SK Channels Research Articles

Combined SK and K2P channel block cardioverts atrial fibrillation in 82% of cases, doubling the efficacy of single channel block

Abstract Background Inhibiting the atrial-selective SK and K2P channels represents a promising strategy for cardioverting atrial fibrillation (AF), but its efficacy has not been evaluated in a large human cohort. Objectives This study employed in-silico trials in 1000 virtual patients (i.e., clinical trials conducted in a population of virtual human subjects through modelling and simulation) to estimate the efficacy and safety of three pharmacological interventions: single and combined SK and K2P channel block. Methods A large cohort of 1000 virtual patients was developed based on human data, to cover the electrophysiological, anatomical and structural variability encountered in clinical practice. The subset of virtual patients with AF maintenance underwent pharmacological cardioversion (Figure). Results Sustained AF was observed in 654 (65%) virtual patients. In this cohort, cardioversion efficacies of 33% (213 of 654), 43% (278 of 654) and 82% (534 of 654) were obtained for single SK, single K2P and combined channel block, respectively (Figure). The cardioversion efficacy was proportional to an increase in tissue refractoriness (increase of 45.2±43.0, 71.0±55.3 and 133.0±48.4 ms in the atrial refractory period) and depended on the ionic current profile. Virtual patients cardioverted by SK channel block presented lower K2P densities (0.008±0.002 vs. 0.006±0.002 S/mF; non-cardioverted vs. cardioverted virtual patients, respectively). Similarly, a lower SK density favoured the success of K2P channel inhibition (1.87±0.24 vs. 1.46±0.5 S/mF; non-cardioverted vs. cardioverted virtual patients, respectively). Both ionic currents had a crucial role on atrial repolarization, and thus, a synergism resulted from the polypharmacological strategy (i.e., higher efficacy than the additive effects of single channel block). All three interventions, including the multi-channel block, preserved the atrial electrophysiological function (i.e., conduction velocity and calcium transient dynamics) and thus, the atrial contractile properties (safety). Conclusion In-silico trials identify the polypharmacological SK+K2P channel block as an effective and safe strategy for AF management.

BA6b9, a novel sk4 K + channel blocker, attenuates atrial fibrillation substrate in rats with reduced ejection fraction

Abstract Background Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as novel druggable targets for efforts to treat AF. Methods and Findings We examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat post-myocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Interpretation Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Pharmacological characterization of alpha adrenoceptor-mediated motor responses in the rat colon.

Inhibitory neuromuscular transmission in the gastrointestinal tract is mediated by intrinsic nitrergic and purinergic neurons. Purines activate G protein-coupled receptor P2Y1 receptors, increasing intracellular Ca2+ that activates small conductance calcium-activated potassium (SKCa) channels. Little is known about the effect of adrenergic receptor activation on intestinal smooth muscle. In vascular tissue, stimulation of α-adrenoceptors causes smooth muscle contraction, while their effect on intestinal tissue is poorly understood. This study aimed to pharmacologically characterize the effect of α-adrenoceptor activation in the rat colon, which shares similar inhibitory pathways to the human colon. Muscle bath experiments were performed with the rat proximal, mid, and distal colon oriented both circularly and longitudinally. The α1-adrenoceptor agonist phenylephrine (PE) (10-8-10-5 M) evoked concentration-dependent relaxations of the intestinal smooth muscle from all regions and orientations. However, in the mid-circular colon at low PE concentrations, a contraction sensitive to 10-5 M phentolamine (non-selective α-adrenoceptor blocker), the neural blocker tetrodotoxin (TTX; 10-6 M), and atropine (10-6 M) was recorded. PE-induced relaxations were insensitive to TTX (10-6 M) and the nonselective β-adrenoceptor blocker propranolol (10-6 M). In contrast, PE-induced relaxations were blocked by phentolamine (10-5 M), prazosin (10-6 M) (α1-adrenoceptor blocker), and RS17053 (10-6 M) (α1A-blocker), but not by yohimbine (10-6 M) (α2-adrenoceptor blocker). Apamin (10-6 M), a SKCa channel blocker, abolished PE-induced relaxations. Contractile responses in the circular muscle of the mid colon could be attributed to α-adrenoceptors located on enteric cholinergic neurons. Stimulation of α1A-adrenoreceptors activates SKCa channels to cause smooth muscle relaxation, which constitutes a signaling pathway that shares similarities with P2Y1 receptors.

