A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein.

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

The renal vasodilatation from ß-adrenergic activation in vivo in rats is not driven by KV7 and BKCa channels.

The mechanisms behind renal vasodilatation elicited by stimulation of ß-adrenergic receptors are not clarified. As several classes of K channels are potentially activated, we tested the hypothesis that KV7 and BKCa channels contribute to the decreased renal vascular tone in vivo and in vitro. Changes in renal blood flow (RBF) during ß-adrenergic stimulation were measured in anaesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. The ß-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 and BKCa channels affected the ß-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the ß-adrenergic vasorelaxation. However, inhibiting BKCa channels was equally effective in reducing the ß-adrenergic vasorelaxation. The ß-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking KV7.1 channels. As opposed to rats, inhibition of KV7 channels did not affect the murine ß-adrenergic vasorelaxation. Although inhibition and activation of KV7 channels or BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected ß-adrenergic vasodilatation. In isolated segmental arteries, however, inhibition of KV7 and BKCa channels significantly reduced the ß-adrenergic vasorelaxation, indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data illustrates the challenge in extrapolating results from in vitro to in vivo conditions.

Generation of an induced pluripotent stem cell line from patient with atrial fibrillation with KCNQ1 p.Ser209Pro mutation.

Gain-of-function mutations in the KCNQ1 gene can cause atrial fibrillation. In this study, we generated an induced stem cell line (GRCHJUi001) from one member of an atrial fibrillation family line, whom had heterozygous mutation in the KCNQ1 gene c.625 T > C (p.Ser209Pro), and the cell line showed maintenance of stem cells characterized by morphology, normal karyotype, and pluripotency.

Imprinted gene alterations in the kidneys of growth restricted offspring may be mediated by a long non-coding RNA.

Altered epigenetic mechanisms have been previously reported in growth restricted offspring whose mothers experienced environmental insults during pregnancy in both human and rodent studies. We previously reported changes in the expression of the DNA methyltransferase Dnmt3a and the imprinted genes Cdkn1c (Cyclin-dependent kinase inhibitor 1C) and Kcnq1 (Potassium voltage-gated channel subfamily Q member 1) in the kidney tissue of growth restricted rats whose mothers had uteroplacental insufficiency induced on day 18 of gestation, at both embryonic day 20 (E20) and postnatal day 1 (PN1). To determine the mechanisms responsible for changes in the expression of these imprinted genes, we investigated DNA methylation of KvDMR1, an imprinting control region (ICR) that includes the promoter of the antisense long non-coding RNA Kcnq1ot1 (Kcnq1 opposite strand/antisense transcript 1). Kcnq1ot1 expression decreased by 51% in growth restricted offspring compared to sham at PN1. Interestingly, there was a negative correlation between Kcnq1ot1 and Kcnq1 in the E20 growth restricted group (Spearman's ρ = 0.014). No correlation was observed between Kcnq1ot1 and Cdkn1c expression in either group at any time point. Additionally, there was a 11.25% decrease in the methylation level at one CpG site within KvDMR1 ICR. This study, together with others in the literature, supports that long non-coding RNAs may mediate changes seen in tissues of growth restricted offspring.

Mechanistic understanding of KCNQ1 activating polyunsaturated fatty acid analogs.

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.

Mechanistic insights into robust cardiac IKs potassium channel activation by aromatic polyunsaturated fatty acid analogues.

