exposed dyad that becomes buried during MD simulations, correlates with weak or absent activity against K V 1.x channels. Therefore, structure determination coupled with MD simulations, may be used to predict whether new sequences belonging to the ShKT family may act as potassium channel blockers., (© 2023 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Sea anemone venoms are complex mixtures of biologically active compounds, including disulfide-rich peptides, some of which have found applications as research tools, and others as therapeutic leads. Our recent transcriptomic and proteomic studies of the Australian sea anemone Telmatactis stephensoni identified a transcript for a peptide designated Tst2. Tst2 is a 38-residue peptide showing sequence similarity to peptide toxins known to interact with a range of ion channels (Na V , TRPV1, K V and Ca V ). Recombinant Tst2 (rTst2, which contains an additional Gly at the N-terminus) was produced by periplasmic expression in Escherichia coli, enabling the production of both unlabelled and uniformly 13 C, 15 N-labelled peptide for functional assays and structural studies. The LC-MS profile of the recombinant Tst2 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds from its six cysteine residues. The solution structure of rTst2 was determined using multidimensional NMR spectroscopy and revealed that rTst2 adopts an inhibitor cystine knot (ICK) structure. rTst2 was screened using various functional assays, including patch-clamp electrophysiological and cytotoxicity assays. rTst2 was inactive against voltage-gated sodium channels (Na V ) and the human voltage-gated proton (hHv1) channel. rTst2 also did not possess cytotoxic activity when assessed against Drosophila melanogaster flies. However, the recombinant peptide at 100 nM showed >50% inhibition of the transient receptor potential subfamily V member 1 (TRPV1) and slight (∼10%) inhibition of transient receptor potential subfamily A member 1 (TRPA1). Tst2 is thus a novel ICK inhibitor of the TRPV1 channel., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the results reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Cardiovascular (CV) morbidity and mortality, and perpetuated synovial angiogenesis have been associated with RA. In our study we evaluated angiogenic factors in relation to vascular inflammation and function, and clinical markers in RA patients undergoing 1-year tofacitinib therapy., Methods: Thirty RA patients treated with either 5 mg or 10 mg twice daily tofacitinib were included in a 12-month follow-up study. Eventually, 26 patients completed the study and were included in data analysis. Levels of various angiogenic cytokines (TNF-α, IL-6), growth factors [VEGF, basic fibroblast (bFGF), epidermal (EGF), placental (PlGF)], cathepsin K (CathK), CXC chemokine ligand 8 (CXCL8), galectin-3 (Gal-3) and N-terminal prohormone brain natriuretic peptide (NT-proBNP) were determined at baseline, and at 6 and 12 months after initiating tofacitinib treatment. In order to assess flow-mediated vasodilation, common carotid intima-media thickness (ccIMT) and carotid-femoral pulse-wave velocity, ultrasonography was performed. Synovial and aortic inflammation was also assessed by 18F-fluorodeoxyglucose-PET/CT., Results: One-year tofacitinib therapy significantly decreased IL-6, VEGF, bFGF, EGF, PlGF and CathK, while it increased Gal-3 production (P < 0.05). bFGF, PlGF and NT-proBNP levels were higher, while platelet-endothelial cell adhesion molecule 1 (PECAM-1) levels were lower in RF-seropositive patients (P < 0.05). TNF-α, bFGF and PlGF correlated with post-treatment synovial inflammation, while aortic inflammation was rather dependent on IL-6 and PECAM-1 as determined by PET/CT (P < 0.05). In the correlation analyses, NT-proBNP, CXCL8 and Cath variables correlated with ccIMT (P < 0.05)., Conclusions: Decreasing production of bFGF, PlGF or IL-6 by 1-year tofacitinib therapy potentially inhibits synovial and aortic inflammation. Although NT-proBNP, CXCL8 and CathK were associated with ccIMT, their role in RA-associated atherosclerosis needs to be further evaluated., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Seven new peptides denominated CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito and their primary structures were determined. The molecular weights ranged between 3760.4 Da and 4357.9 Da, containing 32 to 39 amino acid residues with three putative disulfide bridges. The comparison of amino acid sequences with known potassium scorpion toxins (KTx) and phylogenetic analysis revealed that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to the α-KTx 10.x subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), and CboK7 (α-KTx 2.24) bear > 95% amino acid similarity with members of the α-KTx 2.x subfamily, and CboK5 is identical to Ce3 toxin (α-KTx 2.10). Electrophysiological assays demonstrated that except CboK1, all six other peptides blocked the Kv1.2 channel with Kd values in the picomolar range (24-763 pM) and inhibited the Kv1.3 channel with comparatively less potency (Kd values between 20-171 nM). CboK3 and CboK4 inhibited less than 10% and CboK7 inhibited about 42% of Kv1.1 currents at 100 nM concentration. Among all, CboK7 showed out-standing affinity for Kv1.2 (Kd = 24 pM), as well as high selectivity over Kv1.3 (850-fold) and Kv1.1 (~6000-fold). These characteristics of CboK7 may provide a framework for developing tools to treat Kv1.2-related channelopathies.