Atomistic mechanisms of the regulation of small-conductance Ca2+-activated K+ channel (SK2) by PIP2

Small-conductance Ca2+-activated K+ channels (SK, KCa2) are gated solely by intracellular microdomain Ca2+. The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca2+-CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias.

Effect of fibroblasts small- conductance Ca2+ -activated potassium channel subtype 2 (SK2) on myocardial fibrosis in pressure overload mouse

Studies have shown that Small conductance Ca2 + −activated K+ (SK) channel are expressed in fibroblasts. We aimed to determine the expression of SK2 channels in cardiac fibroblasts during myocardial hypertrophy and investigate its relationship with fibrotic remodeling. Myocardial hypertrophy and fibrotic remodeling induced by transverse aortic constriction (TAC) were assessed by echocardiography, Masson's trichrome staining and Western blot. Knockdown and overexpression of the SK2 protein were used to assess relationship between SK2 expression in fibroblasts and myocardial fibrosis. There is a positive correlation between myocardial fibrosis and SK2 channel protein expression during the development of myocardial hypertrophy. The differentiation and secretion of fibroblasts in mice with cardiac hypertrophy are enhanced, and the expression of SK2 channel protein is increased. Manipulating SK2 levels in fibroblasts can either promote or inhibit their differentiation and secretory function. Knocking down SK2 reduces the up-regulation of TGF β1, p-Smad2/3/GAPDH, p-p38/GAPDH, p-ERK1/2/GAPDH, and p-JNK/GAPDH proteins induced by Ang II in cardiac fibroblasts without significantly affecting total protein levels. AAV9-SK2-RNAi injection in mice improves cardiac function. Collectively, our study suggests that the expression of the SK2 channel is significantly increased in fibroblasts of mice with myocardial hypertrophy, potentially impacting myocardial fibrosis remodeling via the TGF-β signaling pathway.

Mild neonatal hypoxia disrupts adult hippocampal learning and memory and is associated with CK2-mediated dysregulation of synaptic calcium-activated potassium channel KCNN2.

Although nearly half of preterm survivors display persistent neurobehavioral dysfunction including memory impairment without overt gray matter injury, the underlying mechanisms of neuronal or glial dysfunction, and their relationship to commonly observed cerebral white matter injury are unclear. We developed a mouse model to test the hypothesis that mild hypoxia during preterm equivalence is sufficient to persistently disrupt hippocampal neuronal maturation related to adult cellular mechanisms of learning and memory. Methods: Neonatal (P2) mice were exposed to mild hypoxia (8%O 2 ) for 30 min and evaluated for acute injury responses or survived until adulthood for assessment of learning and memory and hippocampal neurodevelopment. Neonatal mild hypoxia resulted in clinically relevant oxygen desaturation and tachycardia without bradycardia and was not accompanied by cerebral gray or white matter injury. Neonatal hypoxia exposure was sufficient to cause hippocampal learning and memory deficits and abnormal maturation of CA1 neurons that persisted into adulthood. This was accompanied by reduced hippocampal CA3-CA1 synaptic strength and LTP and reduced synaptic activity of calcium-sensitive SK2 channels, key regulators of spike timing dependent neuroplasticity, including LTP. Structural illumination microscopy revealed reduced synaptic density, but intact SK2 localization at the synapse. Persistent loss of SK2 activity was mediated by altered casein kinase 2 (CK2) signaling. Clinically relevant mild hypoxic exposure in the neonatal mouse is sufficient to produce morphometric and functional disturbances in hippocampal neuronal maturation independently of white matter injury. Additionally, we describe a novel persistent mechanism of potassium channel dysregulation after neonatal hypoxia. Collectively our findings suggest an unexplored explanation for the broad spectrum of neurobehavioral, cognitive and learning disabilities that paradoxically persist into adulthood without overt gray matter injury after preterm birth.