Voltage-gated potassium (KV) channels are important regulators of cellular excitability and control action potential repolarization in the heart and brain. KV channel mutations lead to disordered cellular excitability. Loss-of-function mutations, for example, result in membrane hyperexcitability, a characteristic of epilepsy and cardiac arrhythmias. Interventions intended to restore KV channel function have strong therapeutic potential in such disorders. Polyunsaturated fatty acids (PUFAs) and PUFA analogues comprise a class of KV channel activators with potential applications in the treatment of arrhythmogenic disorders such as long QT syndrome (LQTS). LQTS is caused by a loss-of-function of the cardiac IKs channel - a tetrameric potassium channel complex formed by KV7.1 and associated KCNE1 protein subunits. We have discovered a set of aromatic PUFA analogues that produce robust activation of the cardiac IKs channel, and a unique feature of these PUFA analogues is an aromatic, tyrosine head group. We determine the mechanisms through which tyrosine PUFA analogues exert strong activating effects on the IKs channel by generating modified aromatic head groups designed to probe cation-pi interactions, hydrogen bonding, and ionic interactions. We found that tyrosine PUFA analogues do not activate the IKs channel through cation-pi interactions, but instead do so through a combination of hydrogen bonding and ionic interactions.

Differential expression of KCNQ1 and ATP4A genes according to the sex and age in the stomach.

We compared the expression of six genes in stomach tissue samples between healthy men and women in different age groups to study sexually dimorphic gene expression. Real-Time RT-PCR was used to compare gene expression between men and women. Our results showed that the expression of KCNQ1 (p = 0.01) was significantly higher in non-menopausal women compared to post-menopausal women. In addition, the expression level of the ATP4A gene in men under 35 years was significantly higher than in men above 50 (p = 0.026). Sexually and age dimorphic gene expression in some genes throughout life may affect gastric function.

Generation of a human embryonic stem cell line WAe009-A-79 carrying a long QT syndrome mutation in KCNQ1.

The voltage-gated potassium channel KvLQT1 encoded by KCNQ1 plays an important role in the repolarization of myocardial action potentials. KCNQ1 mutations can cause Long QT syndrome type 1 (LQT1), which is considered to be the most common causative gene of LQT. In this study, we established a human embryonic stem cell line KCNQ1L114P/+ (WAe009-A-79) carrying a LQT1 related mutation in KCNQ1. The WAe009-A-79 line maintains the morphology, pluripotency, and normal karyotype of stem cells, and can differentiate into all three germ layers in vivo.

The membrane electric field regulates the PIP2-binding site to gate the KCNQ1 channel.

Voltage-dependent ion channels underlie the propagation of action potentials and other forms of electrical activity in cells. In these proteins, voltage sensor domains (VSDs) regulate opening and closing of the pore through the displacement of their positive-charged S4 helix in response to the membrane voltage. The movement of S4 at hyperpolarizing membrane voltages in some channels is thought to directly clamp the pore shut through the S4-S5 linker helix. The KCNQ1 channel (also known as Kv7.1), which is important for heart rhythm, is regulated not only by membrane voltage but also by the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2). KCNQ1 requires PIP2 to open and to couple the movement of S4 in the VSD to the pore. To understand the mechanism of this voltage regulation, we use cryogenic electron microscopy to visualize the movement of S4 in the human KCNQ1 channel in lipid membrane vesicles with a voltage difference across the membrane, i.e., an applied electric field in the membrane. Hyperpolarizing voltages displace S4 in such a manner as to sterically occlude the PIP2-binding site. Thus, in KCNQ1, the voltage sensor acts primarily as a regulator of PIP2 binding. The voltage sensors' influence on the channel's gate is indirect through the reaction sequence: voltage sensor movement → alter PIP2 ligand affinity → alter pore opening.

Human Sinoatrial Node Pacemaker Activity: Role of the Slow Component of the Delayed Rectifier K+ Current, IKs.

