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Translational Stroke Research Jun 2024Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We...
Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC of 7 nM, reduced Ca signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke.
Topics: Animals; Intermediate-Conductance Calcium-Activated Potassium Channels; Ischemic Stroke; Male; Mice, Inbred C57BL; Mice; Microglia; Drug Repositioning; Infarction, Middle Cerebral Artery; Acetamides; Potassium Channel Blockers; Trityl Compounds
PubMed: 37088858
DOI: 10.1007/s12975-023-01152-6 -
Channels (Austin, Tex.) Dec 2023The voltage-gated potassium channel K1.3 is an important therapeutic target for the treatment of autoimmune and neuroinflammatory diseases. The recent structures of... (Review)
Review
The voltage-gated potassium channel K1.3 is an important therapeutic target for the treatment of autoimmune and neuroinflammatory diseases. The recent structures of K1.3, Shaker-IR (wild-type and inactivating W434F mutant) and an inactivating mutant of rat K1.2-K2.1 paddle chimera (KChim-W362F+S367T+V377T) reveal that the transition of voltage-gated potassium channels from the open-conducting conformation into the non-conducting inactivated conformation involves the rupture of a key intra-subunit hydrogen bond that tethers the selectivity filter to the pore helix. Breakage of this bond allows the side chains of residues at the external end of the selectivity filter (Tyr447 and Asp449 in K1.3) to rotate outwards, dilating the outer pore and disrupting ion permeation. Binding of the peptide dalazatide (ShK-186) and an antibody-ShK fusion to the external vestibule of K1.3 narrows and stabilizes the selectivity filter in the open-conducting conformation, although K efflux is blocked by the peptide occluding the pore through the interaction of ShK-Lys22 with the backbone carbonyl of K1.3-Tyr447 in the selectivity filter. Electrophysiological studies on ShK and the closely-related peptide HmK show that ShK blocks K1.3 with significantly higher potency, even though molecular dynamics simulations show that ShK is more flexible than HmK. Binding of the anti-K1.3 nanobody A0194009G09 to the turret and residues in the external loops of the voltage-sensing domain enhances the dilation of the outer selectivity filter in an exaggerated inactivated conformation. These studies lay the foundation to further define the mechanism of slow inactivation in K channels and can help guide the development of future K1.3-targeted immuno-therapeutics.
Topics: Animals; Rats; Biological Transport; Cell Line; Molecular Conformation; Molecular Dynamics Simulation; Potassium Channels, Voltage-Gated
PubMed: 37695839
DOI: 10.1080/19336950.2023.2253104 -
The Journal of General Physiology Oct 2023The KCNQ1 channel is important for the repolarization phase of the cardiac action potential. Loss of function mutations in KCNQ1 can cause long QT syndrome (LQTS), which...
The KCNQ1 channel is important for the repolarization phase of the cardiac action potential. Loss of function mutations in KCNQ1 can cause long QT syndrome (LQTS), which can lead to cardiac arrythmia and even sudden cardiac death. We have previously shown that polyunsaturated fatty acids (PUFAs) and PUFA analogs can activate the cardiac KCNQ1 channel, making them potential therapeutics for the treatment of LQTS. PUFAs bind to KCNQ1 at two different binding sites: one at the voltage sensor (Site I) and one at the pore (Site II). PUFA interaction at Site I shifts the voltage dependence of the channel to the left, while interaction at Site II increases maximal conductance. The PUFA analogs, linoleic-glycine and linoleic-tyrosine, are more effective than linoleic acid at Site I, but less effective at Site II. Using both simulations and experiments, we find that the larger head groups of linoleic-glycine and linoleic-tyrosine interact with more residues than the smaller linoleic acid at Site I. We propose that this will stabilize the negatively charged PUFA head group in a position to better interact electrostatically with the positively charges in the voltage sensor. In contrast, the larger head groups of linoleic-glycine and linoleic-tyrosine compared with linoleic acid prevent a close fit of these PUFA analogs in Site II, which is more confined. In addition, we identify several KCNQ1 residues as critical PUFA-analog binding residues, thereby providing molecular models of specific interactions between PUFA analogs and KCNQ1. These interactions will aid in future drug development based on PUFA-KCNQ1 channel interactions.
