Mechanisms Underlying C-type Inactivation in Kv Channels: Lessons From Structures of Human Kv1.3 and Fly Shaker-IR Channels.

Voltage-gated potassium (Kv) channels modulate the function of electrically-excitable and non-excitable cells by using several types of "gates" to regulate ion flow through the channels. An important gating mechanism, C-type inactivation, limits ion flow by transitioning Kv channels into a non-conducting inactivated state. Here, we highlight two recent papers, one on the human Kv1.3 channel and the second on the Drosophila Shaker Kv channel, that combined cryogenic electron microscopy and molecular dynamics simulation to define mechanisms underlying C-type inactivation. In both channels, the transition to the non-conducting inactivated conformation begins with the rupture of an intra-subunit hydrogen bond that fastens the selectivity filter to the pore helix. The freed filter swings outwards and gets tethered to an external residue. As a result, the extracellular end of the selectivity filter dilates and K+ permeation through the pore is impaired. Recovery from inactivation may entail a reversal of this process. Such a reversal, at least partially, is induced by the peptide dalazatide. Binding of dalazatide to external residues in Kv1.3 frees the filter to swing inwards. The extracellular end of the selectivity filter narrows allowing K+ to move in single file through the pore typical of conventional knock-on conduction. Inter-subunit hydrogen bonds that stabilize the outer pore in the dalazatide-bound structure are equivalent to those in open-conducting conformations of Kv channels. However, the intra-subunit bond that fastens the filter to the pore-helix is absent, suggesting an incomplete reversal of the process. These mechanisms define how Kv channels self-regulate the flow of K+ by changing the conformation of the selectivity filter.

Imaging Kv1.3 Expressing Memory T Cells as a Marker of Immunotherapy Response.

Immune checkpoint inhibitors have shown great promise, emerging as a new pillar of treatment for cancer; however, only a relatively small proportion of recipients show a durable response to treatment. Strategies that reliably differentiate durably-responding tumours from non-responsive tumours are a critical unmet need. Persistent and durable immunological responses are associated with the generation of memory T cells. Effector memory T cells associated with tumour response to immune therapies are characterized by substantial upregulation of the potassium channel Kv1.3 after repeated antigen stimulation. We have developed a new Kv1.3 targeting radiopharmaceutical, [18F]AlF-NOTA-KCNA3P, and evaluated whether it can reliably differentiate tumours successfully responding to immune checkpoint inhibitor (ICI) therapy targeting PD-1 alone or combined with CLTA4. In a syngeneic colon cancer model, we compared tumour retention of [18F]AlF-NOTA-KCNA3P with changes in the tumour immune microenvironment determined by flow cytometry. Imaging with [18F]AlF-NOTA-KCNA3P reliably differentiated tumours responding to ICI therapy from non-responding tumours and was associated with substantial tumour infiltration of T cells, especially Kv1.3-expressing CD8+ effector memory T cells.

Histone acetylome-wide associations in immune cells from individuals with active Mycobacterium tuberculosis infection.

Host cell chromatin changes are thought to play an important role in the pathogenesis of infectious diseases. Here we describe a histone acetylome-wide association study (HAWAS) of an infectious disease, on the basis of genome-wide H3K27 acetylation profiling of peripheral blood granulocytes and monocytes from persons with active Mycobacterium tuberculosis (Mtb) infection and healthy controls. We detected >2,000 differentially acetylated loci in either cell type in a Singapore Chinese discovery cohort (n = 46), which were validated in a subsequent multi-ethnic Singapore cohort (n = 29), as well as a longitudinal cohort from South Africa (n = 26), thus demonstrating that HAWAS can be independently corroborated. Acetylation changes were correlated with differential gene expression. Differential acetylation was enriched near potassium channel genes, including KCNJ15, which modulates apoptosis and promotes Mtb clearance in vitro. We performed histone acetylation quantitative trait locus (haQTL) analysis on the dataset and identified 69 candidate causal variants for immune phenotypes among granulocyte haQTLs and 83 among monocyte haQTLs. Our study provides proof-of-principle for HAWAS to infer mechanisms of host response to pathogens.

Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug.

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide-Kv1.3). Both the apo-Kv1.3 and dalazatide-Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K+ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide-Kv1.3, binding of dalazatide to the channel's outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K+ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3's transition into the drug-blocked state.

Core-Shell Structured Antimicrobial Nanofiber Dressings Containing Herbal Extract and Antibiotics Combination for the Prevention of Biofilms and Promotion of Cutaneous Wound Healing.

