Calmodulin Binding Research Articles

Calmodulin Modulates the Gating Properties of Voltage-Dependent Anion Channel from Rat Brain Mitochondria.

Calmodulin (CaM) is a key signaling protein that plays a decisive role in mitochondrial Ca2+ homeostasis and signaling and modulates the mitochondrial membrane properties. We propose that voltage-dependent anion channel 1 (VDAC1), one of the most abundant outer mitochondrial membrane (OMM) proteins, could be its possible target or site of action. VDAC1 is known to play a crucial role in the mitochondrial Ca2+ signaling mechanism. Bilayer electrophysiology experiments show that CaM significantly reduces VDAC1's conductivity and modulates its gating as well as permeability properties. Also, spectrofluorimetric analysis indicates the possibility of binding CaM with VDAC1. Theoretical analysis of fluorescence data shows that the aforementioned protein-protein interaction is not linear, but rather it is a complex nonlinear process. In VDAC1, CaM binding site has been predicted using various bioinformatics tools. It is proposed that CaM could interact with VDAC1's outer-loop region and regulate its gating properties. Our findings suggest that VDAC1-CaM interaction could play a crucial role in the transport of ions and metabolites through the OMM and the regulation of the mitochondrial Ca2+ signaling mechanism through alteration of VDAC1's gating and conductive properties.

EjCaM7 and EjCAMTA3 synergistically alleviate chilling-induced lignification in loquat fruit by repressing the expression of lignin biosynthesis genes

Flesh lignification is a typical chilling injury (CI) symptom of loquat fruit during cold storage. Calcium chloride (CaCl2) could enhance chilling tolerance in loquat fruit, but the molecular mechanism is still not illuminated. In this study, CaCl2 treatment could decrease the activities and genes expression of peroxidase (POD) laccase (LAC), and inhibit the increase in firmness and lignin accumulation, thereby alleviating chilling-induced lignification in loquat fruit. Additionally, CaCl2 enhanced the calmodulin (CaM) content, free Ca2+ distribution, EjCaM7, and CaM binding transcription activator 3 (EjCAMTA3) expression in loquat fruit during cold storage, indicating Ca2+/CaM-CAMTA might be played a key role in response to cold stress. Moreover, the EjCAMTA3 and EjCaM7 proteins were identified and characterized from loquat fruit. EjCAMTA3 bound to the CG-box in EjPOD64 and EjLAC12-like promoters to repress their transcription. Further analysis revealed that EjCaM7 interacted with EjCAMTA3 in a Ca2+-dependent manner, and this interaction enhanced the EjCAMTA3-mediated transcriptional repression of EjPOD64 and EjLAC12-like genes. Taken together, these findings suggested that CaCl2 treatment alleviates chilling-induced lignification in loquat fruit, which probably via the synergistic role of EjCaM7 and EjCAMTA3 in the modulation of EjPOD64 and EjLAC12-like genes expression, leading to the repression of lignin polymerization.

Low androgen level impairs erectile function of rat by regulating the Ng/CaN/AKT/eNOS pathway in penile corpus cavernosum.

The mechanism by which low androgen status inhibit erectile function has not yet been clearly elucidated. Neurogranin (Ng) is a Ca2+ -sensitive calmodulin binding protein that is expressed in endothelial cells and regulates eNOS function. To investigate whether low androgen status inhibit erectile function by regulating the Ng/CaN/AKT/eNOS pathway in the penile cavernous tissue of rats. Thirty-six 8-week-old male Sprague-Dawley rats were randomly divided into six groups as follows (n=6): 4-week control group (4w-control), 4-week castration group (4w-cast), 4-week castration+testosterone replacement group (4w-cast+T), 8-week control group (8w-control), 8-week castration group (8w-cast), and 8-week castration+testosterone replacement group (8w-cast+T). Four weeks and eight weeks after surgery, the ratio of the maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP) was examined. The level of NO and the expression of Ng, calcineurin (CaN), AKT, p-AKT(S473), eNOS, and p-eNOS(Ser1177) in the penile cavernous tissue of each group were determined. Ng and CaN were mainly expressed in the membrane and cytoplasm of endothelial cells and smooth muscle cells in the penile cavernous tissue of rats. The ICPmax/MAP and the concentration of NO in the cast group were significantly lower than those in the control group and cast+T replacement group (p<0.01). The expression of Ng and the ratios of p-AKT/AKT and p-eNOS/eNOS in the penile cavernous tissue of rats in the cast group were significantly lower than those in the control group and cast+T replacement group (p<0.01). The expression of CaN in the penile cavernous tissue of rats in the cast group was significantly increased compared with that in the control group and the cast+T replacement group (p<0.01). Inhibiting the expression of Ng and subsequently upregulating the expression of CaN in the rat penile cavernous tissue was one of the upstream mechanisms of low androgen status inhibiting erectile function by inhibiting the AKT/eNOS signaling pathway.