Topics: Humans; KCNQ1 Potassium Channel; Potassium Channels, Voltage-Gated; Heart; Fatty Acids, Unsaturated; Long QT Syndrome; Mutation; Linoleic Acids
PubMed: 37526928
DOI: 10.1085/jgp.202313339 -
The Journal of Neuroscience : the... Sep 2023Gain-of-function (GOF) pathogenic variants in the potassium channels KCNQ2 and KCNQ3 lead to hyperexcitability disorders such as epilepsy and autism spectrum disorders....
Gain-of-function (GOF) pathogenic variants in the potassium channels KCNQ2 and KCNQ3 lead to hyperexcitability disorders such as epilepsy and autism spectrum disorders. However, the underlying cellular mechanisms of how these variants impair forebrain function are unclear. Here, we show that the R201C variant in KCNQ2 has opposite effects on the excitability of two types of mouse pyramidal neurons of either sex, causing hyperexcitability in layer 2/3 (L2/3) pyramidal neurons and hypoexcitability in CA1 pyramidal neurons. Similarly, the homologous R231C variant in KCNQ3 leads to hyperexcitability in L2/3 pyramidal neurons and hypoexcitability in CA1 pyramidal neurons. However, the effects of KCNQ3 gain-of-function on excitability are specific to superficial CA1 pyramidal neurons. These findings reveal a new level of complexity in the function of KCNQ2 and KCNQ3 channels in the forebrain and provide a framework for understanding the effects of gain-of-function variants and potassium channels in the brain. KCNQ2/3 gain-of-function (GOF) variants lead to severe forms of neurodevelopmental disorders, but the mechanisms by which these channels affect neuronal activity are poorly understood. In this study, using a series of transgenic mice we demonstrate that the same KCNQ2/3 GOF variants can lead to either hyperexcitability or hypoexcitability in different types of pyramidal neurons [CA1 vs layer (L)2/3]. Additionally, we show that expression of the recurrent KCNQ2 GOF variant R201C in forebrain pyramidal neurons could lead to seizures and SUDEP. Our data suggest that the effects of KCNQ2/3 GOF variants depend on specific cell types and brain regions, possibly accounting for the diverse range of phenotypes observed in individuals with KCNQ2/3 GOF variants.
Topics: Animals; Mice; Gain of Function Mutation; KCNQ2 Potassium Channel; Mice, Transgenic; Neurodevelopmental Disorders; Potassium Channels; Prosencephalon; Pyramidal Cells; KCNQ3 Potassium Channel
PubMed: 37607817
DOI: 10.1523/JNEUROSCI.0980-23.2023 -
BMC Research Notes Sep 2023Insulin secretion is regulated by ATP-sensitive potassium (K) channels in pancreatic beta-cells. Peroxisome proliferator-activated receptors (PPAR) α ligands are...
OBJECTIVE
Insulin secretion is regulated by ATP-sensitive potassium (K) channels in pancreatic beta-cells. Peroxisome proliferator-activated receptors (PPAR) α ligands are clinically used to treat dyslipidemia. A PPARα ligand, fenofibrate, and PPARγ ligands troglitazone and 15-deoxy-∆-prostaglandin J2 are known to close K channels and induce insulin secretion. The recently developed PPARα ligand, pemafibrate, became a new entry for treating dyslipidemia. Because pemafibrate is reported to improve glucose intolerance in mice treated with a high fat diet and a novel selective PPARα modulator, it may affect K channels or insulin secretion.
RESULTS
The effect of fenofibrate (100 µM) and pemafibrate (100 µM) on insulin secretion from MIN6 cells was measured by using batch incubation for 10 and 60 min in low (2 mM) and high (10 mM) glucose conditions. The application of fenofibrate for 10 min significantly increased insulin secretion in low glucose conditions. Pemafibrate failed to increase insulin secretion in all of the conditions experimented in this study. The K channel activity was measured by using whole-cell patch clamp technique. Although fenofibrate (100 µM) reduced the K channel current, the same concentration of pemafibrate had no effect. Both fenofibrate and pemafibrate had no effect on insulin mRNA expression.