Background and Purpose: Despite its contradictory clinical performance, remdesivir (Veklury®) has a pivotal role in COVID-19 therapy. Possible contributions of the vehicle, sulfobutylether-β-cyclodextrin (SBECD) to Veklury® effects have been overlooked. The powder and solution formulations of Veklury® are treated equivalently despite their different vehicle content. Our objective was to study Veklury® effects on initial membrane-coupled events of SARS-CoV-2 infection focusing on the cholesterol depletion-mediated role of SBECD., Experimental Approach: Using time-correlated flow cytometry and quantitative three-dimensional confocal microscopy, we studied early molecular events of SARS-CoV-2-host cell membrane interactions., Key Results: Veklury® and different cholesterol-depleting cyclodextrins (CDs) reduced binding of the spike receptor-binding domain (RBD) to ACE2 and spike trimer internalization for Wuhan-Hu-1, Delta and Omicron variants. Correlations of these effects with cholesterol-dependent changes in membrane structure and decreased lipid raft-dependent ACE2-TMPRSS2 interaction establish that SBECD is not simply a vehicle but also an effector along with remdesivir due to its cholesterol-depleting potential. Veklury® solution inhibited RBD binding more efficiently due to its twice higher SBECD content. The CD-induced inhibitory effects were more prominent at lower RBD concentrations and in cells with lower endogenous ACE2 expression, indicating that the supportive CD actions can be even more pronounced during in vivo infection when viral load and ACE expression are typically low., Conclusion and Implications: Our findings call for the differentiation of Veklury® formulations in meta-analyses of clinical trials, potentially revealing neglected benefits of the solution formulation, and also raise the possibility of adjuvant cyclodextrin (CD) therapy, even at higher doses, in COVID-19., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Voltage-gated K+ channels have distinct gates that regulate ion flux: the activation gate (A-gate) formed by the bundle crossing of the S6 transmembrane helices and the slow inactivation gate in the selectivity filter. These two gates are bidirectionally coupled. If coupling involves the rearrangement of the S6 transmembrane segment, then we predict state-dependent changes in the accessibility of S6 residues from the water-filled cavity of the channel with gating. To test this, we engineered cysteines, one at a time, at S6 positions A471, L472, and P473 in a T449A Shaker-IR background and determined the accessibility of these cysteines to cysteine-modifying reagents MTSET and MTSEA applied to the cytosolic surface of inside-out patches. We found that neither reagent modified either of the cysteines in the closed or the open state of the channels. On the contrary, A471C and P473C, but not L472C, were modified by MTSEA, but not by MTSET, if applied to inactivated channels with open A-gate (OI state). Our results, combined with earlier studies reporting reduced accessibility of residues I470C and V474C in the inactivated state, strongly suggest that the coupling between the A-gate and the slow inactivation gate is mediated by rearrangements in the S6 segment. The S6 rearrangements are consistent with a rigid rod-like rotation of S6 around its longitudinal axis upon inactivation. S6 rotation and changes in its environment are concomitant events in slow inactivation of Shaker KV channels., (© 2023 Szanto et al.)

Every cell biological textbook teaches us that the main role of the plasma membrane is to separate cells from their neighborhood to allow for a controlled composition of the intracellular space. The mostly hydrophobic nature of the cell membrane presents an impenetrable barrier for most hydrophilic molecules larger than 1 kDa. On the other hand, cell-penetrating peptides (CPPs) are capable of traversing this barrier without compromising membrane integrity, and they can do so on their own or coupled to cargos. Coupling biologically and medically relevant cargos to CPPs holds great promise of delivering membrane-impermeable drugs into cells. If the cargo is able to interact with certain cell types, uptake of the CPP-drug complex can be tailored to be cell-type-specific. Besides outlining the major membrane penetration pathways of CPPs, this review is aimed at deciphering how properties of the membrane influence the uptake mechanisms of CPPs. By summarizing an extensive body of experimental evidence, we argue that a more ordered, less flexible membrane structure, often present in the very diseases planned to be treated with CPPs, decreases their cellular uptake. These correlations are not only relevant for understanding the cellular biology of CPPs, but also for rationally improving their value in translational or clinical applications.