The pacemaker activity of the sinoatrial node (SAN) has been studied extensively in animal species but is virtually unexplored in humans. Here we assess the role of the slowly activating component of the delayed rectifier K+ current (IKs) in human SAN pacemaker activity and its dependence on heart rate and ß-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and ß-subunits of the IKs channel, respectively. KCNQ1/KCNE1 currents were recorded both during a traditional voltage clamp and during an action potential (AP) clamp with human SAN-like APs. Forskolin (10 µmol/L) was used to increase the intracellular cAMP level, thus mimicking ß-adrenergic stimulation. The experimentally observed effects were evaluated in the Fabbri-Severi computer model of an isolated human SAN cell. Transfected HEK-293 cells displayed large IKs-like outward currents in response to depolarizing voltage clamp steps. Forskolin significantly increased the current density and significantly shifted the half-maximal activation voltage towards more negative potentials. Furthermore, forskolin significantly accelerated activation without affecting the rate of deactivation. During an AP clamp, the KCNQ1/KCNE1 current was substantial during the AP phase, but relatively small during diastolic depolarization. In the presence of forskolin, the KCNQ1/KCNE1 current during both the AP phase and diastolic depolarization increased, resulting in a clearly active KCNQ1/KCNE1 current during diastolic depolarization, particularly at shorter cycle lengths. Computer simulations demonstrated that IKs reduces the intrinsic beating rate through its slowing effect on diastolic depolarization at all levels of autonomic tone and that gain-of-function mutations in KCNQ1 may exert a marked bradycardic effect during vagal tone. In conclusion, IKs is active during human SAN pacemaker activity and has a strong dependence on heart rate and cAMP level, with a prominent role at all levels of autonomic tone.

The role of native cysteine residues in the oligomerization of KCNQ1 channels.

KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1.

Endocannabinoids enhance hKV7.1/KCNE1 channel function and shorten the cardiac action potential and QT interval.

BACKGROUND: Genotype-positive patients who suffer from the cardiac channelopathy Long QT Syndrome (LQTS) may display a spectrum of clinical phenotypes, with often unknown causes. Therefore, there is a need to identify factors influencing disease severity to move towards an individualized clinical management of LQTS. One possible factor influencing the disease phenotype is the endocannabinoid system, which has emerged as a modulator of cardiovascular function. In this study, we aim to elucidate whether endocannabinoids target the cardiac voltage-gated potassium channel KV7.1/KCNE1, which is the most frequently mutated ion channel in LQTS. METHODS: We used two-electrode voltage clamp, molecular dynamics simulations and the E4031 drug-induced LQT2 model of ex-vivo guinea pig hearts. FINDINGS: We found a set of endocannabinoids that facilitate channel activation, seen as a shifted voltage-dependence of channel opening and increased overall current amplitude and conductance. We propose that negatively charged endocannabinoids interact with known lipid binding sites at positively charged amino acids on the channel, providing structural insights into why only specific endocannabinoids modulate KV7.1/KCNE1. Using the endocannabinoid ARA-S as a prototype, we show that the effect is not dependent on the KCNE1 subunit or the phosphorylation state of the channel. In guinea pig hearts, ARA-S was found to reverse the E4031-prolonged action potential duration and QT interval. INTERPRETATION: We consider the endocannabinoids as an interesting class of hKV7.1/KCNE1 channel modulators with putative protective effects in LQTS contexts. FUNDING: ERC (No. 850622), Canadian Institutes of Health Research, Canada Research Chairs and Compute Canada, Swedish National Infrastructure for Computing.

The second PI(3,5)P2 binding site in the S0 helix of KCNQ1 stabilizes PIP2-at the primary PI1 site with potential consequences on intermediate-to-open state transition.

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.

Mechanism of external K+ sensitivity of KCNQ1 channels.

KCNQ1 voltage-gated K+ channels are involved in a wide variety of fundamental physiological processes and exhibit the unique feature of being markedly inhibited by external K+. Despite the potential role of this regulatory mechanism in distinct physiological and pathological processes, its exact underpinnings are not well understood. In this study, using extensive mutagenesis, molecular dynamics simulations, and single-channel recordings, we delineate the molecular mechanism of KCNQ1 modulation by external K+. First, we demonstrate the involvement of the selectivity filter in the external K+ sensitivity of the channel. Then, we show that external K+ binds to the vacant outermost ion coordination site of the selectivity filter inducing a diminution in the unitary conductance of the channel. The larger reduction in the unitary conductance compared to whole-cell currents suggests an additional modulatory effect of external K+ on the channel. Further, we show that the external K+ sensitivity of the heteromeric KCNQ1/KCNE complexes depends on the type of associated KCNE subunits.