Hypersensitivity-Related Genes HSR201 and HSR203J Are Regulated by Calmodulin-Binding Protein 60-Type Transcription Factors and Required for Pathogen Signal-Induced Salicylic Acid Synthesis.

Salicylic acid (SA) plays a key role in plant resistance to pathogens. In Arabidopsis, the isochorismate synthase pathway mainly contributes to pathogen-induced SA synthesis, and the expression of SA synthesis genes is activated by two calmodulin (CaM)-binding protein 60 (CBP60)-type transcription factors, CBP60g and SARD1. In tobacco, the mechanisms underlying SA synthesis remain largely unknown. SA production is induced by wounding in tobacco plants in which the expression of two stress-related mitogen-activated protein kinases is suppressed. Using this phenomenon, we identified genes whose expression is associated with SA synthesis. One of the genes, NtCBP60g, showed 23% amino acid sequence identity with CBP60g. Transient overexpression of NtCBP60g as well as NtSARD1, a tobacco homolog of SARD1, induced SA accumulation in Nicotiana benthamiana leaves. NtCBP60g and NtSARD1 bound CaM, and CaM enhanced SA accumulation induced by NtCBP60g and NtSARD1. Conversely, mutations in NtCBP60g and NtSARD1 that abolished CaM binding reduced their ability to induce SA. Expression profiling and promoter analysis identified two hypersensitivity-related genes, HSR201 and HSR203J as the targets of NtCBP60g and NtSARD1. Virus-induced gene silencing of both NtCBP60g and NtSARD1 homologs compromised SA accumulation and the expression of HSR201 and HSR203J homologs, which were induced by a pathogen-derived elicitor in N. benthamiana leaves. Moreover, elicitor-induced SA accumulation was compromised by silencing of the HSR201 homolog and the HSR203J homolog. These results suggested that HSR201 and HSR203J are regulated by NtCBP60g and NtSARD1 and are required for elicitor-induced SA synthesis.

Title: Jealous protons sour another happy marriage; the story of how TRPV5 and PI(4,5)P2 split up.

TRPV5 is a highly selective calcium channel that finetunes urinary calcium excretion by reabsorbing calcium from the pro-urine. New structural findings show how PTH and pH control TRPV5 activity by altering the binding of endogenous ligands calmodulin and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2).

Ectopic expression of BrIQD35 promotes drought stress tolerance in Nicotiana benthamiana.

The plant IQD gene family is responsive to a variety of stresses. In this study, we studied the structural features and functions of the gene BrIQD35 in Chinese cabbage, a member of the IQD gene family. BrIQD35 was cloned and shown to contain an IQ motif. Transient expression of BrIQD35 indicated that it was localized on the plasma membrane and was significantly upregulated under drought and salt stress in Chinese cabbage. To further identify the function of BrIQD35, it was heterologously overexpressed in Nicotiana benthamiana. Although there was no significant difference between BrIQD35-overexpressed and wild-type (WT) plants under salt stress, WT N. benthamiana showed more wilting than the BrIQD35-overexpressed plants under drought stress. Since the IQ motif has been annotated as a CaM binding site, yeast two-hybrid assays were used to explore the interaction between BrIQD35 and CaM. The results indicated that BrIQD35 interacts weakly with CaMb, but not with CaMa, suggesting that BrIQD35 may function through the Ca2+ -CaMb pathway. The findings reveal a novel gene involved in drought tolerance, which is important for plant breeding and quality improvement for Chinese cabbage.

Calcium Mediated Cold Acclimation in Plants: Underlying Signaling and Molecular Mechanisms.

