Novel insights into the modulation of the voltage-gated potassium channel KV1.3 activation gating by membrane ceramides.

Membrane lipids extensively modulate the activation gating of voltage-gated potassium channels (KV), 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 KV1.3 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 KV1.3. 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.

Functional characterization of cell-free expressed Kv1.3 channel using a voltage-sensitive fluorescent dye.

Using a cell-free expression system, we produced the Kv1.3 protein embedded in one step within detergent micelles. The protein was then purified and relipidated into mixed lipid bilayers. These proteoliposomes held an average of 0.8 protein per liposome. We examined channel forming activity using an oxonol VI fluorescent probe and verified its inhibition using margatoxin and ShK toxins. This assay was automatized and optimized so as to get a Z' statistical factor acceptable for venom fraction screening. We obtained a sensible amount of membrane protein using the cell-free assay, that proved to be active when embedded in liposomes. These findings emphasize the quality of the cell-free produced KV1.3 proteoliposomes and the usefulness of a fluorescent probe. This method can benefit the field of channel characterization, as well as provide tools for the development of new inhibitors, so as to reinforce our therapeutic arsenal against autoimmune diseases.

Human immunodeficiency virus protein Tat induces oligodendrocyte injury by enhancing outward K+ current conducted by KV1.3.

Brain white matter damage is frequently detected in patients infected with human immunodeficiency virus type 1 (HIV-1). White matter is composed of neuronal axons sheathed by oligodendrocytes (Ols), the myelin-forming cells in central nervous system. Ols are susceptible to HIV-1 viral trans-activator of transcription (Tat) and injury of Ols results in myelin sheath damage. It has been demonstrated that activation of voltage-gated K+ (KV) channels induces cell apoptosis and Ols predominantly express K+ channel KV1.3. It is our hypothesis that Tat injures Ols via activation of KV1.3. To test this hypothesis, we studied the involvement of KV1.3 in Tat-induced Ol/myelin injury both in vitro and ex vivo. Application of Tat to primary rat Ol cultures enhanced whole-cell KV1.3 current recorded under voltage clamp configuration and confirmed by specific KV1.3 antagonists Margatoxin (MgTx) and 5-(4-phenoxybutoxy) psoralen (PAP). The Tat enhancement of KV1.3 current was associated with Tat-induced Ol apoptosis, which was blocked by MgTx and PAP or by siRNA knockdown of KV1.3 gene. The Tat-induced Ol injury was validated in cultured rat brain slices, particularly in corpus callosum and striatum, that incubation of the slices with Tat resulted in myelin damage and reduction of myelin basic protein which were also blocked by aforementioned KV1.3 antagonists. Further studies revealed that Tat interacts with KV1.3 as determined by protein pull-down of recombinant GST-Tat with KV1.3 expressed in rat brains and HEK293 cells. Such protein-protein interaction may alter channel protein phosphorylation, resultant channel activity and consequent Ol/myelin injury. Taken together, these results demonstrate an involvement of KV1.3 in Tat- induced Ol/myelin injury, a potential mechanism for the pathogenesis of HIV-1-associated white matter damage.

Trabectedin modulates macrophage polarization in the tumor-microenvironment. Role of KV1.3 and KV1.5 channels.

Immune cells have an important role in the tumor-microenvironment. Macrophages may tune the immune response toward inflammatory or tolerance pathways. Tumor-associated macrophages (TAM) have a string of immunosuppressive functions and they are considered a therapeutic target in cancer. This study aimed to analyze the effects of trabectedin, an antitumor agent, on the tumor-microenvironment through the characterization of the electrophysiological and molecular phenotype of macrophages. Experiments were performed using the whole-cell configuration of the patch-clamp technique in resident peritoneal mouse macrophages. Trabectedin does not directly interact with KV1.5 and KV1.3 channels, but their treatment (16 h) with sub-cytotoxic concentrations of trabectedin increased their KV current due to an upregulation of KV1.3 channels. In vitro generated TAM (TAMiv) exhibited an M2-like phenotype. TAMiv generated a small KV current and express high levels of M2 markers. K+ current from TAMs isolated from tumors generated in mice is a mixture of KV and KCa, and in TAM isolated from tumors generated in trabectedin-treated mice, the current is mostly driven by KCa. We conclude that the antitumor capacity of trabectedin is not only due to its effects on tumor cells, but also to the modulation of the tumor microenvironment, due, at least in part, to the modulation of the expression of different macrophage ion channels.

Immunosuppressive effects of new thiophene-based KV1.3 inhibitors.