Optimized tight binding between the S1 segment and KCNE3 is required for the constitutively open nature of the KCNQ1-KCNE3 channel complex.

Tetrameric voltage-gated K+ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K+ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel at physiological voltages by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the KCNQ1-KCNE3-calmodulin complex structure, we thoroughly surveyed amino acid residues on KCNE3 and the S1 segment of the KCNQ1 voltage sensor facing each other. By changing the side-chain bulkiness of these interacting amino acid residues (volume scanning), we found that the distance between the S1 segment and KCNE3 is elaborately optimized to achieve the constitutive activity. In addition, we identified two pairs of KCNQ1 and KCNE3 mutants that partially restored constitutive activity by co-expression. Our work suggests that tight binding of the S1 segment and KCNE3 is crucial for controlling the voltage sensor domains.

Poor semen parameters are associated with abnormal methylation of imprinted genes in sperm DNA.

BACKGROUND: Altered sperm DNA methylation patterns of imprinted genes as well as certain spermatogenesis-related genes has been proposed as a possible mechanism of male subfertility. Some reports suggest that there is an elevated risk of congenital diseases, associated with imprinted genes, in children conceived via intra-cytoplasmic sperm injection, due to the involvement of spermatozoa with aberrant imprinted genes obtained from infertile men. METHODS: In this study, the DNA methylation status of the promoter regions of six imprinted genes, namely potassium voltage-gated channel subfamily Q member 1 (KCNQ1), maternally expressed gene 3 (MEG3), insulin-like growth factor 2 (IGF-2), KCNQ1 overlapping transcript 1 (KCNQ1OT1), mesoderm specific transcript (MEST), and paternally expressed gene 3 (PEG3), were detected by a next generation sequencing-based multiple methylation-specific polymerase chain reaction analysis of sperm samples obtained from 166 men who sought fertility evaluation in our Reproductive Medicine Center. Thereafter, the semen samples were classified into subgroups according to sperm motility and DNA integrity status. RESULTS: As compared to the normozoospermic group, the samples of the asthenospermic group exhibited significant hypermethylation in two CpG sites of IGF-2 and significant hypomethylation in one CpG site of KCNQ1 as well as three CpG sites of MEST (P < 0.05). However, we did not observe any significant differences in the overall methylation levels of these six imprinted genes (P > 0.05). Additionally, we found that 111 of 323 CpG sites were hypomethylated in the group with DNA fragmentation index (DFI) ≥ 30% as compared to the group with DFI < 30% (P < 0.05). In this case, there were significant differences in the overall methylation levels of MEG3, IGF-2, MEST, and PEG3 (P < 0.05), but not in that of KCNQ1OT1 and KCNQ1 (P > 0.05). Hence, aberrant methylation patterns of imprinted genes were more prevalent in males with poor sperm quality, especially in those with severe sperm DNA damage. CONCLUSION: In conclusion, abnormal DNA methylation of some CpG sites of imprinted genes are associated with poor sperm quality, including asthenospermia and severe sperm DNA impairment.

Disruption of a Conservative Motif in the C-Terminal Loop of the KCNQ1 Channel Causes LQT Syndrome.

We identified a single nucleotide variation (SNV) (c.1264A > G) in the KCNQ1 gene in a 5-year-old boy who presented with a prolonged QT interval. His elder brother and mother, but not sister and father, also had this mutation. This missense mutation leads to a p.Lys422Glu (K422E) substitution in the Kv7.1 protein that has never been mentioned before. We inserted this substitution in an expression plasmid containing Kv7.1 cDNA and studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1, using the whole-cell configuration of the patch-clamp technique. Expression of the mutant Kv7.1 channel in both homo- and heterozygous conditions in the presence of auxiliary subunit KCNE1 results in a significant decrease in tail current densities compared to the expression of wild-type (WT) Kv7.1 and KCNE1. This study also indicates that K422E point mutation causes a dominant negative effect. The mutation was not associated with a trafficking defect; the mutant channel protein was confirmed to localize at the cell membrane. This mutation disrupts the poly-Lys strip in the proximal part of the highly conserved cytoplasmic A−B linker of Kv7.1 that was not shown before to be crucial for channel functioning.