Exposure of plants to low temperatures adversely affects plant growth, development, and productivity. Plant response to cold stress is an intricate process that involves the orchestration of various physiological, signaling, biochemical, and molecular pathways. Calcium (Ca2+) signaling plays a crucial role in the acquisition of several stress responses, including cold. Upon perception of cold stress, Ca2+ channels and/or Ca2+ pumps are activated, which induces the Ca2+ signatures in plant cells. The Ca2+ signatures spatially and temporally act inside a plant cell and are eventually decoded by specific Ca2+ sensors. This series of events results in the molecular regulation of several transcription factors (TFs), leading to downstream gene expression and withdrawal of an appropriate response by the plant. In this context, calmodulin binding transcription activators (CAMTAs) constitute a group of TFs that regulate plant cold stress responses in a Ca2+ dependent manner. The present review provides a catalog of the recent progress made in comprehending the Ca2+ mediated cold acclimation in plants.

Structural basis of TRPV5 regulation by physiological and pathophysiological modulators.

SUMMARYTransient receptor potential vanilloid 5 (TRPV5) is a kidney-specific Ca2+-selective ion channel that plays a key role in Ca2+ homeostasis. The basal activity of TRPV5 is balanced through activation by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and inhibition by Ca2+-bound calmodulin (CaM). Parathyroid hormone (PTH), the key extrinsic regulator of Ca2+ homeostasis, increases the activity of TRPV5 via protein kinase A (PKA)-mediated phosphorylation. Metabolic acidosis leads to reduced TRPV5 activity independent of PTH, causing hypercalciuria. Using cryoelectron microscopy (cryo-EM), we show that low pH inhibits TRPV5 by precluding PI(4,5)P2 activation. We capture intermediate conformations at low pH, revealing a transition from open to closed state. In addition, we demonstrate that PI(4,5)P2 is the primary modulator of channel gating, yet PKA controls TRPV5 activity by preventing CaM binding and channel inactivation. Our data provide detailed molecular mechanisms for regulation of TRPV5 by two key extrinsic modulators, low pH and PKA.

Transcriptome-wide identification and computational insights into protein modeling and docking of CAMTA transcription factors in Eleusine coracana L (finger millet)

Calmodulin-binding transcription activator (CAMTA) are a group of transcription factors that are known to perform various important biological functions in plants. Here, we report 7 putative CAMTA transcription factors identified from finger millet transcriptome data. They were further analyzed for physicochemical properties, subcellular localization, conserved domains and motifs, Gene Ontology (GO) terms, phylogeny, 3D structure prediction and CAMTA-Ca2+-Calmodulin interaction through protein-protein docking. All EcCAMTAs were found to be localized in the nucleus and possessed a calmodulin binding domain (CaMBD). GO results indicated the involvement of CAMTAs in DNA binding and protein binding molecular functions. Phylogenetic analysis classified EcCAMTA genes into 3-subgroups. 3D-structure of CAMTA proteins was elucidated through ab-initio protein modeling and its interaction with Calmodulin was investigated by docking studies. Our study provides molecular insight into the structure and function of CAMTA genes in finger millet and also highlights the role of omics-based in-silico approaches for identification of novel gene families in the absence of a reference genome or annotated database. This being the first study of CAMTA transcription factor family in finger millet, it could serve as a resource for further studies of CAMTA genes either in finger millet or other related millets and cereal crops.

Arabidopsis CAMTA3/SR1 is involved in drought stress tolerance and ABA signaling

Calcium/calmodulin signals are important for various cellular and physiological activities in plants. Calmodulin binding transcription activators also named Signal Responsive (SR) proteins belong to an important calcium/calmodulin-dependent transcription factor family that plays critical roles in stress responses. However, the role of SRs in abscisic acid (ABA) regulated plant responses to drought stress is largely unknown. Here, we characterized the role of Arabidopsis SR1 in drought stress tolerance and ABA response by analyzing the phenotypes of SR1 knockout and SR1-overexpression plants. sr1 mutants which accumulate salicylic acid (SA) were found more sensitive to drought stress and showed a higher water loss rate as compared with wild-type. By contrast, SR1-overexpression lines exhibited increased drought tolerance and less water loss than wild-type. Furthermore, sr1 mutants showed reduced ABA response in seed germination, root elongation, and stomatal closure, while SR1-overexpression lines displayed more sensitive to ABA than wild-type. In addition, the drought-sensitive and ABA-insensitive phenotypes of sr1 mutants were recovered by diminishing SA accumulation via knockouts of SA synthesizer ICS1 or activator PAD4, or through expression of SA-degrading enzyme NahG. Some drought/ABA-responsive genes exhibited differentially expressed in sr1 mutants and SR1-overexpression plants. These results suggest that SR1 plays a positive role in drought stress tolerance and ABA response, and drought/ABA responses are antagonized by SA accumulation that is negatively regulated by SR1.