Background: The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni., Results: We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity., Conclusions: Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni., (© 2023. The Author(s).)

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H + ) channel (H V 1, K d ≈ 26 μM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the K V 1.3 channel. We, therefore, tested ClGBI directly on hK V 1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hH V 1 ( K d ≈ 72 μM). We then further investigated ClGBI selectivity on the hK V 1.1, hK V 1.4-IR, hK V 1.5, hK V 10.1, hK V 11.1, hK Ca 3.1, hNa V 1.4, and hNa V 1.5 channels. Our results show that, besides H V 1 and K V 1.3, all other off-target channels were inhibited by ClGBI, with K d values ranging from 12 to 894 μM. Based on our comprehensive data, ClGBI has to be considered a non-selective hH V 1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b + /Ly6G + ) and Mo-MDSCs (CD11b + /Ly6C + ) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b + /Ly6G + PMN-MDSCs and CD11b + /Ly6C + /F4/80 - /MHCII - Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H + channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H + selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H + currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.

A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus . Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K + channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K + channel hK V 1.2 with high affinity (K d = 65 nM). The conductance-voltage relationship of K V 1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin-channel interaction. Cm39 also inhibits the Ca 2+ -activated K Ca 2.2 and K Ca 3.1 channels, with K d = 502 nM, and K d = 58 nM, respectively. However, the peptide does not inhibit hK V 1.1, hK V 1.3, hK V 1.4, hK V 1.5, hK V 1.6, hK V 11.1, mK Ca 1.1 K + channels or the hNa V 1.5 and hNa V 1.4 Na + channels at 1 μM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels.

The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer's and Parkinson's diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC 50 ∼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [ 64 Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [ 68 Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [ 68 Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC 50 ∼ 6 μM), was also tested. Significantly increased uptake of [ 68 Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood-brain barrier disruption. PET imaging also showed accumulation of [ 68 Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.

Most species of bee are capable of delivering a defensive sting which is often painful. A solitary lifestyle is the ancestral state of bees and most extant species are solitary, but information on bee venoms comes predominantly from studies on eusocial species. In this study we investigated the venom composition of the Australian great carpenter bee, Xylocopa aruana Ritsema, 1876. We show that the venom is relatively simple, composed mainly of one small amphipathic peptide (XYTX 1 -Xa1a), with lesser amounts of an apamin homologue (XYTX 2 -Xa2a) and a venom phospholipase-A 2 (PLA 2 ). XYTX 1 -Xa1a is homologous to, and shares a similar mode-of-action to melittin and the bombilitins, the major components of the venoms of the eusocial Apis mellifera (Western honeybee) and Bombus spp. (bumblebee), respectively. XYTX 1 -Xa1a and melittin directly activate mammalian sensory neurons and cause spontaneous pain behaviours in vivo, effects which are potentiated in the presence of venom PLA 2 . The apamin-like peptide XYTX 2 -Xa2a was a relatively weak blocker of small conductance calcium-activated potassium (K Ca ) channels and, like A. mellifera apamin and mast cell-degranulating peptide, did not contribute to pain behaviours in mice. While the composition and mode-of-action of the venom of X. aruana are similar to that of A. mellifera, the greater potency, on mammalian sensory neurons, of the major pain-causing component in A. mellifera venom may represent an adaptation to the distinct defensive pressures on eusocial Apidae., (© 2022. The Author(s).)