Topics: Animals; Mice; Fenofibrate; PPAR alpha; Ligands; Insulin Secretion; Glucose; KATP Channels; Adenosine Triphosphate
PubMed: 37697384
DOI: 10.1186/s13104-023-06489-7 -
The Journal of Clinical Investigation Mar 2024Ca2+-activated BK channels in renal intercalated cells (ICs) mediate luminal flow-induced K+ secretion (FIKS), but how ICs sense increased flow remains uncertain. We...
Ca2+-activated BK channels in renal intercalated cells (ICs) mediate luminal flow-induced K+ secretion (FIKS), but how ICs sense increased flow remains uncertain. We examined whether PIEZO1, a mechanosensitive Ca2+-permeable channel expressed in the basolateral membranes of ICs, is required for FIKS. In isolated cortical collecting ducts (CCDs), the mechanosensitive cation-selective channel inhibitor GsMTx4 dampened flow-induced increases in intracellular Ca2+ concentration ([Ca2+]i), whereas the PIEZO1 activator Yoda1 increased [Ca2+]i and BK channel activity. CCDs from mice fed a high-K+ (HK) diet exhibited a greater Yoda1-dependent increase in [Ca2+]i than CCDs from mice fed a control K+ diet. ICs in CCDs isolated from mice with a targeted gene deletion of Piezo1 in ICs (IC-Piezo1-KO) exhibited a blunted [Ca2+]i response to Yoda1 or increased flow, with an associated loss of FIKS in CCDs. Male IC-Piezo1-KO mice selectively exhibited an increased blood [K+] in response to an oral K+ bolus and blunted urinary K+ excretion following a volume challenge. Whole-cell expression of BKα subunit was reduced in ICs of IC-Piezo1-KO mice fed an HK diet. We conclude that PIEZO1 mediates flow-induced basolateral Ca2+ entry into ICs, is upregulated in the CCD in response to an HK diet, and is necessary for FIKS.
Topics: Male; Mice; Animals; Kidney Tubules, Collecting; Large-Conductance Calcium-Activated Potassium Channels; Calcium; Nephrons; Kidney; Ion Channels
PubMed: 38426496
DOI: 10.1172/JCI174806 -
Proceedings of the National Academy of... Oct 2023Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K) to diffuse across a hydrophobic...
Voltage-gated potassium channels (Kv) are tetrameric membrane proteins that provide a highly selective pathway for potassium ions (K) to diffuse across a hydrophobic cell membrane. These unique voltage-gated cation channels detect changes in membrane potential and, upon activation, help to return the depolarized cell to a resting state during the repolarization stage of each action potential. The Kv3 family of potassium channels is characterized by a high activation potential and rapid kinetics, which play a crucial role for the fast-spiking neuronal phenotype. Mutations in the Kv3.1 channel have been shown to have implications in various neurological diseases like epilepsy and Alzheimer's disease. Moreover, disruptions in neuronal circuitry involving Kv3.1 have been correlated with negative symptoms of schizophrenia. Here, we report the discovery of a novel positive modulator of Kv3.1, investigate its biophysical properties, and determine the cryo-EM structure of the compound in complex with Kv3.1. Structural analysis reveals the molecular determinants of positive modulation in Kv3.1 channels by this class of compounds and provides additional opportunities for rational drug design for the treatment of associated neurological disorders.
Topics: Humans; Neurons; Potassium Channels, Voltage-Gated; Potassium Channels; Action Potentials; Membrane Proteins
PubMed: 37812700
DOI: 10.1073/pnas.2220029120 -
Cellular Physiology and Biochemistry :... Mar 2024Anomalous expression of potassium channels in cancer tissues is associated with several cancer hallmarks that support deregulated proliferation and tumor progression.... (Review)
Review
Anomalous expression of potassium channels in cancer tissues is associated with several cancer hallmarks that support deregulated proliferation and tumor progression. Ion channels seem to influence cell proliferation; however, the crucial molecular mechanisms involved remain elusive. Some results show how extracellular mitogenic signals modulate ion channel activity through intracellular secondary messengers. It is relevant because we are beginning to understand how potassium channels can affect the proliferative capacity of cells, either in normal mitogen-dependent proliferation or in mitogen-unresponsive proliferation. Calciumdependent potassium channels have been implicated in cell cycle signaling in many cancerous cell lines. In particular, the so-called intermediate conductance KCa3.1 (IKCa) is reported to play a significant role in uncontrolled cell cycle signaling, among other malignant processes driven by cancer hallmarks. In addition to these features, this channel can be subjected to specific pharmacological regulation, making it a promising cornerstone for understanding the signaling behavior of several types of cancer and as a target for chemotherapeutic approaches. This review is dedicated to the connection of KCa3.1 activity, in canonical and non-canonical ways, to the cell cycle signaling, including the cooperation with calcium channels to generate calcium signals and its role as a mediator of proliferative signals.
