Common PIEZO1 Allele in African Populations Causes RBC Dehydration and Attenuates Plasmodium Infection.

Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro. The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance.

Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.

Kca3.1-Related Cellular Signalling Involved in Cancer Proliferation.

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.

Ca2+-permeable AMPA receptor-dependent silencing of neurons by KCa3.1 channels during epileptiform activity.

Ca2+-activated KCa3.1 channels are known to contribute to slow afterhyperpolarization in pyramidal neurons of several brain areas, while Ca2+-permeable AMPA receptors (CP-AMPARs) may provide a subthreshold source of Ca2+ elevation in the cytoplasm. The functionality of these two types of channels has also been shown to be altered by epileptic disorders. However, the link between KCa3.1 channels and CP-AMPARs is poorly understood, and their potential interaction in epilepsy remains unclear. Here, we address this issue by overexpressing the KCNN4 gene, which encodes the KCa3.1 channel, using patch clamp, imaging, and channel blockers in an in vitro model of epilepsy in neuronal culture. We show that KCNN4 overexpression causes strong hyperpolarization and substantial silencing of neurons during epileptiform activity events, which also prevents KCNN4-positive neurons from firing action potentials (APs) during experimentally induced status epilepticus. Intracellular blocker application experiments showed that the amplitude of hyperpolarization was strongly dependent on CP-AMPARs, but not on NMDA receptors. Taken together, our data strongly suggest that subthreshold Ca2+ elevation produced by CP-AMPARs can trigger KCa3.1 channels to hyperpolarize neurons and protect them from seizures.

Endothelial KCa3.1 and KCa2.3 Mediate S1P (Sphingosine-1-Phosphate)-Dependent Vasodilation and Blood Pressure Homeostasis.

BACKGROUND: S1P (sphingosine-1-phosphate) has been reported to possess vasodilatory properties, but the underlying pathways are largely unknown. METHODS: Isolated mouse mesenteric artery and endothelial cell models were used to determine S1P-induced vasodilation, intracellular calcium, membrane potentials, and calcium-activated potassium channels (KCa2.3 and KCa3.1 [endothelial small- and intermediate-conductance calcium-activated potassium channels]). Effect of deletion of endothelial S1PR1 (type 1 S1P receptor) on vasodilation and blood pressure was evaluated. RESULTS: Mesenteric arteries subjected to acute S1P stimulation displayed a dose-dependent vasodilation response, which was attenuated by blocking endothelial KCa2.3 or KCa3.1 channels. In cultured human umbilical vein endothelial cells, S1P stimulated immediate membrane potential hyperpolarization following activation of KCa2.3/KCa3.1 with elevated cytosolic Ca2+. Further, chronic S1P stimulation enhanced expression of KCa2.3 and KCa3.1 in human umbilical vein endothelial cells in dose- and time-dependent manners, which was abolished by disrupting either S1PR1-Ca2+ signaling or downstream Ca2+-activated calcineurin/NFAT (nuclear factor of activated T-cells) signaling. By combination of bioinformatics-based binding site prediction and chromatin immunoprecipitation assay, we revealed in human umbilical vein endothelial cells that chronic activation of S1P/S1PR1 promoted NFATc2 nuclear translocation and binding to promoter regions of KCa2.3 and KCa3.1 genes thus to upregulate transcription of these channels. Deletion of endothelial S1PR1 reduced expression of KCa2.3 and KCa3.1 in mesenteric arteries and exacerbated hypertension in mice with angiotensin II infusion. CONCLUSIONS: This study provides evidence for the mechanistic role of KCa2.3/KCa3.1-activated endothelium-dependent hyperpolarization in vasodilation and blood pressure homeostasis in response to S1P. This mechanistic demonstration would facilitate the development of new therapies for cardiovascular diseases associated with hypertension.

Identification of PGAM5 as a Mammalian Protein Histidine Phosphatase that Plays a Central Role to Negatively Regulate CD4(+) T Cells.