Burn wounds are susceptible to microbial invasion from both resident and exogenous bacteria, which becomes a critical public health issue and causes substantial economic burden. There is a perceived demand to produce new antimicrobial wound dressings that hinder bacterial colonization while accelerating the healing process and hence would provide an improved standard of care for patients. Since ancient times, herbal extracts from medicinally important plants have extensively been used for treating burn injuries. This work reports the utility of electrospun nanofibers containing plant extracts and antibiotics combination as a multifunctional scaffold for treating second-degree burns. First, we determined the various components of plant extracts from Gymnema sylvestre by two different processing methods and their synergism with minocycline antibiotics. Then, we prepared core-shell nanofibrous dressings with poly-ε-caprolactone/gelatin laden with minocycline hydrochloride as a shell and gelatin infused with G. sylvestre extracts (ultrasound-assisted extracts and cold macerated extracts) as the core using coaxial electrospinning. The electrospun nanofibers displayed a smooth, continuous, and bead-free morphology with adequate wettability. The presence of extract components in the core-shell nanofibers resulted in enhanced mechanical properties when compared to pristine mats. The core-shell structures resulted in sustained release of the bioactive components when compared to nanofiber blends. Core-shell nanofiber mats containing plant extracts and antibiotic combinations displayed potent antimicrobial and antibiofilm properties while promoting the spread and proliferation of skin cells when compared to pristine mats. In a porcine model of cutaneous second-degree burns, we showed that wounds treated with the antimicrobial dressing improved re-epithelialization and collagen organization in comparison to untreated wounds.

Modulation of Lymphocyte Potassium Channel KV1.3 by Membrane-Penetrating, Joint-Targeting Immunomodulatory Plant Defensin.

We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated KV1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to KV1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for KV1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with 18F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10-100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.

Contributions of natural products to ion channel pharmacology.

Covering: Up to 2020Ion channels are a vast super-family of membrane proteins that play critical physiological roles in excitable and non-excitable cells. Their biomedical importance makes them valuable and attractive drug targets for neurological, cardiovascular, gastrointestinal and metabolic diseases, and for cancer therapy and immune modulation. Current therapeutics target only a minor subset of ion channels, leaving a large unexploited space within the ion channel field. Natural products harnessed from the almost unlimited and diverse universe of compounds within the bioenvironment have been used to modulate channels for decades. In this review we highlight the impact made by natural products on ion channel pharmacology, specifically on K+, NaV and CaV channels, and use case studies to describe the development of ion channel-modulating drugs from natural sources for the treatment of pain, heart disease and autoimmune diseases.

Poly-ε-Caprolactone/Gelatin Hybrid Electrospun Composite Nanofibrous Mats Containing Ultrasound Assisted Herbal Extract: Antimicrobial and Cell Proliferation Study.

Electrospun fibers have emerged as promising materials in the field of biomedicine, due to their superior physical and cell supportive properties. In particular, electrospun mats are being developed for advanced wound dressing applications. Such applications require the firers to possess excellent antimicrobial properties in order to inhibit potential microbial colonization from resident and non-resident bacteria. In this study, we have developed Poly-ε-Caprolactone /gelatin hybrid composite mats loaded with natural herbal extract (Gymnema sylvestre) to prevent bacterial colonization. As-spun scaffolds exhibited good wettability and desirable mechanical properties retaining their fibrous structure after immersing them in phosphate buffered saline (pH 7.2) for up to 30 days. The initial burst release of Gymnema sylvestre prevented the colonization of bacteria as confirmed by the radial disc diffusion assay. Furthermore, the electrospun mats promoted cellular attachment, spreading and proliferation of human primary dermal fibroblasts and cultured keratinocytes, which are crucial parenchymal cell-types involved in the skin recovery process. Overall these results demonstrated the utility of Gymnema sylvestre impregnated electrospun PCL/Gelatin nanofibrous mats as an effective antimicrobial wound dressing.

Antimicrobial properties and biocompatibility of electrospun poly-ε-caprolactone fibrous mats containing Gymnema sylvestre leaf extract.