Voltage-gated potassium channel KV1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca2+ homeostasis. Here, we present the structure-activity relationship, KV1.3 inhibition, and immunosuppressive effects of new thiophene-based KV1.3 inhibitors with nanomolar potency on K+ current in T-lymphocytes and KV1.3 inhibition on Ltk- cells. The new KV1.3 inhibitor trans-18 inhibited KV1.3 -mediated current in phytohemagglutinin (PHA)-activated T-lymphocytes with an IC50 value of 26.1 nM and in mammalian Ltk- cells with an IC50 value of 230 nM. The KV1.3 inhibitor trans-18 also had nanomolar potency against KV1.3 in Xenopus laevis oocytes (IC50 = 136 nM). The novel thiophene-based KV1.3 inhibitors impaired intracellular Ca2+ signaling as well as T-cell activation, proliferation, and colony formation.

Inhibition of inflammatory cytokine production and proliferation in macrophages by Kunitz-type inhibitors from Echinococcus granulosus.

The genus Echinococcus of cestode parasites includes important pathogens of humans and livestock animals. Transcriptomic and genomic studies on E. granulosus and E. multilocularis uncovered striking expansion of monodomain Kunitz proteins. This expansion is accompanied by the specialization of some family members away from the ancestral protease inhibition function to fulfill cation channel blockade functions. Since cation channels are involved in immune processes, we tested the effects on macrophage physiology of two E. granulosus Kunitz-type inhibitors of voltage-activated cation channels (Kv) that are close paralogs. Both inhibitors, EgKU-1 and EgKU-4, inhibited production of the Th1/Th17 cytokine subunit IL-12/23p40 by macrophages stimulated with the TLR4 agonist LPS. In addition, EgKU-4 but not EgKU-1 inhibited production of the inflammatory cytokine IL-6. These activities were not displayed by EgKU-3, a family member that is a protease inhibitor without known activity on cation channels. EgKU-4 potently inhibited macrophage proliferation in response to M-CSF, whereas EgKU-1 displayed similar activity but with much lower potency, similar to EgKU-3. We discuss structural differences, including a heavily cationic C-terminal extension present in EgKU-4 but not in EgKU-1, that may explain the differential activities of the two close paralogs.

Targeting inflammatory cytokine storm to fight against COVID-19 associated severe complications.

Such testing and trying time probably never seen before in the human history. The novel coronavirus disease abbreviated as COVID-19 is the ongoing health crisis which entered into human life in late December 2019. The ease of transmission between humans and the undetectability in early stage makes COVID-19 frightening and unprecedented. The disease is characterised by pneumonia progressing to breathing difficulty, acute respiratory distress syndrome (ARDS) and multi-organ failure. Clinical studies suggest excessive release of inflammatory mediators leads to cytokine storm, a phenomenon which appears to be potentially life-threatening in COVID-19. Across the globe, when the world authorities are grappling to contain the virus, our review provides a glimpse on structure, pathophysiology of the virus and further sheds light on various clinical complications associated with the disease in order to open up/raise new horizons to explore various possible theoretical targets for COVID-19. The review also portrays a question and debates: Can targeting cytokine storm can be a feasible approach to combat COVID-19?

Geraniol targets KV1.3 ion channel and exhibits anti-inflammatory activity in vitro and in vivo.

Naturally occurring monoterpenes are known for their various pharmacological activities including anti-inflammation. KV1.3 ion channel is a voltage-gated potassium channel and has been validated as a drug target for autoimmune and chronic inflammatory diseases like psoriasis. Here we experimentally test the direct interaction between monoterpenes and KV1.3 ion channel. Our electrophysiological analysis determined that monoterpenes (geraniol, nerol, ß-citronellol, citral and linalool) have inhibitory effects on KV1.3 ion channel. Representatively, geraniol reversibly blocked KV1.3 currents in a voltage-dependent manner with an IC50 of 490.50 ±â€¯1.04 µM at +40 mV in HEK293T cells. At the effective concentrations, geraniol also inhibited cytokine secretion of activated human T cells, including IL-2, TNF-α and IFN-γ. In an imiquimod-induced psoriasis-like animal model, geraniol administration significantly reduced psoriasis area and severity index scores, ameliorated the deteriorating histopathology and decreased the degree of splenomegaly. Together, our findings not only suggest that monoterpenes may serve as lead molecules for the development of KV1.3 inhibitors, but also indicate that geraniol could be considered as a promising therapeutic candidate to treat autoimmune diseases.

Determining the target of membrane sterols on voltage-gated potassium channels.

Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel Kv1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing Kv1.3 and Kv10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-ß-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.