Sigma-1 receptor modulation fine-tunes KV1.5 channels and impacts pulmonary vascular function.

KV1.5 channels are key players in the regulation of vascular tone and atrial excitability and their impairment is associated with cardiovascular diseases including pulmonary arterial hypertension (PAH) and atrial fibrillation (AF). Unfortunately, pharmacological strategies to improve KV1.5 channel function are missing. Herein, we aimed to study whether the chaperone sigma-1 receptor (S1R) is able to regulate these channels and represent a new strategy to enhance their function. By using different electrophysiological and molecular techniques in X. laevis oocytes and HEK293 cells, we demonstrate that S1R physically interacts with KV1.5 channels and regulate their expression and function. S1R induced a bimodal regulation of KV1.5 channel expression/activity, increasing it at low concentrations and decreasing it at high concentrations. Of note, S1R agonists (PRE084 and SKF10047) increased, whereas the S1R antagonist BD1047 decreased, KV1.5 expression and activity. Moreover, PRE084 markedly increased KV1.5 currents in pulmonary artery smooth muscle cells and attenuated vasoconstriction and proliferation in pulmonary arteries. We also show that both KV1.5 channels and S1R, at mRNA and protein levels, are clearly downregulated in samples from PAH and AF patients. Moreover, the expression of both genes showed a positive correlation. Finally, the ability of PRE084 to increase KV1.5 function was preserved under sustained hypoxic conditions, as an in vitro PAH model. Our study provides insight into the key role of S1R in modulating the expression and activity of KV1.5 channels and highlights the potential role of this chaperone as a novel pharmacological target for pathological conditions associated with KV1.5 channel dysfunction.

Encapsulation of Azotobacter vinelandii ATCC 12837 in Alginate-Na Beads as a Tomato Seedling Inoculant.

Encapsulation is an immobilization method characterized by restricting microbial cells to a delimited area while preserving their metabolic viability. This technique represents an alternative to improve the adaptive capacity of bacteria in the face of interactions with native microorganisms and environmental factors that limit their inoculation. This study aimed to evaluate the effect of Azotobacter vinelandii ATCC 12837 encapsulated in alginate-Na beads as an inoculant of tomato (Solanum Lycopersicum L) seedlings. Two inoculation treatments were carried out: liquid and encapsulated, and the control without microorganisms. Physiological variables, microbial viability, and the presence of A. vinelandii were determined by qPCR. Inoculation with A. vinelandii in liquid and encapsulated form favored seedling growth. Plants with the encapsulated inoculum significantly increased germination percentage (20%), stem diameter (38%), seedling height (34%), root length (69%), NO3 concentration (41%), and Na (30%); compared to the control. Encapsulation of A. vinelandii in alginate-Na macrocapsules allowed its establishment in the rhizosphere and was corroborated by viable count and molecular methods. The viability of the bacteria was maintained for 28 days using both inoculation methods, and not detected in the control treatment.

IQM-PC332, a Novel DREAM Ligand with Antinociceptive Effect on Peripheral Nerve Injury-Induced Pain.

Neuropathic pain is a form of chronic pain arising from damage of the neural cells that sense, transmit or process sensory information. Given its growing prevalence and common refractoriness to conventional analgesics, the development of new drugs with pain relief effects constitutes a prominent clinical need. In this respect, drugs that reduce activity of sensory neurons by modulating ion channels hold the promise to become effective analgesics. Here, we evaluated the mechanical antinociceptive effect of IQM-PC332, a novel ligand of the multifunctional protein downstream regulatory element antagonist modulator (DREAM) in rats subjected to chronic constriction injury of the sciatic nerve as a model of neuropathic pain. IQM-PC332 administered by intraplantar (0.01-10 µg) or intraperitoneal (0.02-1 µg/kg) injection reduced mechanical sensitivity by ≈100% of the maximum possible effect, with ED50 of 0.27 ± 0.05 µg and 0.09 ± 0.01 µg/kg, respectively. Perforated-patch whole-cell recordings in isolated dorsal root ganglion (DRG) neurons showed that IQM-PC332 (1 and 10 µM) reduced ionic currents through voltage-gated K+ channels responsible for A-type potassium currents, low, T-type, and high voltage-activated Ca2+ channels, and transient receptor potential vanilloid-1 (TRPV1) channels. Furthermore, IQM-PC332 (1 µM) reduced electrically evoked action potentials in DRG neurons from neuropathic animals. It is suggested that by modulating multiple DREAM-ion channel signaling complexes, IQM-PC332 may serve a lead compound of novel multimodal analgesics.

Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2.

The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.

Nuclear abnormalities in umbilical cord blood lymphocytes of newborns from the Ahome and Guasave municipalities in Sinaloa, Mexico.

AIM: We measured the frequency of nuclear abnormalities of 210 blood samples from the umbilical cord, since human fetuses are exposed to environmental mixtures of pesticides that induce DNA damage. METHODS: The determinations were made through the micronucleus assay test in lymphocytes from the umbilical cord blood of newborns whose mothers live in Ahome (n = 105) and Guasave (n = 105), Sinaloa, Mexico. RESULTS: The average frequency of anomalies in 1000 cells were, respectively: micronucleus 0.4 vs. 2.9, pyknotic cells 18.3 vs. 109.2, chromatin condensation 7.7 vs. 150.1, karyolitic cells 1.8 vs. 24.4, and binucleated cells 4.9 vs. 74.6. The calculated Pearson correlation factors of nuclear abnormality frequencies between both municipalities were low and negative, suggesting that they did not correlate between the Ahome and Guasave newborns and indicating a higher number of mothers exposed in Guasave. CONCLUSION: Our data suggest that monitoring nuclear abnormalities in umbilical cord blood samples could be a useful tool to identify transplacental mutagens perfusion that is being discharged into the local environment.

KV1.5-KVß1.3 Recycling Is PKC-Dependent.

KV1.5 channel function is modified by different regulatory subunits. KVß1.3 subunits assemble with KV1.5 channels and induce a fast and incomplete inactivation. Inhibition of PKC abolishes the KVß1.3-induced fast inactivation, decreases the amplitude of the current KV1.5-KVß1.3 and modifies their pharmacology likely due to changes in the traffic of KV1.5-KVß1.3 channels in a PKC-dependent manner. In order to analyze this hypothesis, HEK293 cells were transfected with KV1.5-KVß1.3 channels, and currents were recorded by whole-cell configuration of the patch-clamp technique. The presence of KV1.5 in the membrane was analyzed by biotinylation techniques, live cell imaging and confocal microscopy approaches. PKC inhibition resulted in a decrease of 33 ± 7% of channels in the cell surface due to reduced recycling to the plasma membrane, as was confirmed by confocal microscopy. Live cell imaging indicated that PKC inhibition almost abolished the recycling of the KV1.5-KVß1.3 channels, generating an accumulation of channels into the cytoplasm. All these results suggest that the trafficking regulation of KV1.5-KVß1.3 channels is dependent on phosphorylation by PKC and, therefore, they could represent a clinically relevant issue, mainly in those diseases that exhibit modifications in PKC activity.

Pharmacological Approaches for the Modulation of the Potassium Channel KV4.x and KChIPs.

Ion channels are macromolecular complexes present in the plasma membrane and intracellular organelles of cells. Dysfunction of ion channels results in a group of disorders named channelopathies, which represent an extraordinary challenge for study and treatment. In this review, we will focus on voltage-gated potassium channels (KV), specifically on the KV4-family. The activation of these channels generates outward currents operating at subthreshold membrane potentials as recorded from myocardial cells (ITO, transient outward current) and from the somata of hippocampal neurons (ISA). In the heart, KV4 dysfunctions are related to Brugada syndrome, atrial fibrillation, hypertrophy, and heart failure. In hippocampus, KV4.x channelopathies are linked to schizophrenia, epilepsy, and Alzheimer's disease. KV4.x channels need to assemble with other accessory subunits (ß) to fully reproduce the ITO and ISA currents. ß Subunits affect channel gating and/or the traffic to the plasma membrane, and their dysfunctions may influence channel pharmacology. Among KV4 regulatory subunits, this review aims to analyze the KV4/KChIPs interaction and the effect of small molecule KChIP ligands in the A-type currents generated by the modulation of the KV4/KChIP channel complex. Knowledge gained from structural and functional studies using activators or inhibitors of the potassium current mediated by KV4/KChIPs will better help understand the underlying mechanism involving KV4-mediated-channelopathies, establishing the foundations for drug discovery, and hence their treatments.