Topics: Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mitogens; Neoplasms; Cell Proliferation; Ion Channels
PubMed: 38623063
DOI: 10.33594/000000688 -
Molecular Psychiatry Sep 2023The pathogenesis of schizophrenia is believed to involve combined dysfunctions of many proteins including microtubule-associated protein 6 (MAP6) and Kv3.1 voltage-gated...
The pathogenesis of schizophrenia is believed to involve combined dysfunctions of many proteins including microtubule-associated protein 6 (MAP6) and Kv3.1 voltage-gated K (Kv) channel, but their relationship and functions in behavioral regulation are often not known. Here we report that MAP6 stabilizes Kv3.1 channels in parvalbumin-positive (PV+ ) fast-spiking GABAergic interneurons, regulating behavior. MAP6 and Kv3.1 mice display similar hyperactivity and avoidance reduction. Their proteins colocalize in PV+ interneurons and MAP6 deletion markedly reduces Kv3.1 protein level. We further show that two microtubule-binding modules of MAP6 bind the Kv3.1 tetramerization domain with high affinity, maintaining the channel level in both neuronal soma and axons. MAP6 knockdown by AAV-shRNA in the amygdala or the hippocampus reduces avoidance or causes hyperactivity and recognition memory deficit, respectively, through elevating projection neuron activity. Finally, knocking down Kv3.1 or disrupting the MAP6-Kv3.1 binding in these brain regions causes avoidance reduction and hyperactivity, consistent with the effects of MAP6 knockdown. Thus, disrupting this conserved cytoskeleton-membrane interaction in fast-spiking neurons causes different degrees of functional vulnerability in various neural circuits.
Topics: Mice; Animals; Neurons; Potassium Channels, Voltage-Gated; Cytoskeleton; Microtubules; Emotions; Shaw Potassium Channels
PubMed: 37833406
DOI: 10.1038/s41380-023-02286-7 -
Proceedings of the National Academy of... Aug 2023Endothelial cells (ECs) line the lumen of all blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and...
Endothelial cells (ECs) line the lumen of all blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intravascular flow, activate transient receptor potential vanilloid 4 (TRPV4) channels, which stimulate small (SK3)- and intermediate (IK)-conductance Ca-activated potassium channels in ECs to produce vasodilation. Whether physiological vasodilators also modulate the surface abundance of these ion channels in ECs to elicit functional responses is unclear. Here, we show that ACh and intravascular flow stimulate rapid anterograde trafficking of an intracellular pool of SK3 channels in ECs of resistance-size arteries, which increases surface SK3 protein more than two-fold. In contrast, ACh and flow do not alter the surface abundance of IK or TRPV4 channels. ACh triggers SK3 channel trafficking by activating TRPV4-mediated Ca influx, which stimulates Rab11A, a Rab GTPase associated with recycling endosomes. Superresolution microscopy data demonstrate that SK3 trafficking specifically increases the size of surface SK3 clusters which overlap with TRPV4 clusters. We also show that Rab11A-dependent trafficking of SK3 channels is an essential contributor to vasodilator-induced SK current activation in ECs and vasorelaxation. In summary, our data demonstrate that vasodilators activate Rab11A, which rapidly delivers an intracellular pool of SK3 channels to the vicinity of surface TRPV4 channels in ECs. This trafficking mechanism increases surface SK3 cluster size, elevates SK3 current density, and produces vasodilation. These data also demonstrate that SK3 and IK channels are differentially regulated by trafficking-dependent and -independent signaling mechanisms in endothelial cells.
Topics: Vasodilator Agents; TRPV Cation Channels; Endothelial Cells; Small-Conductance Calcium-Activated Potassium Channels; Arteries; Vasodilation; Acetylcholine; Endothelium, Vascular
PubMed: 37494394
DOI: 10.1073/pnas.2303238120