27-Hydroxycholesterol acts on estrogen receptor α expressed by POMC neurons in the arcuate nucleus to modulate feeding behavior.

Oxysterols are metabolites of cholesterol that regulate cholesterol homeostasis. Among these, the most abundant oxysterol is 27-hydroxycholesterol (27HC), which can cross the blood-brain barrier. Because 27HC functions as an endogenous selective estrogen receptor modulator, we hypothesize that 27HC binds to the estrogen receptor α (ERα) in the brain to regulate energy balance. Supporting this view, we found that delivering 27HC to the brain reduced food intake and activated proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (POMCARH) in an ERα-dependent manner. In addition, we observed that inhibiting brain ERα, deleting ERα in POMC neurons, or chemogenetic inhibition of POMCARH neurons blocked the anorexigenic effects of 27HC. Mechanistically, we further revealed that 27HC stimulates POMCARH neurons by inhibiting the small conductance of the calcium-activated potassium (SK) channel. Together, our findings suggest that 27HC, through its interaction with ERα and modulation of the SK channel, inhibits food intake as a negative feedback mechanism against a surge in circulating cholesterol.

Small conductance calcium-activated potassium channel contributes to stress induced endothelial dysfunctions

Patients with Takotsubo syndrome displayed endothelial dysfunction, but underlying mechanisms have not been fully clarified. This study aimed to explore molecular signalling responsible for catecholamine excess induced endothelial dysfunction.Human cardiac microvascular endothelial cells were challenged by epinephrine to mimic catecholamine excess. Patch clamp, FACS, ELISA, PCR, and immunostaining were employed for the study.Epinephrine (Epi) enhanced small conductance calcium-activated potassium channel current (ISK1–3) through activating α1 adrenoceptor. Phenylephrine enhanced edothelin-1 (ET-1) and reactive oxygen species (ROS) production, and the effects involved contribution of ISK1–3. H2O2 enhanced ISK1–3 and ET-1 production. Enhancing ISK1–3 caused a hyperpolarization, which increases ROS and ET-1 production. BAPTA partially reduced phenylephrine-induced enhancement of ET-1 and ROS, suggesting that α1 receptor activation can enhance ROS/ET-1 generation in both calcium-dependent and calcium-independent ways.The study demonstrates that high concentration catecholamine can activate SK1–3 channels through α1 receptor-ROS signalling and increase ET-1 production, facilitating vasoconstriction.

Dysfunction of Small-Conductance Ca2+-Activated Potassium (SK) Channels Drives Amygdala Hyperexcitability and Neuropathic Pain Behaviors: Involvement of Epigenetic Mechanisms.

Neuroplasticity in the amygdala and its central nucleus (CeA) is linked to pain modulation and pain behaviors, but cellular mechanisms are not well understood. Here, we addressed the role of small-conductance Ca2+-activated potassium (SK) channels in pain-related amygdala plasticity. The facilitatory effects of the intra-CeA application of an SK channel blocker (apamin) on the pain behaviors of control rats were lost in a neuropathic pain model, whereas an SK channel activator (NS309) inhibited pain behaviors in neuropathic rats but not in sham controls, suggesting the loss of the inhibitory behavioral effects of amygdala SK channels. Brain slice electrophysiology found hyperexcitability of CeA neurons in the neuropathic pain condition due to the loss of SK channel-mediated medium afterhyperpolarization (mAHP), which was accompanied by decreased SK2 channel protein and mRNA expression, consistent with a pretranscriptional mechanisms. The underlying mechanisms involved the epigenetic silencing of the SK2 gene due to the increased DNA methylation of the CpG island of the SK2 promoter region and the change in methylated CpG sites in the CeA in neuropathic pain. This study identified the epigenetic dysregulation of SK channels in the amygdala (CeA) as a novel mechanism of neuropathic pain-related plasticity and behavior that could be targeted to control abnormally enhanced amygdala activity and chronic neuropathic pain.