Use of a Humanized Anti-CD6 Monoclonal Antibody (Itolizumab) in Elderly Patients with Moderate COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a recent outbreak of coronavirus disease (COVID-19). In Cuba, the first case of COVID-19 was reported on March 11, 2020. Elderly individuals with multiple comorbidities are particularly susceptible to adverse clinical outcomes in the course of SARS-CoV-2 infection. During the outbreak, a local transmission event took place in a nursing home in Villa Clara province, Cuba, in which 19 elderly residents tested positive for SARS-CoV-2. METHODS: Based on the increased susceptibility to cytokine release syndrome, inducing respiratory and systemic complications in this population, 19 patients were included in an expanded access clinical trial to receive itolizumab, an anti-CD6 monoclonal antibody. RESULTS: All patients had underlying medical conditions. The product was well tolerated. After the first dose, the course of the disease was favorable, and 18 of the 19 patients (94.7%) were discharged clinically recovered with negative real-time reverse transcription polymerase chain reaction test results at 13 days. After one dose of itolizumab, circulating IL-6 decreased within the first 24-48 h in patients with high baseline values, whereas in patients with low levels, this concentration remained over low values. To preliminarily assess the effect of itolizumab, a control group was selected among the Cuban COVID-19 patients that did not receive immunomodulatory therapy. The control subjects were well matched regarding age, comorbidities, and severity of the disease. The percentage of itolizumab-treated, moderately ill patients who needed to be admitted to the intensive care unit was only one-third of that of the control group not treated with itolizumab. Additionally, treatment with itolizumab reduced the risk of death 10 times as compared with the control group. CONCLUSION: This study corroborates that the timely use of itolizumab in combination with other antivirals reduces COVID-19 disease worsening and mortality. The humanized antibody itolizumab emerges as a therapeutic alternative for patients with COVID-19. Our results suggest the possible use of itolizumab in patients with cytokine release syndrome from other pathologies.

An anti-CD6 monoclonal antibody (itolizumab) reduces circulating IL-6 in severe COVID-19 elderly patients.

BACKGROUND: Since the COVID-19 outbreak an unprecedented challenge for healthcare systems around the world has been placed. In Cuba, the first case of COVID-19 was reported on March 11. Elderly with multiple comorbidities have been the most risky population. Although most patients present a mild to moderate disease, some have developed severe symptoms. One of the possible mechanisms underlying rapid disease progression is a cytokine storm, in which interleukin (IL) -6 seems to be a major mediator. Itolizumab is a humanized recombinant anti-CD6 monoclonal antibody (MAb), with the ability of reducing serum interferon gamma (INF-γ), tumour necrosis factor alpha (TNFα) and IL-6. Based on these previous results in patients with psoriasis and rheumatoid arthritis, an expanded access clinical trial was approved by the Cuban regulatory agency for COVID-19 critically, severely and moderately ill patients. RESULTS: We show here a short kinetic of IL-6 serum concentration in the first 24 COVID-19 patients treated with itolizumab. Most of patients were elderly with multiple comorbidities. We found that with one itolizumab dose, the circulating IL-6 decreased in critically and severely ill patients, whereas in moderately ill patients the values didn't rise as compared to their low baseline levels. CONCLUSION: These findings suggest that itolizumab could be an attractive therapeutic option to decrease the negative outcome of the cytokine storm in COVID-19 patients. TRIAL REGISTRATION: CECMED IIC RD-EC 179, RPCEC00000311. Registered 4 May 2020 - Retrospectively registered, http://rpcec.sld.cu/ensayos/RPCEC00000311-Sp or http://rpcec.sld.cu/trials/RPCEC00000311-En.

Differential effect of Androctonus australis hector venom components on macrophage KV channels: electrophysiological characterization.

Neurotoxins of scorpion venoms modulate ion channels. Voltage-gated potassium (KV) channels regulate the membrane potential and are involved in the activation and proliferation of immune cells. Macrophages are key components of the inflammatory response induced by scorpion venom. The present study was undertaken to investigate the effect of Androctonus australis hector (Aah) venom on KV channels in murine resident peritoneal macrophages. The cytotoxicity of the venom was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) -based assay and electrophysiological recordings were performed using the whole-cell patch clamp technique. High doses of Aah venom (50, 125, 250 and 500 µg/ml) significantly decreased cell viability, while concentrations of 0.1-25 µg/ml were not cytotoxic towards peritoneal macrophages. Electrophysiological data revealed a differential block of KV current between resting and LPS-activated macrophages. Aah venom significantly reduced KV current amplitude by 62.5 ± 4.78% (n = 8, p < 0.05), reduced the use-dependent decay of the current, decreased the degree of inactivation and decelerated the inactivation process of KV current in LPS-activated macrophages. Unlike cloned KV1.5 channels, Aah venom exerted a similar blocking effect on KV1.3 compared to KV current in LPS-activated macrophages, along with a hyperpolarizing shift in the voltage dependence of KV1.3 inactivation, indicating a direct mechanism of current inhibition by targeting KV1.3 subunits. The obtained results, demonstrating that Aah venom differentially targets KV channels in macrophages, suggest differential outcomes for their inhibitions, and that further investigations of scorpion venom immunomodulatory potential are required.