Chemical shift assignments of calmodulin bound to the β-subunit of a retinal cyclic nucleotide-gated channel (CNGB1)

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679–702) binds tightly to Ca2+-bound CaM, which promotes Ca2+-induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222).

The disordered C-terminal tail of TRPA1 is required for calmodulin binding and desensitization

TRPA1 is a non-selective cation channel that has been Identified as a potential drug target for persistent pain since it is required for thermal and mechanical hypersensitivity. While TRPA1 channel gating by direct agonists has been resolved, the pathways that control TRPA1 channel regulation are not well characterized. Increases in intracellular calcium control both TRPA1 potentiation and desensitization, but the mechanisms that govern these processes are poorly understood. Our research shows that calmodulin is required for TRPA1 channel desensitization, and TRPA1 lacks intrinsic channel desensitization capability unlike other ion channels.

Reactions of Recombinant Neuronal Nitric Oxide Synthase with Redox Cycling Xenobiotics: A Mechanistic Study

Neuronal nitric oxide synthase (nNOS) catalyzes single-electron reduction of quinones (Q), nitroaromatic compounds (ArNO2) and aromatic N-oxides (ArN → O), and is partly responsible for their oxidative stress-type cytotoxicity. In order to expand a limited knowledge on the enzymatic mechanisms of these processes, we aimed to disclose the specific features of nNOS in the reduction of such xenobiotics. In the absence or presence of calmodulin (CAM), the reactivity of Q and ArN → O increases with their single-electron reduction midpoint potential (E17). ArNO2 form a series with lower reactivity. The calculations according to an “outer-sphere” electron transfer model show that the binding of CAM decreases the electron transfer distance from FMNH2 to quinone by 1–2 Å. The effects of ionic strength point to the interaction of oxidants with a negatively charged protein domain close to FMN, and to an increase in accessibility of the active center induced by high ionic strength. The multiple turnover experiments of nNOS show that, in parallel with reduced FAD-FMN, duroquinone reoxidizes the reduced heme, in particular its Fe2+-NO form. This finding may help to design the heme-targeted bioreductively activated agents and contribute to the understanding of the role of P-450-type heme proteins in the bioreduction of quinones and other prooxidant xenobiotics.

Calmodulin binding to the dehydrogenase domain of NADPH oxidase 5 alters its oligomeric state

Superoxide generated by NADPH Oxidase 5 (Nox5) is regulated by Ca2+ through the interaction of its self-contained Ca2+ binding domain and dehydrogenase domain (DH). Recently, calmodulin (CaM) has been reported to enhance the Ca2+ sensitivity of Nox5 by binding to the CaM-binding domain sequence (CMBD), in which the interaction between CaM and Nox5 is largely unclear. Here, we used the CMBD peptide and truncated DH constructs, and separately studied their interaction with CaM by fluorescence, calorimetry, and dynamic light scattering. Our results revealed that each half-domain of CaM binds one CMBD peptide with a binding constant near 106 M-1 and a binding enthalpy change of −3.81 kcal/mol, consistent with an extended 1:2 CaM:CMBD structure. However, the recombinant truncated DH proteins exist as oligomers, possibly trimer and tetramer. The oligomeric states are concentration and salt dependent. CaM binding appears to stabilize the DH dimer complexed with CaM. The thermodynamics of CaM binding to the DH is comparable to the peptide-based study except that the near unity binding stoichiometry and a large conformational change were observed. Our result suggests that the oligomeric states of Nox5, mediated by its DH domain and CaM, may be important for its superoxide-generating activity.

Genome-wide in silico characterization and stress induced expression analysis of BcL-2 associated athanogene (BAG) family in Musa spp.