Catecholamine induces endothelial dysfunction via Angiotensin II and intermediate conductance calcium activated potassium channel

Endothelial dysfunction contributes to the pathogenesis of Takotsubo syndrome (TTS). However, the exact mechanism underlying endothelial dysfunction in the setting of TTS has not been completely clarified. This study aims to investigate the roles of angiotensin II (Ang II) and intermediate-conductance Ca2+-activated K+ (SK4) channels in catecholamine-induced endothelial dysfunction. Human cardiac microvascular endothelial cells (HCMECs) were exposed to 100 µM epinephrine (Epi), mimicking the setting of TTS. Epi treatment increased the ET-1 concentration and reduced NO levels in HCMECs. Importantly, the effects of Epi were found to be mitigated in the presence of Ang II receptor blockers. Furthermore, Ang II mimicked Epi effects on ET-1 and NO production. Additionally, Ang II inhibited tube formation and increased cell apoptosis. The effects of Ang II could be reversed by an SK4 activator NS309 and mimicked by an SK4 channel blocker TRAM-34. Ang II also inhibited the SK4 channel current (ISK4) without affecting its expression level. Ang II could depolarize the cell membrane potential. Ang II promoted ROS release and reduced protein kinase A (PKA) expression. A ROS blocker prevented Ang II effect on ISK4. The PKA activator Sp-8-Br-cAMPS increased SK4 channel currents. Epinephrine enhanced the activity of ACE by activating the α1 receptor/Gq/PKC signal pathway, thereby promoting the secretion of Ang II. The study suggested that high-level catecholamine can increase Ang II release from endothelial cells by α1 receptors/Gq/PKC signal pathway. Ang II can inhibit SK4 channel current by increasing ROS generation and reducing PKA expression, thereby contributing to endothelial dysfunction.

GluN2A- and GluN2B-containing pre-synaptic N-methyl-d-aspartate receptors differentially regulate action potential-evoked Ca2+ influx via modulation of SK channels.

N-methyl-d-aspartate receptors (NMDARs) play a pivotal role in synaptic plasticity. While the functional role of post-synaptic NMDARs is well established, pre-synaptic NMDAR (pre-NMDAR) function is largely unexplored. Different pre-NMDAR subunit populations are documented at synapses, suggesting that subunit composition influences neuronal transmission. Here, we used electrophysiological recordings at Schaffer collateral-CA1 synapses partnered with Ca2+ imaging and glutamate uncaging at boutons of CA3 pyramidal neurones to reveal two populations of pre-NMDARs that contain either the GluN2A or GluN2B subunit. Activation of the GluN2B population decreases action potential-evoked Ca2+ influx via modulation of small-conductance Ca2+-activated K+ channels, while activation of the GluN2A population does the opposite. Critically, the level of functional expression of the subunits is subject to homeostatic regulation, bidirectionally affecting short-term facilitation, thus providing a capacity for a fine adjustment of information transfer. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons.