Venom resistance mechanisms in centipede show tissue specificity.

Venomous animals utilize venom glands to secrete and store powerful toxins for intraspecific and/or interspecific antagonistic interactions, implying that tissue-specific resistance is essential for venom glands to anatomically separate toxins from other tissues. Here, we show the mechanism of tissue-specific resistance in centipedes (Scolopendra subspinipes mutilans), where the splice variant of the receptor repels its own toxin. Unlike the well-known resistance mechanism by mutation in a given exon, we found that the KCNQ1 channel is highly expressed in the venom gland as a unique splice variant in which the pore domain and transmembrane domain six, partially encoded by exon 6 (rather than 7 as found in other tissues), contain eleven mutated residues. Such a splice variant is sufficient to gain resistance to SsTx (a lethal toxin for giant prey capture) in the venom gland due to a partially buried binding site. Therefore, the tissue-specific KCNQ1 modification confers resistance to the toxins, establishing a safe zone in the venom-storing/secreting environment.

Purification and membrane interactions of human KCNQ1100-370 potassium ion channel.

KCNQ1 (Kv7.1 or KvLQT1) is a voltage-gated potassium ion channel that is involved in the ventricular repolarization following an action potential in the heart. It forms a complex with KCNE1 in the heart and is the pore forming subunit of slow delayed rectifier potassium current (Iks). Mutations in KCNQ1, leading to a dysfunctional channel or loss of activity have been implicated in a cardiac disorder, long QT syndrome. In this study, we report the overexpression, purification, biochemical characterization of human KCNQ1100-370, and lipid bilayer dynamics upon interaction with KCNQ1100-370. The recombinant human KCNQ1 was expressed in Escherichia coli and purified into n-dodecylphosphocholine (DPC) micelles. The purified KCNQ1100-370 was biochemically characterized by SDS-PAGE electrophoresis, western blot and nano-LC-MS/MS to confirm the identity of the protein. Circular dichroism (CD) spectroscopy was utilized to confirm the secondary structure of purified protein in vesicles. Furthermore, 31P and 2H solid-state NMR spectroscopy in DPPC/POPC/POPG vesicles (MLVs) indicated a direct interaction between KCNQ100-370 and the phospholipid head groups. Finally, a visual inspection of KCNQ1100-370 incorporated into MLVs was confirmed by transmission electron microscopy (TEM). The findings of this study provide avenues for future structural studies of the human KCNQ1 ion channel to have an in depth understanding of its structure-function relationship.

Structural and electrophysiological basis for the modulation of KCNQ1 channel currents by ML277.

The KCNQ1 ion channel plays critical physiological roles in electrical excitability and K+ recycling in organs including the heart, brain, and gut. Loss of function is relatively common and can cause sudden arrhythmic death, sudden infant death, epilepsy and deafness. Here, we report cryogenic electron microscopic (cryo-EM) structures of Xenopus KCNQ1 bound to Ca2+/Calmodulin, with and without the KCNQ1 channel activator, ML277. A single binding site for ML277 was identified, localized to a pocket lined by the S4-S5 linker, S5 and S6 helices of two separate subunits. Several pocket residues are not conserved in other KCNQ isoforms, explaining specificity. MD simulations and point mutations support this binding location for ML277 in open and closed channels and reveal that prevention of inactivation is an important component of the activator effect. Our work provides direction for therapeutic intervention targeting KCNQ1 loss of function pathologies including long QT interval syndrome and seizures.