Programmed cell death (PCD) is a genetically controlled process for the selective removal of damaged cells. Though understanding about plant PCD has improved over years, the mechanisms are yet to be fully deciphered. Among the several molecular players of PCD in plants, B cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) family of co-chaperones are evolutionary conserved and regulate cell death, growth and development. In this study, we performed a genome-wide in silico analysis of the MusaBAG gene family in a globally important fruit crop banana. Thirteen MusaBAG genes were identified, out of which MusaBAG1, 7 and 8 genes were found to have multiple copies. MusaBAG genes were distributed on seven out of 11 chromosomes in banana. Except for one paralog of MusaBAG8 all the other 12 proteins have characteristic BAG domain. MusaBAG1, 2 and 4 have an additional ubiquitin-like domain whereas MusaBAG5-8 have a calmodulin binding motif. Most of the MusaBAG proteins were predicted to be localized in the nucleus and mitochondria or chloroplast. The in silico cis-regulatory element analysis suggested regulation associated with photoperiodic control, abiotic and biotic stress. The phylogenetic analysis revealed 2 major clusters. Digital gene expression analysis and quantitative real-time RT-PCR depicted the differential expression pattern of MusaBAG genes under abiotic and biotic stress conditions. Further studies are warranted to uncover the role of each of these proteins in growth, PCD and stress responses so as to explore them as candidate genes for engineering transgenic banana plants with improved agronomic traits.

DR1 Activation Inhibits the Proliferation of Vascular Smooth Muscle Cells through Increasing Endogenous H2S in Diabetes.

Tissue ischemia and hypoxia caused by the abnormal proliferation of smooth muscle cells (SMCs) in the diabetic state is an important pathological basis for diabetic microangiopathy. Studies in recent years have shown that the chronic complications of diabetes are related to the decrease of endogenous hydrogen sulfide (H2S) in diabetic patients, and it has been proven that H2S can inhibit the proliferation of vascular SMCs (VSMCs). Our study showed that the endogenous H2S content and the expression of cystathionine gamma-lyase (CSE), which is the key enzyme of H2S production, were decreased in arterial SMCs of diabetic mice. The expression of PCNA and Cyclin D1 was increased, and the expression of p21 was decreased in the diabetic state. After administration of dopamine 1-like receptors (DR1) agonist SKF38393 and exogenous H2S donor NaHS, the expression of CSE was increased and the change in proliferation-related proteins caused by diabetes was reversed. It was further verified by cell experiments that SKF38393 activated calmodulin (CaM) by increasing the intracellular calcium ([Ca2+]i) concentration, which activated the CSE/H2S pathway, enhancing the H2S content in vivo. We also found that SKF38393 and NaHS inhibited insulin-like growth factor-1 (IGF-1)/IGF-1R and heparin-binding EGF-like growth factor (HB-EGF)/EGFR, as well as their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways. Taken together, our results suggest that DR1 activation up-regulates the CSE/H2S system by increasing Ca2+-CaM binding, which inhibits the IGF-1/IGF-1R and HB-EGF/EGFR pathways, thereby decreasing their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways to achieve the effect of inhibiting HG-induced VSMCs proliferation.

Hepatitis B virus X protein enhances liver cancer cell migration by regulating calmodulin-associated actin polymerization

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), which is a highly aggressive cancer. HBV X protein (HBx), one of four HBV gene products, plays pivotal roles in the development and metastasis of HCC. It has been reported that HBx induces liver cancer cell migration and reorganizes actin cytoskeleton, however the molecular basis for actin cytoskeleton reorganization remains obscure. In this study, we for the first time report that HBx promotes actin polymerization and liver cancer cell migration by regulating calcium modulated protein, calmodulin (CaM). HBx physically interacts with CaM to control the level of phosphorylated cofilin, an actin depolymerizing factor. Mechanistically, HBx interacts with CaM, liberates Hsp90 from its inhibitory partner CaM, and increases the activity of Hsp90, thus activating LIMK1/cofilin pathway. Interestingly, the interaction between HBx and CaM is calcium-dependent and requires the CaM binding motif on HBx. These results indicate that HBx modulates CaM which plays a regulatory role in Hsp90/LIMK1/cofilin pathway of actin reorganization, suggesting a new mechanism of HBV-induced HCC metastasis specifically derived by HBx.