Pacemaking activity in substantia nigra dopaminergic neurons is generated by the coordinated activity of a variety of distinct somatodendritic voltage- and calcium-gated ion channels. We investigated whether these functional interactions could arise from a common localization in macromolecular complexes where physical proximity would allow for efficient interaction and co-regulations. For that purpose, we immunopurified six ion channel proteins involved in substantia nigra neuron autonomous firing to identify their molecular interactions. The ion channels chosen as bait were Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 channel proteins, and the methods chosen to determine interactions were co-immunoprecipitation analyzed through immunoblot and mass spectrometry as well as proximity ligation assay. A macromolecular complex composed of Cav1.3, HCN, and SK3 channels was unraveled. In addition, novel potential interactions between SK3 channels and sclerosis tuberous complex (Tsc) proteins, inhibitors of mTOR, and between HCN4 channels and the pro-degenerative protein Sarm1 were uncovered. In order to demonstrate the presence of these molecular interactions in situ, we used proximity ligation assay (PLA) imaging on midbrain slices containing the substantia nigra, and we could ascertain the presence of these protein complexes specifically in substantia nigra dopaminergic neurons. Based on the complementary functional role of the ion channels in the macromolecular complex identified, these results suggest that such tight interactions could partly underly the robustness of pacemaking in dopaminergic neurons.

A combination of chlorzoxazone and folic acid improves recognition memory, anxiety and depression in SCA3-84Q mice.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is reported to be the most common type of autosomal dominant cerebellar ataxia (ADCA). SCA3 patients suffer from a progressive decline in motor coordination and other disease-associated symptoms. Moreover, recent studies have reported that SCA3 patients also exhibit symptoms of cerebellar cognitive affective syndrome (CCAS). We previously observed signs of CCAS in mouse model of SCA3. Particularly, SCA3-84Q mice suffer from anxiety, recognition memory decline, and also exhibit signs of low mood and aversion to activity. Here we studied the effect of long-term injections of SK channels activator chlorzoxazone (CHZ) together and separately with the folic acid (FA) on the cerebellar Purkinje cell (PC) firing and histology, and also on the motor and cognitive functions as well as mood alterations in SCA3-84Q hemizygous transgenic mice. We realized that both CHZ and CHZ-FA combination had similar positive effect on pure cerebellum impairments including PC firing precision, PC histology, and motor performance in SCA3-84Q mice. However, only the CHZ-FA combination, but not CHZ, had significantly ameliorated the signs of anxiety and depression, and also noticeably improved recognition memory in SCA3-84Q mice. Our results suggest that the combination therapy for both ataxia and non-motor symptoms is required for the complex treatment of ADCA.

Store-operated calcium entry is necessary for sustained K+-induced capillary-to-arteriole dilation in the brain

Neurovascular coupling (NVC) is a physiological process that directs blood flow to the most active regions of the brain. The extensive network of capillaries in the brain acts as a sensory web that detects local neuronal activity and orchestrates the dilation of upstream arterioles to meet the brain’s metabolic demands. Activation of inwardly rectifying K+ (KIR) channels on brain capillary endothelial cells (cECs) by modest concentrations of extracellular K+ initiates retrograde propagating hyperpolarizing signals that induce vasodilation and increase blood flow. Brain capillaries exhibit diverse and dynamic patterns of Ca2+ signaling in vivo, but little is known about their origins or physiological function. Store-operated Ca2+ entry (SOCE) is fundamental to Ca2+homeostasis in non-excitable cells, but the contribution of SOCE to capillary-to-arteriole dilation has not been explored. Here, we report that brain cECs express the endoplasmic reticulum-resident Ca2+ sensing protein stromal interaction molecule 1 ( Stim1) and the Ca2+-selective Orai1 and Orai3 channels. Ca2+ imaging experiments using brain cECs from transgenic mice expressing GCaMP8 in the endothelium or loaded with Fluo-4 showed that these cells exhibited robust SOCE that was blocked by the Orai1 inhibitor Synta66. Using EC-specific knockout (ecKO) mice, we found that Stim1 and Orai1 but not Orai3 are required for SOCE. Ex vivo brain microvascular preparations consisting of cannulated and pressured parenchymal arterioles with intact capillary beds were used to examine the role of SOCE in capillary-to-arteriole dilation. Arteriole dilation in response to a train of K+ pulses applied to capillaries exhibited dramatic run-down when preparations were treated with Snyta66 or when tissues from Stim1-ecKO mice were used. Preparations from Orai3-ecKO mice did not demonstrate run-down. Moreover, inhibition of Ca2+-activated K+ (SK and IK) channels also induced run-down, suggesting that SOCE activates SK and IK channels to boost KIR-induced hyperpolarizing signals to dilate upstream arterioles during multiple stimulations. We conclude that brain cECs exhibit SOCE that requires Stim1 and Orai1. Our data further show that brain cEC SOCE is necessary for capillary-to-arteriole dilation during repeated stimulations, suggesting an essential role for SOCE in NVC. R35HL155008, R33NS115132, and P20GM130459 (to SE). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