Whereas phosphorylation of serine, threonine, and tyrosine is exceedingly well characterized, the role of histidine phosphorylation in mammalian signaling is largely unexplored. Here we show that phosphoglycerate mutase family 5 (PGAM5) functions as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic histidine on nucleoside diphosphate kinase B (NDPK-B). By dephosphorylating NDPK-B, PGAM5 negatively regulates CD4(+) T cells by inhibiting NDPK-B-mediated histidine phosphorylation and activation of the K(+) channel KCa3.1, which is required for TCR-stimulated Ca(2+) influx and cytokine production. Using recently developed monoclonal antibodies that specifically recognize phosphorylation of nitrogens at the N1 (1-pHis) or N3 (3-pHis) positions of the imidazole ring, we detect for the first time phosphoisoform-specific regulation of histidine-phosphorylated proteins in vivo, and we link these modifications to TCR signaling. These results represent an important step forward in studying the role of histidine phosphorylation in mammalian biology and disease.

ACYP2 functions as an innovative nano-therapeutic target to impede the progression of hepatocellular carcinoma by inhibiting the activity of TERT and the KCNN4/ERK pathway.

An increasing body of evidence suggests that acylphosphatase-2 (ACYP2) polymorphisms are correlated with an increased susceptibility to a range of malignancies. Nevertheless, its potential functions, molecular mechanisms in hepatocellular carcinoma (HCC) and whether it can be act as a therapeutic target remain uninvestigated. Herein, ACYP2 was found to be lowly expressed in HCC and was negatively correlated with tumor size, tumor differentiation, microvascular invasion and the prognosis of HCC patients. Functional investigations revealed that overexpression of ACYP2 inhibited the proliferation and metastasis of HCC cells while promoting apoptosis; knockdown of ACYP2 had the exact opposite effect. Additionally, it was observed that ACYP2 was distributed in both the cytoplasm and nucleus of HCC cells. According to the mechanistic studies, the expression of potassium calcium-activated channel subfamily N member 4 (KCNN4) was negatively regulated by cytoplasmic ACYP2, resulting in the inhibition of K+ outflow and subsequent inactivation of the ERK pathway, which impeded the growth and metastasis of HCC. Furthermore, the activity of telomerase reverse transcriptase (TERT) was inhibited by nuclear ACYP2, leading to the reduction in length of telomeres and consequent reversal of HCC cell immortalization. Additionally, a novel targeted nanotherapy strategy was developed wherein the pcDNA-ACYP2 vector was encapsulated within polyetherimide nanoparticles (PEI/NPs), which were subsequently coated with HCC cell membranes (namely pcDNA/PEI/NPs@M). Safety and targeting characteristics abound for these nanocomposites, in both subcutaneous graft tumor models and orthotopic mouse models, they inhibited the progression of HCC by impeding TERT activity and the KCNN4/ERK pathway. In conclusion, our research identifies novel molecular mechanisms involving cytoplasmic and nuclear ACYP2 that inhibit the progression of HCC. Moreover, pcDNA/PEI/NPs@M represents a targeted therapeutic strategy for HCC that holds great promising.

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.

The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells.

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

Transcriptional Up-Regulation of FBXW7 by KCa1.1 K+ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model.

The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.

Regulation of T Lymphocyte Functions through Calcium Signaling Modulation by Nootkatone.

Recent advancements in understanding the intricate molecular mechanisms underlying immunological responses have underscored the critical involvement of ion channels in regulating calcium influx, particularly in inflammation. Nootkatone, a natural sesquiterpenoid found in Alpinia oxyphylla and various citrus species, has gained attention for its diverse pharmacological properties, including anti-inflammatory effects. This study aimed to elucidate the potential of nootkatone in modulating ion channels associated with calcium signaling, particularly CRAC, KV1.3, and KCa3.1 channels, which play pivotal roles in immune cell activation and proliferation. Using electrophysiological techniques, we demonstrated the inhibitory effects of nootkatone on CRAC, KV1.3, and KCa3.1 channels in HEK293T cells overexpressing respective channel proteins. Nootkatone exhibited dose-dependent inhibition of channel currents, with IC50 values determined for each channel. Nootkatone treatment did not significantly affect cell viability, indicating its potential safety for therapeutic applications. Furthermore, we observed that nootkatone treatment attenuated calcium influx through activated CRAC channels and showed anti-proliferative effects, suggesting its role in regulating inflammatory T cell activation. These findings highlight the potential of nootkatone as a natural compound for modulating calcium signaling pathways by targeting related key ion channels and it holds promise as a novel therapeutic agent for inflammatory disorders.

Corticotropin Releasing Factor Mediates KCa3.1 Inhibition, Hyperexcitability, and Seizures in Acquired Epilepsy.