In vitro cytotoxicity and genotoxicity of Furia®180 SC (zeta-cypermethrin) and Bulldock 125®SC (ß-cyfluthrin) pyrethroid insecticides in human peripheral blood lymphocytes.

In the present study, human peripheral blood lymphocytes were exposed in vitro to 0, 6, 12, 18, 24, and 30 µg/mL Furia®180 SC (zeta-cypermethrin) and 0, 6.3, 12.5, 18.8, 25, and 31.3 µg/mL Bulldock®125 SC (ß-cyfluthrin). Exposure to 32 µg/mL bleomycin for 24 h served as a positive control. The cytotoxic and genotoxic effects of each insecticide were analyzed using alkaline comet and trypan blue dye exclusion assays. DNA damage was evaluated through three genotoxicity parameters: tail length (TL), tail moment (TM) and tail intensity (TI). Furia®180 SC and Bulldock®125 SC pyrethroid insecticides and bleomycin significantly increased DNA damage in a concentration-dependent manner. Bulldock®125 SC induced more DNA damage than Furia. Lymphocyte viability did not change after exposure to different concentrations of the two pyrethroid insecticides and bleomycin. Moreover, genotoxic results demonstrated that Furia®180 SC and Bulldock®125 SC insecticides caused in vitro DNA damage in human peripheral lymphocytes.

D242N, a KV7.1 LQTS mutation uncovers a key residue for IKs voltage dependence.

KV7.1 and KCNE1 co-assemble to give rise to the IKs current, one of the most important repolarizing currents of the cardiac action potential. Its relevance is underscored by the identification of >500 mutations in KV7.1 and, at least, 36 in KCNE1, that cause Long QT Syndrome (LQTS). The aim of this study was to characterize the biophysical and cellular consequences of the D242N KV7.1 mutation associated with the LQTS. The mutation is located in the S4 transmembrane segment, within the voltage sensor of the KV7.1 channel, disrupting the conserved charge balance of this region. Perforated patch-clamp experiments show that, unexpectedly, the mutation did not disrupt the voltage-dependent activation but it removed the inactivation and slowed the activation kinetics of D242N KV7.1 channels. Biotinylation of cell-surface protein and co-immunoprecipitation experiments revealed that neither plasma membrane targeting nor co-assembly between KV7.1 and KCNE1 was altered by the mutation. However, the association of D242N KV7.1 with KCNE1 strongly shifted the voltage dependence of activation to more depolarized potentials (+50mV), hindering IKs current at physiologically relevant membrane potentials. Both functional and computational analysis suggest that the clinical phenotype of the LQTS patients carrying the D242N mutation is due to impaired action potential adaptation to exercise and, in particular, to increase in heart rate. Moreover, our data identify D242 aminoacidic position as a potential residue involved in the KCNE1-mediated regulation of the voltage dependence of activation of the KV7.1 channel.

Functional assembly of Kv7.1/Kv7.5 channels with emerging properties on vascular muscle physiology.

OBJECTIVE: Voltage-dependent K(+) (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. APPROACH AND RESULTS: Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. CONCLUSIONS: Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.

Modulation of voltage-dependent and inward rectifier potassium channels by 15-epi-lipoxin-A4 in activated murine macrophages: implications in innate immunity.