Wound care management presents one of the substantial and tenacious challenges to the healthcare systems worldwide. Microbial colonization and subsequent biofilm formation after injury have garnered much attention, as there is an appreciable correlation between biofilms formation and delayed healing in chronic wounds. Nanotechnology has emerged as a potential platform for the management of treating acute and chronic wounds. This study presents the utility of electrospun nanofiber mats containing a natural extract (Gymnema sylvestre) that averts biofilm formation but supports human dermal fibroblasts (hDFs) attachment. The scaffolds exhibited good wettability, enhanced mechanical properties and contact mediated inhibition of Gram-positive and Gram-negative bacteria. MTS viability assay and confocal imaging further confirmed that the natural extract loaded mats remained non-cytotoxic for hDFs. Overall these findings evidenced the suitability of the Gymnema sylvestre (GS) functionalized electrospun poly-ε-caprolactone (PCL) nanofibers, as an effective wound dressing with broad spectrum anti-bacterial properties.

Isolation of Human Peripheral Blood T-Lymphocytes.

Peripheral blood is the most common source of T-lymphocytes for in vitro culture. Here, we present a simple and standardized method for small- or large-scale isolation of viable T-lymphocytes and other mononuclear cells from fresh peripheral blood or buffy coat blood samples using the density gradient centrifugation. T-cells obtained using the protocol described here can be used for a variety of downstream analysis, including cellular, molecular, and functional assays.

A Laboratory Model to Study T-Cell Motility.

Regulated migration of T-lymphocytes through high endothelial venules and secondary lymphoid organs is necessary for an adaptive immune response. Uncontrolled trafficking of T-cells is implicated in many pathological conditions, including autoimmune disorders, such as psoriasis and inflammatory bowel disease. T-cell migration is regulated mainly by the αLß2 integrin receptor LFA-1, which interacts primarily with its cognate ligand ICAM-1 expressed on the endothelium. This interaction triggers a plethora of downstream signaling pathways, which are not fully understood. Thus, in order to dissect the signal transduction processes at molecular levels and phenotypic changes in migrating T-cells, a laboratory model mimicking T-cell motility is important. Here, we describe a simple and highly reproducible in vitro model to study T-cell migration.

Quantification of T-Cell Migratory Phenotypes Using High-Content Analysis.

The exploration screening of phenotypic changes in motile T-cells within a signaling environment has always been an arduous task due to the sheer population of these microscopic cells. In recent years, High-Content Analysis (HCA) has gained epochal momentum and has allowed for a wider range of quantitative multiplexed cell-based assays in the field of lymphocyte signaling. In this chapter, we consolidate our understanding and describe the technical approach and methodology to quantify T-cell migratory phenotypes using HCA. Optimizations to be adopted to generate high-quality cytological images of motile T-cells and subsequent analysis using HCA are detailed as well.

Three-Dimensional Structured Illumination Microscopy (3D-SIM) to Dissect Signaling Cross-Talks in Motile T-Cells.

Visualization of signal transduction events in T-cells has always been a challenge due to their miniscule size. Recent advancement in super-resolution microscopy techniques presents many new opportunities to navigate the spatial and temporal signaling cross-talks in motile T-cells. Here, we provide technical details, optimal conditions, and critical practical considerations that need to be taken into account during cell handling, sample preparation, and image acquisition of motile T-cells for performing three-dimensional structured illumination microscopy (3D-SIM).

Live Cell Imaging and Analysis to Capture T-Cell Motility in Real-Time.

T-lymphocytes are the principle coordinators of the immune defense system and play a major role in the protection of our body against infections, intruders of non-self, and malignancies. To mount an immune response, T-cells need to be effectively employed to tissue sites of infection or inflammation and establish contacts with antigen-presenting cells (APCs) or malignant cells. Understanding how T-cells navigate toward their recruitment sites would offer new therapeutic opportunities. Advancement in the hardware and software upgrades of microscopy technology has created several efficient and easy-to-operate live cell imaging platforms. In this protocol, we present a generalized and simple-to-follow protocol for live cell imaging of migrating T-cells, which can also be adopted to visualize real-time tracking of intracellular signaling events.

GapmeR-Mediated Gene Silencing in Motile T-Cells.

Gene silencing is an important method to study gene functions in health and diseases. While there are various techniques that are applied to knockdown specific gene(s) of interest, they have certain limitations in application to T-lymphocytes. T-cells are "hard-to-transfect" cells and are recalcitrant to transfection reagents. Here, we describe the use of novel cell-permeating antisense molecules, called "GapmeR", to knockdown specific gene(s) in human primary T-cells.

Extracellular K+ Dampens T Cell Functions: Implications for Immune Suppression in the Tumor Microenvironment.