Endogenous ether lipids differentially promote tumor aggressiveness by regulating the SK3 channel

SK3 channels are potassium channels found to promote tumor aggressiveness. We have previously demonstrated that SK3 is regulated by synthetic ether lipids, but the role of endogenous ether lipids is unknown. Here, we have studied the role of endogenous alkyl- and alkenyl-ether lipids on SK3 channels and on the biology of cancer cells. Experiments revealed that the suppression of alkylglycerone phosphate synthase or plasmanylethanolamine desaturase 1, which are key enzymes for alkyl- and alkenyl-ether-lipid synthesis, respectively, decreased SK3 expression by increasing micro RNA (miR)-499 and miR-208 expression, leading to a decrease in SK3-dependent calcium entry, cell migration, and matrix metalloproteinase 9–dependent cell adhesion and invasion. We identified several ether lipids that promoted SK3 expression and found a differential role of alkyl- and alkenyl-ether lipids on SK3 activity. The expressions of alkylglycerone phosphate synthase, SK3, and miR were associated in clinical samples emphasizing the clinical consistency of our observations. To our knowledge, this is the first report showing that ether lipids differentially control tumor aggressiveness by regulating an ion channel. This insight provides new possibilities for therapeutic interventions, offering clinicians an opportunity to manipulate ion channel dysfunction by adjusting the composition of ether lipids.

Identification of Riluzole derivatives as novel calmodulin inhibitors with neuroprotective activity by a joint synthesis, biosensor, and computational guided strategy

The development of new molecules for the treatment of calmodulin related cardiovascular or neurodegenerative diseases is an interesting goal. In this work, we introduce a novel strategy with four main steps: (1) chemical synthesis of target molecules, (2) Förster Resonance Energy Transfer (FRET) biosensor development and in vitro biological assay of new derivatives, (3) Cheminformatics models development and in vivo activity prediction, and (4) Docking studies. This strategy is illustrated with a case study. Firstly, a series of 4-substituted Riluzole derivatives 1–3 were synthetized through a strategy that involves the construction of the 4-bromoriluzole framework and its further functionalization via palladium catalysis or organolithium chemistry. Next, a FRET biosensor for monitoring Ca2+-dependent CaM-ligands interactions has been developed and used for the in vitro assay of Riluzole derivatives. In particular, the best inhibition (80%) was observed for 4-methoxyphenylriluzole 2b. Besides, we trained and validated a new Networks Invariant, Information Fusion, Perturbation Theory, and Machine Learning (NIFPTML) model for predicting probability profiles of in vivo biological activity parameters in different regions of the brain. Next, we used this model to predict the in vivo activity of the compounds experimentally studied in vitro. Last, docking study conducted on Riluzole and its derivatives has provided valuable insights into their binding conformations with the target protein, involving calmodulin and the SK4 channel. This new combined strategy may be useful to reduce assay costs (animals, materials, time, and human resources) in the drug discovery process of calmodulin inhibitors.

Investigating the Impact of Electrostatic Interactions on Calmodulin Binding and Ca2+-Dependent Activation of the Calcium-Gated Potassium SK4 Channel.

Ca2+ binding to the ubiquitous Ca2+ sensing protein calmodulin (CaM) activates the intermediate conductance Ca2+-activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K+ current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K+ currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca2+-dependent gating process.