Temporal lobe epilepsy (TLE), the most common focal seizure disorder in adults, can be instigated in experimental animals by convulsant-induced status epilepticus (SE). Principal hippocampal neurons from SE-experienced epileptic male rats (post-SE neurons) display markedly augmented spike output compared with neurons from nonepileptic animals (non-SE neurons). This enhanced firing results from a cAMP-dependent protein kinase A-mediated inhibition of KCa3.1, a subclass of Ca2+-gated K+ channels generating the slow afterhyperpolarizing Ca2+-gated K+ current (IsAHP). The inhibition of KCa3.1 in post-SE neurons leads to a marked reduction in amplitude of the IsAHP that evolves during repetitive firing, as well as in amplitude of the associated Ca2+-dependent component of the slow afterhyperpolarization potential (KCa-sAHP). Here we show that KCa3.1 inhibition in post-SE neurons is induced by corticotropin releasing factor (CRF) through its Type 1 receptor (CRF1R). Acute application of CRF1R antagonists restores KCa3.1 activity in post-SE neurons, normalizing KCa-sAHP/IsAHP amplitudes and neuronal spike output, without affecting these variables in non-SE neurons. Moreover, pharmacological antagonism of CRF1Rs in vivo reduces the frequency of spontaneous recurrent seizures in post-SE chronically epileptic rats. These findings may provide a new vista for treating TLE.SIGNIFICANCE STATEMENT Epilepsy, a common neurologic disorder, often develops following a brain insult. Identifying key cellular mechanisms underlying acquired epilepsy is critical for developing effective antiepileptic therapies. In an experimental model of acquired epilepsy, principal hippocampal neurons manifest hyperexcitability because of downregulation of KCa3.1, a subtype of Ca2+-gated K+ ion channels. We show that KCa3.1 downregulation is mediated by corticotropin releasing factor (CRF) acting through its Type 1 receptor (CRF1R). Congruently, acute application of selective CRF1R antagonists restores KCa3.1 channel activity, leading to normalization of neuronal excitability. In the same model, injection of a CRF1R antagonist to epileptic animals markedly decreases the frequency of electrographic seizures. Therefore, targeting CRF1Rs may provide a new strategy in the treatment of acquired epilepsy.

Aggressive migration in acidic pH of a glioblastoma cancer stem cell line in vitro is independent of ASIC and KCa3.1 ion channels, but involves phosphoinositide 3-kinase.

The microenvironment of proliferative and aggressive tumours, such as the brain tumour glioblastoma multiforme (GBM), is often acidic, hypoxic, and nutrient deficient. Acid-sensing ion channels (ASICs) are proton-sensitive Na+ channels that have been proposed to play a role in pH sensing and in modulation of cancer cell migration. We previously reported that primary glioblastoma stem cells (GSCs), which grow as multicellular tumour spheroids, express functional ASIC1a and ASIC3, whereas ASIC2a is downregulated in GSCs. Using a 2.5D migration assay, here we report that acidic pH dramatically increased migration of GSCs of the pro-neural subtype. Pharmacological blockade as well as CRISPR-Cas9-mediated gene knock-out of ASIC1a or stable overexpression of ASIC2a, however, revealed that neither ASIC1a nor ASIC3, nor downregulation of ASIC2a, mediated the aggressive migration at acidic pH. Therefore, we tested the role of two other proteins previously implicated in cancer cell migration: the Ca2+-activated K+ channel KCa3.1 (KCNN4) and phosphoinositide 3-kinase (PI3K). While pharmacological blockade of KCa3.1 did also not affect migration, blockade of PI3K decreased migration at acidic pH to control levels. In summary, our study reveals a strongly enhanced migration of GSCs at acidic pH in vitro and identifies PI3K as an important mediator of this effect.

Pharmacology of Small- and Intermediate-Conductance Calcium-Activated Potassium Channels.

The three small-conductance calcium-activated potassium (KCa2) channels and the related intermediate-conductance KCa3.1 channel are voltage-independent K+ channels that mediate calcium-induced membrane hyperpolarization. When intracellular calcium increases in the channel vicinity, it calcifies the flexible N lobe of the channel-bound calmodulin, which then swings over to the S4-S5 linker and opens the channel. KCa2 and KCa3.1 channels are highly druggable and offer multiple binding sites for venom peptides and small-molecule blockers as well as for positive- and negative-gating modulators. In this review, we briefly summarize the physiological role of KCa channels and then discuss the pharmacophores and the mechanism of action of the most commonly used peptidic and small-molecule KCa2 and KCa3.1 modulators. Finally, we describe the progress that has been made in advancing KCa3.1 blockers and KCa2.2 negative- and positive-gating modulators toward the clinic for neurological and cardiovascular diseases and discuss the remaining challenges.