A novel assay to assess the effects of estrogen on the cardiac calmodulin binding equilibrium

AimsThe Ca2+-binding protein calmodulin (CaM) modulates numerous target proteins but is produced insufficiently to bind all of them, generating a limiting CaM equilibrium. Menopause increases cardiac morbidity; however, it is unknown if the cardiac CaM equilibrium is affected by estrogen. We devised an assay to assess the effects of ovariectomy and estrogen treatment on the cardiac CaM equilibrium. Materials and methodsSprague-Dawley rats received sham surgery or ovariectomy, followed by 2-week treatment with vehicle or 17β-estradiol. Ca2+-saturated left ventricular (LV) lysates were processed through CaM sepharose columns, which retained CaM-binding proteins unoccupied by endogenous CaM. Eluants therefrom were subjected to a competitive binding assay against purified CaM and a CaM biosensor to assess the amounts of unoccupied CaM-binding sites. LV cellular composition was assessed by immunohistochemistry. Key findingsLV eluants processed from sham animals reduce biosensor response by ~32%, indicating baseline presence of unoccupied CaM-binding sites and a limiting CaM equilibrium. Ovariectomy exacerbates the limiting CaM equilibrium, reducing biosensor response by ~65%. 17β-estradiol treatment equalizes the difference between sham and ovariectomized animals. These changes reflect whole tissue responses and are not mirrored by changes in total surface areas of cardiomyocytes and fibroblasts. Consistently, Ca2+-dependent, but not Ca2+-independent, interaction between CaM and the cardiac inositol trisphosphate receptor (IP3R) is reduced following ovariectomy and is restored by subsequent 17β-estradiol treatment. SignificanceOur assay provides a new parameter to assess tissue CaM equilibrium. The exacerbated limiting CaM equilibrium following estrogen loss may contribute to cardiac morbidity and is prevented by estrogen treatment.

Orai1 Boosts SK3 Channel Activation.

Simple SummaryBreast, colon, and prostate cancer account for about a third of cancer cases and a fifth of cancer deaths. At the molecular level, one reason for the development of cancer is the dysfunction or altered co-regulation of cellular proteins. In this study, we focused on the co-regulation of ion channels, specifically the prominent Ca2+ ion channel Orai1 and the Ca2+ activated K+ ion channel SK3. It has recently been reported that their interplay promotes the growth of breast and colon cancer cells, but the molecular determinants for their co-regulation have remained elusive. In this study, we set out to characterize their interplay and the crucial regions therefore required. Moreover, we found that the function of prostate cancer cells is also controlled by the interplay of Ca2+ and the Ca2+ sensitive K+ channels. Our findings provide a better understanding of the co-regulation of these ion channels, which could be used in the future for the development of novel therapeutics.The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been reported to increase cytosolic Ca2+ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K+ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca2+ levels that decrease SK3 K+ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3–CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role in SK3–Orai1 regulation. Under physiological ionic conditions, STIM1 is able to impede SK3–Orai1 interplay by significantly decreasing their co-localization. Forced STIM1–Orai1 activity and associated Ca2+ influx promote SK3 K+ currents. The dynamic regulation of Orai1 to boost endogenous SK3 channels was also determined in the human prostate cancer cell line LNCaP.

Spontaneous seizure and memory loss in mice expressing an epileptic encephalopathy variant in the calmodulin-binding domain of Kv7.2

Epileptic encephalopathy (EE) is characterized by seizures that respond poorly to antiseizure drugs, psychomotor delay, and cognitive and behavioral impairments. One of the frequently mutated genes in EE is KCNQ2, which encodes the Kv7.2 subunit of voltage-gated Kv7 potassium channels. Kv7 channels composed of Kv7.2 and Kv7.3 are enriched at the axonal surface, where they potently suppress neuronal excitability. Previously, we reported that the de novo dominant EE mutation M546V in human Kv7.2 blocks calmodulin binding to Kv7.2 and axonal surface expression of Kv7 channels via their intracellular retention. However, whether these pathogenic mechanisms underlie epileptic seizures and behavioral comorbidities remains unknown. Here, we report conditional transgenic cKcnq2+/M547V mice, in which expression of mouse Kv7.2-M547V (equivalent to human Kv7.2-M546V) is induced in forebrain excitatory pyramidal neurons and astrocytes. These mice display early mortality, spontaneous seizures, enhanced seizure susceptibility, memory impairment, and repetitive behaviors. Furthermore, hippocampal pathology shows widespread neurodegeneration and reactive astrocytes. This study demonstrates that the impairment in axonal surface expression of Kv7 channels is associated with epileptic seizures, cognitive and behavioral deficits, and neuronal loss in KCNQ2-related EE.