Glioblastoma (GBM) is the most aggressive glial tumor, where ion channels, including K Ca 1.1, are candidates for new therapeutic options. Since the auxiliary subunits linked to K Ca 1.1 in GBM are largely unknown we used electrophysiology combined with pharmacology and gene silencing to address the functional expression of K Ca 1.1/β subunits complexes in both primary tumor cells and in the glioblastoma cell line U-87 MG. The pattern of the sensitivity (activation/inhibition) of the whole-cell currents to paxilline, lithocholic acid, arachidonic acid, and iberiotoxin; the presence of inactivation of the whole-cell current along with the loss of the outward rectification upon exposure to the reducing agent DTT collectively argue that K Ca 1.1/β3 complex is expressed in U-87 MG. Similar results were found using human primary glioblastoma cells isolated from patient samples. Silencing the β3 subunit expression inhibited carbachol-induced Ca 2+ transients in U-87 MG thereby indicating the role of the K Ca 1.1/β3 in the Ca 2+ signaling of glioblastoma cells. Functional expression of the K Ca 1.1/β3 complex, on the other hand, lacks cell cycle dependence. We suggest that the K Ca 1.1/β3 complex may have diagnostic and therapeutic potential in glioblastoma in the future., (© 2022. The Author(s).)

Cyclodextrins, representing a versatile family of cyclic oligosaccharides, have extensive pharmaceutical applications due to their unique truncated cone-shaped structure with a hydrophilic outer surface and a hydrophobic cavity, which enables them to form non-covalent host-guest inclusion complexes in pharmaceutical formulations to enhance the solubility, stability and bioavailability of numerous drug molecules. As a result, cyclodextrins are mostly considered as inert carriers during their medical application, while their ability to interact not only with small molecules but also with lipids and proteins is largely neglected. By forming inclusion complexes with cholesterol, cyclodextrins deplete cholesterol from cellular membranes and thereby influence protein function indirectly through alterations in biophysical properties and lateral heterogeneity of bilayers. In this review, we summarize the general chemical principles of direct cyclodextrin-protein interactions and highlight, through relevant examples, how these interactions can modify protein functions in vivo, which, despite their huge potential, have been completely unexploited in therapy so far. Finally, we give a brief overview of disorders such as Niemann-Pick type C disease, atherosclerosis, Alzheimer's and Parkinson's disease, in which cyclodextrins already have or could have the potential to be active therapeutic agents due to their cholesterol-complexing or direct protein-targeting properties., Competing Interests: L.S. is currently employed by the Cyclodextrin R & D Laboratory Ltd. The remaining authors declare no conflict of interest. The funders had no role in the writing of the manuscript.

The Kv1.3 channel has become a therapeutic target for the treatment of various diseases. Several Kv1.3 channel blockers have been characterized from scorpion venom; however, extensive studies require amounts of toxin that cannot be readily obtained directly from venoms. The Pichia pastoris expression system provides a cost-effective approach to overcoming the limitations of chemical synthesis and E. coli recombinant expression. In this work, we developed an efficient system for the production of three potent Kv1.3 channel blockers from different scorpion venoms: Vm24, AnTx, and Ts6. Using the Pichia system, these toxins could be obtained in sufficient quantities (Vm24 1.6 mg/L, AnTx 46 mg/L, and Ts6 7.5 mg/L) to characterize their biological activity. A comparison was made between the activity of tagged and untagged recombinant peptides. Tagged Vm24 and untagged AnTx are nearly equivalent to native toxins in blocking Kv1.3 (Kd = 4.4 pM and Kd = 0.72 nM, respectively), whereas untagged Ts6 exhibits a 53-fold increase in Kd (Kd = 29.1 nM) as compared to the native peptide. The approach described here provides a method that can be optimized for toxin production to develop more selective and effective Kv1.3 blockers with therapeutic potential.

Upregulation of the voltage-gated potassium channel K V 1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of K V 1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer , binds K V 1.3 with high affinity (IC 50 of 45 pM) and selectivity (2000-fold for K V 1.3 over K V 1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against K V 1.3 (IC 50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (K V 1.2, K V 1.4, K V 1.5, K V 1.6, K Ca 1.1 and K Ca 3.1), as well as selectivity against heteromeric channels assembled from K V 1.3/K V 1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged K V 1.3 shows co-localization of Cy5-HsTX1[R14A] and K V 1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect K V 1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of K V 1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify K V 1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing K V 1.3, with broad applicability in fundamental investigations of K V 1.3 biology, and the validation of novel disease indications where K V 1.3 inhibition may be of therapeutic value.