Ion Channels in Glioma Malignancy.

Brain tumors come in many types and differ greatly in outcome. They are classified by the cell of origin (astrocytoma, ependymoma, meningioma, medulloblastoma, glioma), although more recently molecular markers are used in addition to histology. Brain tumors are graded (from I to IV) to measure their malignancy. Glioblastoma, one of the most common adult primary brain tumors, displays the highest malignancy (grade IV), and median survival of about 15 months. Main reasons for poor outcome are incomplete surgical resection, due to the highly invasive potential of glioblastoma cells, and chemoresistance that commonly develops during drug treatment. An important role in brain tumor malignancy is played by ion channels. The Ca2+-activated K+ channels of large and intermediate conductance, KCa3.1 and KCa1.1, and the volume-regulated anion channel, whose combined activity results in the extrusion of KCl and osmotic water, control cell volume, and in turn migration, invasion, and apoptotic cell death. The transient receptor potential (TRP) channels and low threshold-activated Ca (T-type) channels have equally critical role in brain tumor malignancy, as dysregulated Ca2+ signals heavily impact on glioma cell proliferation, migration, invasion. The review provides an overview of the current evidence involving these channels in brain tumor malignancy, and the application of these insights in the light of future prospects for experimental and clinical practice.

Channelopathy of small- and intermediate-conductance Ca2+-activated K+ channels.

Small- and intermediate-conductance Ca2+-activated K+ (KCa2.x/KCa3.1 also called SK/IK) channels are gated exclusively by intracellular Ca2+. The Ca2+ binding protein calmodulin confers sub-micromolar Ca2+ sensitivity to the channel-calmodulin complex. The calmodulin C-lobe is constitutively associated with the proximal C-terminus of the channel. Interactions between calmodulin N-lobe and the channel S4-S5 linker are Ca2+-dependent, which subsequently trigger conformational changes in the channel pore and open the gate. KCNN genes encode four subtypes, including KCNN1 for KCa2.1 (SK1), KCNN2 for KCa2.2 (SK2), KCNN3 for KCa2.3 (SK3), and KCNN4 for KCa3.1 (IK). The three KCa2.x channel subtypes are expressed in the central nervous system and the heart. The KCa3.1 subtype is expressed in the erythrocytes and the lymphocytes, among other peripheral tissues. The impact of dysfunctional KCa2.x/KCa3.1 channels on human health has not been well documented. Human loss-of-function KCa2.2 mutations have been linked with neurodevelopmental disorders. Human gain-of-function mutations that increase the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels have been associated with Zimmermann-Laband syndrome and hereditary xerocytosis, respectively. This review article discusses the physiological significance of KCa2.x/KCa3.1 channels, the pathophysiology of the diseases linked with KCa2.x/KCa3.1 mutations, the structure-function relationship of the mutant KCa2.x/KCa3.1 channels, and potential pharmacological therapeutics for the KCa2.x/KCa3.1 channelopathy.

ORAI1 and ORAI2 modulate murine neutrophil calcium signaling, cellular activation, and host defense.

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

Ca2+-Sensitive Potassium Channels.

The Ca2+ ion is used ubiquitously as an intracellular signaling molecule due to its high external and low internal concentration. Many Ca2+-sensing ion channel proteins have evolved to receive and propagate Ca2+ signals. Among them are the Ca2+-activated potassium channels, a large family of potassium channels activated by rises in cytosolic calcium in response to Ca2+ influx via Ca2+-permeable channels that open during the action potential or Ca2+ release from the endoplasmic reticulum. The Ca2+ sensitivity of these channels allows internal Ca2+ to regulate the electrical activity of the cell membrane. Activating these potassium channels controls many physiological processes, from the firing properties of neurons to the control of transmitter release. This review will discuss what is understood about the Ca2+ sensitivity of the two best-studied groups of Ca2+-sensitive potassium channels: large-conductance Ca2+-activated K+ channels, KCa1.1, and small/intermediate-conductance Ca2+-activated K+ channels, KCa2.x/KCa3.1.