Potassium channels modulate macrophage physiology. Blockade of voltage-dependent potassium channels (Kv) by specific antagonists decreases macrophage cytokine production and inhibits proliferation. In the presence of aspirin, acetylated cyclooxygenase-2 loses the activity required to synthesize PGs but maintains the oxygenase activity to produce 15R-HETE from arachidonate. This intermediate product is transformed via 5-LOX into epimeric lipoxins, termed 15-epi-lipoxins (15-epi-lipoxin A4 [e-LXA4]). Kv have been proposed as anti-inflammatory targets. Therefore, we studied the effects of e-LXA4 on signaling and on Kv and inward rectifier potassium channels (Kir) in mice bone marrow-derived macrophages (BMDM). Electrophysiological recordings were performed in these cells by the whole-cell patch-clamp technique. Treatment of BMDM with e-LXA4 inhibited LPS-dependent activation of NF-κB and IκB kinase ß activity, protected against LPS activation-dependent apoptosis, and enhanced the accumulation of the Nrf-2 transcription factor. Moreover, treatment of LPS-stimulated BMDM with e-LXA4 resulted in a rapid decrease of Kv currents, compatible with attenuation of the inflammatory response. Long-term treatment of LPS-stimulated BMDM with e-LXA4 significantly reverted LPS effects on Kv and Kir currents. Under these conditions, e-LXA4 decreased the calcium influx versus that observed in LPS-stimulated BMDM. These effects were partially mediated via the lipoxin receptor (ALX), because they were significantly reverted by a selective ALX receptor antagonist. We provide evidence for a new mechanism by which e-LXA4 contributes to inflammation resolution, consisting of the reversion of LPS effects on Kv and Kir currents in macrophages.

Inverse correlation among organochlorine pesticide levels to total lipid serum contents: a preliminary study in Veracruz, México.

Organochlorine pesticides, due to their hydrophobic nature and persistence, accumulate in tissues rich in lipids, which had been used as a biomarker for environmental pollution. In humans, organochlorine pesticides are continuously circulating and equilibrating among body compartments. The objective of the study was to evaluate the concentrations of organochlorine pesticides in blood serum and compare their levels to the total lipid contents in Veracruz, México inhabitants. Our hypothesis is that concentrations of organochlorine pesticides will increase just as lipid concentrations. Levels of organochlorine pesticides were divided in ascending tertils according to their total lipid content. The linear trend model applied surprisingly reveals that the average level of all organochlorine pesticides decreases as the lipid concentration increases. From one tertil to the next ß-HCH, it shows a decrease of -3.19 mg kg(-1) on lipid basis, pp.'DDE levels decrease by -3.70 mg kg(-1) on lipid basis and pp.'DDT levels decrease -1.13 mg kg(-1) on lipid basis. We conclude that the levels and the orderly sequence of organochlorine pesticide distributions in the blood serum maintain an inverse relationship to total lipid blood serum concentrations.

An update of the classical and novel methods used for measuring fast neurotransmitters during normal and brain altered function.

To understand better the cerebral functions, several methods have been developed to study the brain activity, they could be related with morphological, electrophysiological, molecular and neurochemical techniques. Monitoring neurotransmitter concentration is a key role to know better how the brain works during normal or pathological conditions, as well as for studying the changes in neurotransmitter concentration with the use of several drugs that could affect or reestablish the normal brain activity. Immediate response of the brain to environmental conditions is related with the release of the fast acting neurotransmission by glutamate (Glu), γ-aminobutyric acid (GABA) and acetylcholine (ACh) through the opening of ligand-operated ion channels. Neurotransmitter release is mainly determined by the classical microdialysis technique, this is generally coupled to high performance liquid chromatography (HPLC). Detection of neurotransmitters can be done by fluorescence, optical density, electrochemistry or other detection systems more sophisticated. Although the microdialysis method is the golden technique to monitor the brain neurotransmitters, it has a poor temporal resolution. Recently, with the use of biosensor the drawback of temporal resolution has been improved considerably, however other inconveniences have merged, such as stability, reproducibility and the lack of reliable biosensors mainly for GABA. The aim of this review is to show the important advances in the different ways to measure neurotransmitter concentrations; both with the use of classic techniques as well as with the novel methods and alternant approaches to improve the temporal resolution.

KCNE4-dependent modulation of Kv1.3 pharmacology.