Background: Dying tumor cells release intracellular potassium (K+), raising extracellular K+ ([K+]e) in the tumor microenvironment (TME) to 40-50 mM (high-[K+]e). Here, we investigated the effect of high-[K+]e on T cell functions. Materials and Methods: Functional impacts of high-[K+]e on human T cells were determined by cellular, molecular, and imaging assays. Results: Exposure to high-[K+]e suppressed the proliferation of central memory and effector memory T cells, while T memory stem cells were unaffected. High-[K+]e inhibited T cell cytokine production and dampened antitumor cytotoxicity, by modulating the Akt signaling pathway. High-[K+]e caused significant upregulation of the immune checkpoint protein PD-1 in activated T cells. Although the number of KCa3.1 calcium-activated potassium channels expressed in T cells remained unaffected under high-[K+]e, a novel KCa3.1 activator, SKA-346, rescued T cells from high-[K+]e-mediated suppression. Conclusion: High-[K+]e represents a so far overlooked secondary checkpoint in cancer. KCa3.1 activators could overcome such "ionic-checkpoint"-mediated immunosuppression in the TME, and be administered together with known PD-1 inhibitors and other cancer therapeutics to improve outcomes.

Combating Microbial Contamination with Robust Polymeric Nanofibers: Elemental Effect on the Mussel-Inspired Cross-Linking of Electrospun Gelatin.

Designing biocompatible nanofibrous mats capable of preventing microbial colonization from resident and nosocomial bacteria for an extended period remains an unmet clinical need. In the present work, we designed antibiotic free durable antimicrobial nanofiber mats by taking advantage of synergistic interactions between polydopamine (pDA) and metal ions with varying degree of antimicrobial properties (Ag+, Mg2+, Ca2+, and Zn2+). Microscopic analysis showed successful pDA-mediated cross-linking of the gelatin nanofibers, which further improved by the inclusion of Ag+, Mg2+, and Ca2+ ions as supported by mechanical and thermal studies. Spectroscopic results reinforce the presence of strong interactions between pDA and metal ions in the composite nanofibers, leading to generation of robust polymeric nanofibers. We further showed that strong pDA-Ag interactions attenuated the cell cytotoxicity and anticell proliferative properties of silver ions for immortalized keratinocytes and primary human dermal fibroblasts. pDA-Ca2+/Zn2+ interactions rendered the composite structure sterile against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains, whereas the silver ion-incorporated composite mats displayed broad spectrum antibacterial activity against both Gram-positive/-negative bacteria and yeast strains. We showed that the strong pDA-Ag interactions help retaining long-term antimicrobial activity of the mats for at least 40 days while attenuating mammalian cell cytotoxicity of silver ions for skin cells. Overall, the results suggest the potential of pDA-metal ion interactions for engineering sterile nanofibrous mats and expanding the antibiotic armamentarium against drug-resistant pathogens.

Antimicrobial quaternary ammonium organosilane cross-linked nanofibrous collagen scaffolds for tissue engineering.

INTRODUCTION: In search for cross-linkers with multifunctional characteristics, the present work investigated the utility of quaternary ammonium organosilane (QOS) as a potential cross-linker for electrospun collagen nanofibers. We hypothesized that the quaternary ammonium ions improve the electrospinnability by reducing the surface tension and confer antimicrobial properties, while the formation of siloxane after alkaline hydrolysis could cross-link collagen and stimulate cell proliferation. MATERIALS AND METHODS: QOS collagen nanofibers were electrospun by incorporating various concentrations of QOS (0.1%-10% w/w) and were cross-linked in situ after exposure to ammonium carbonate. The QOS cross-linked scaffolds were characterized and their biological properties were evaluated in terms of their biocompatibility, cellular adhesion and metabolic activity for primary human dermal fibroblasts and human fetal osteoblasts. RESULTS AND DISCUSSION: The study revealed that 1) QOS cross-linking increased the flexibility of otherwise rigid collagen nanofibers and improved the thermal stability; 2) QOS cross-linked mats displayed potent antibacterial activity and 3) the biocompatibility of the composite mats depended on the amount of QOS present in dope solution - at low QOS concentrations (0.1% w/w), the mats promoted mammalian cell proliferation and growth, whereas at higher QOS concentrations, cytotoxic effect was observed. CONCLUSION: This study demonstrates that QOS cross-linked mats possess anti-infective properties and confer niches for cellular growth and proliferation, thus offering a useful approach, which is important for hard and soft tissue engineering and regenerative medicine.