Voltage-clamp fluorometry (VCF) supplies information about the conformational changes of voltage-gated proteins. Changes in the fluorescence intensity of the dye attached to a part of the protein that undergoes a conformational rearrangement upon the alteration of the membrane potential by electrodes constitute the signal. The VCF signal is generated by quenching and dequenching of the fluorescence as the dye traverses various local environments. Here we studied the VCF signal generation, using the Hv1 voltage-gated proton channel as a tool, which shares a similar voltage-sensor structure with voltage-gated ion channels but lacks an ion-conducting pore. Using mutagenesis and lipids added to the extracellular solution we found that the signal is generated by the combined effects of lipids during movement of the dye relative to the plane of the membrane and by quenching amino acids. Our 3-state model recapitulates the VCF signals of the various mutants and is compatible with the accepted model of two major voltage-sensor movements., (© 2022. The Author(s).)

Background: Cardiovascular (CV) morbidity, mortality and metabolic syndrome are associated with rheumatoid arthritis (RA). A recent trial has suggested increased risk of major CV events (MACE) upon the Janus kinase (JAK) inhibitor tofacitinib compared with anti-tumor necrosis factor α (TNF-α) therapy. In our study, we evaluated lipids and other metabolic markers in relation to vascular function and clinical markers in RA patients undergoing one-year tofacitinib therapy. Patients and methods: Thirty RA patients treated with either 5 mg or 10 mg bid tofacitinib were included in a 12-month follow-up study. Various lipids, paraoxonase (PON1), myeloperoxidase (MPO), thrombospondin-1 (TSP-1) and adipokine levels, such as adiponectin, leptin, resistin, adipsin and chemerin were determined. In order to assess flow-mediated vasodilation (FMD), common carotid intima-media thickness (IMT) and arterial pulse-wave velocity (PWV) ultrasonography were performed. Assessments were carried out at baseline, and 6 and 12 months after initiating treatment. Results: One-year tofacitinib therapy significantly increased TC, HDL, LDL, APOA, APOB, leptin, adipsin and TSP-1, while significantly decreasing Lp(a), chemerin, PON1 and MPO levels. TG, lipid indices (TC/HDL and LDL/HDL), adiponectin and resistin showed no significant changes. Numerous associations were found between lipids, adipokines, clinical markers and IMT, FMD and PWV (p < 0.05). Regression analysis suggested, among others, association of BMI with CRP and PWV (p < 0.05). Adipokines variably correlated with age, BMI, CRP, CCP, FMD, IMT and PWV, while MPO, PON1 and TSP-1 variably correlated with age, disease duration, BMI, RF and PWV (p < 0.05). Conclusions: JAK inhibition by tofacitinib exerts balanced effects on lipids and other metabolic markers in RA. Various correlations may exist between metabolic, clinical parameters and vascular pathophysiology during tofacitinib treatment. Complex assessment of lipids, metabolic factors together with clinical parameters and vascular pathophysiology may be utilized in clinical practice to determine and monitor the CV status of patients in relation with clinical response to JAK inhibition.

The hEag1 (Kv10.1) K + channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC 50 = 0.135 μM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K + channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 μM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca 2+ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca 2+ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca 2+ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.

The Cm28 in the venom of Centruroides margaritatus is a short peptide consisting of 27 amino acid residues with a mol wt of 2,820 D. Cm28 has <40% similarity with other known α-KTx from scorpions and lacks the typical functional dyad (lysine-tyrosine) required to block KV channels. However, its unique sequence contains the three disulfide-bond traits of the α-KTx scorpion toxin family. We propose that Cm28 is the first example of a new subfamily of α-KTxs, registered with the systematic number α-KTx32.1. Cm28 inhibited voltage-gated K+ channels KV1.2 and KV1.3 with Kd values of 0.96 and 1.3 nM, respectively. There was no significant shift in the conductance-voltage (G-V) relationship for any of the channels in the presence of toxin. Toxin binding kinetics showed that the association and dissociation rates are consistent with a bimolecular interaction between the peptide and the channel. Based on these, we conclude that Cm28 is not a gating modifier but rather a pore blocker. In a selectivity assay, Cm28 at 150 nM concentration (>100× Kd value for KV1.3) did not inhibit KV1.5, KV11.1, KCa1.1, and KCa3.1 K+ channels; NaV1.5 and NaV1.4 Na+ channels; or the hHV1 H+ channel but blocked ∼27% of the KV1.1 current. In a biological functional assay, Cm28 strongly inhibited the expression of the activation markers interleukin-2 receptor and CD40 ligand in anti-CD3-activated human CD4+ effector memory T lymphocytes. Cm28, due to its unique structure, may serve as a template for the generation of novel peptides targeting KV1.3 in autoimmune diseases., (© 2022 Naseem et al.)