The voltage-dependent potassium channel Kv1.3 is a promising therapeutic target for the treatment of autoimmune and chronic inflammatory disorders. Kv1.3 blockers are effective in treating multiple sclerosis (fampridine) and psoriasis (dalazatide). However, most Kv1.3 pharmacological antagonists are not specific enough, triggering potential side effects and limiting their therapeutic use. Functional Kv are oligomeric complexes in which the presence of ancillary subunits shapes their function and pharmacology. In leukocytes, Kv1.3 associates with KCNE4, which reduces the surface abundance and enhances the inactivation of the channel. This mechanism exerts profound consequences on Kv1.3-related physiological responses. Because KCNE peptides alter the pharmacology of Kv channels, we studied the effects of KCNE4 on Kv1.3 pharmacology to gain insights into pharmacological approaches. To that end, we used margatoxin, which binds the channel pore from the extracellular space, and Psora-4, which blocks the channel from the intracellular side. While KCNE4 apparently did not alter the affinity of either margatoxin or Psora-4, it slowed the inhibition kinetics of the latter in a stoichiometry-dependent manner. The results suggested changes in the Kv1.3 architecture in the presence of KCNE4. The data indicated that while the outer part of the channel mouth remains unaffected, KCNE4 disturbs the intracellular architecture of the complex. Various leukocyte types expressing different Kv1.3/KCNE4 configurations participate in the immune response. Our data provide evidence that the presence of these variable architectures, which affect both the structure of the complex and their pharmacology, should be considered when developing putative therapeutic approaches.

Routing of Kv7.1 to endoplasmic reticulum plasma membrane junctions.

AIM: The voltage-gated Kv7.1 channel, in association with the regulatory subunit KCNE1, contributes to the IKs current in the heart. However, both proteins travel to the plasma membrane using different routes. While KCNE1 follows a classical Golgi-mediated anterograde pathway, Kv7.1 is located in endoplasmic reticulum-plasma membrane junctions (ER-PMjs), where it associates with KCNE1 before being delivered to the plasma membrane. METHODS: To characterize the channel routing to these spots we used a wide repertoire of methodologies, such as protein expression analysis (i.e. protein association and biotin labeling), confocal (i.e. immunocytochemistry, FRET, and FRAP), and dSTORM microscopy, transmission electron microscopy, proteomics, and electrophysiology. RESULTS: We demonstrated that Kv7.1 targeted ER-PMjs regardless of the origin or architecture of these structures. Kv2.1, a neuronal channel that also contributes to a cardiac action potential, and JPHs, involved in cardiac dyads, increased the number of ER-PMjs in nonexcitable cells, driving and increasing the level of Kv7.1 at the cell surface. Both ER-PMj inducers influenced channel function and dynamics, suggesting that different protein structures are formed. Although exhibiting no physical interaction, Kv7.1 resided in more condensed clusters (ring-shaped) with Kv2.1 than with JPH4. Moreover, we found that VAMPs and AMIGO, which are Kv2.1 ancillary proteins also associated with Kv7.1. Specially, VAP B, showed higher interaction with the channel when ER-PMjs were stimulated by Kv2.1. CONCLUSION: Our results indicated that Kv7.1 may bind to different structures of ER-PMjs that are induced by different mechanisms. This variable architecture can differentially affect the fate of cardiac Kv7.1 channels.

Protein kinase C (PKC) activity regulates functional effects of Kvß1.3 subunit on KV1.5 channels: identification of a cardiac Kv1.5 channelosome.

K(v)1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (I(Kur)). The regulatory K(v)ß1.3 subunit converts K(v)1.5 channels from delayed rectifiers with a modest degree of slow inactivation to channels with both fast and slow inactivation components. Previous studies have shown that inhibition of PKC with calphostin C abolishes the fast inactivation induced by K(v)ß1.3. In this study, we investigated the mechanisms underlying this phenomenon using electrophysiological, biochemical, and confocal microscopy approaches. To achieve this, we used HEK293 cells (which lack K(v)ß subunits) transiently cotransfected with K(v)1.5+K(v)ß1.3 and also rat ventricular and atrial tissue to study native α-ß subunit interactions. Immunocytochemistry assays demonstrated that these channel subunits colocalize in control conditions and after calphostin C treatment. Moreover, coimmunoprecipitation studies showed that K(v)1.5 and K(v)ß1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, K(v)ß1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed K(v)ß1.3-induced inactivation, indicating that PKC inhibition causes a dramatic positive shift of the inactivation curve. Our results demonstrate that calphostin C-mediated abolishment of fast inactivation is not due to the dissociation of K(v)1.5 and K(v)ß1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between K(v)1.5, K(v)ß1.3, the receptor for activated C kinase (RACK1), PKCßI, PKCßII, and PKCθ in HEK293 cells. A very similar K(v)1.5 channelosome was found in rat ventricular tissue but not in atrial tissue.