Proteomic analysis of blood serum in bipolar disorder

IntroductionBipolar disorder (BD) often has symptoms similar to other mental disorders (BD), and there are no paraclinical criteria for differential diagnosis. (Geoffroy et al. Bip Dis 2017; 5 7). Published work on MD proteomics is scarce and focused on schizophrenia. (Dmitrieva et al. PeerJ. 2022; 10 e13907). Therefore, it is important to study potential biomarkers of BD using easily accessible material—blood serum (Rhee et al. Transl Psy 2023; 13 44). Identification of proteins involved in the pathogenesis of BD will help in the study of the pathogenetic mechanisms of BD, the development of differential diagnostic methods and pathogenetically based drugs.ObjectivesCarrying out a comparative proteomic analysis of blood serum from patients with BD and healthy individuals to identify potential biomarkersMethodsWe analyzed the protein spectrum of the blood serum of 14 patients with BD who were admitted during a depressive episode at the age of 32 [21;52] years with a disease duration of 8[5;11] years. The control group consisted of 10 mentally and somatically healthy individuals corresponding to the gender and age of the BD group. Blood serum was purified from 14 major proteins using affinity chromatography and separated by electrophoresis using the Laemmli method. After trypsinolysis, proteins were identified using HPLC/mass spectrometry on an Orbitrap instrument. Mass spectrometric analysis was performed on the Advanced Mass Spectrometry Core Facility of Skolkovo Institute of Science and Technology. Protein identification was carried out using the UniProtKB database using the Mascot search engine. The results were tested for significance using the nonparametric Fisher exact test with Yates correction.ResultsIn patients with BD, qualitative mass spectrometry revealed differential expression of 21 neurospecific proteins. Among them: Protein dispatched homolog 3, Ceroid-lipofuscinosis neuronal protein 6, SWI/SNF complex subunit SMARCC1, Neurogenic differentiation factor 4, Protein furry homolog-like, REST corepressor 1 – are involved in the proliferation, development and differentiation of neurons; Hemicentin-2, Dystrophin, Voltage-dependent L-type calcium channel subunit alpha-1D, Syntaxin-binding protein 5, Small conductance calcium-activated potassium channel protein 1– participate in synaptic transmission of ion transport and form receptors.ConclusionsStudying the role of these proteins in BD and their quantitative content in a larger number of patients is promising. This will help in the development of new diagnostic criteria and targets for drug therapy for BD.Support by the Russian Science Foundation grant No. 23-75-00023.Disclosure of InterestNone Declared

Plasma membrane SK2 channel activity regulates migration and chemosensitivity of high-grade serous ovarian cancer cells.

No data are currently available on the functional role of small conductance Ca2+-activated K+ channels (SKCa) in ovarian cancer. Here, we characterized the role of SK2 (KCa2.2) in ovarian cancer cell migration and chemosensitivity. Using the selective non-cell-permeant SK2 inhibitor Lei-Dab7, we identified functional SK2 channels at the plasma membrane, regulating store-operated Ca2+ entry (SOCE) in both cell lines tested (COV504 and OVCAR3). Silencing KCNN2 with short interfering RNA (siRNA), or blocking SK2 activity with Lei-Dab7, decreased cell migration. The more robust effect of KCNN2 knockdown compared to Lei-Dab7 treatment suggested the involvement of functional intracellular SK2 channels in both cell lines. In cells treated with lysophosphatidic acid (LPA), an ovarian cancer biomarker of progression, SK2 channels are a key player of LPA pro-migratory activity but their role in SOCE is abolished. Concerning chemotherapy, SK2 inhibition increased chemoresistance to Taxol® and low KCNN2 mRNA expression was associated with the worst prognosis for progression-free survival in patients with serous ovarian cancer. The dual roles of SK2 mean that SK2 activators could be used as an adjuvant chemotherapy to potentiate treatment efficacy and SK2 inhibitors could be administrated as monotherapy to limit cancer cell dissemination.