Mutations in the KCNQ2 and KCNQ3 genes encoding for Kv 7.2 (KCNQ2; Q2) and Kv 7.3 (KCNQ3; Q3) voltage-dependent K(+) channel subunits, respectively, cause neonatal epilepsies with wide phenotypic heterogeneity. In addition to benign familial neonatal epilepsy (BFNE), KCNQ2 mutations have been recently found in families with one or more family members with a severe outcome, including drug-resistant seizures with psychomotor retardation, electroencephalogram (EEG) suppression-burst pattern (Ohtahara syndrome), and distinct neuroradiological features, a condition that was named "KCNQ2 encephalopathy." In the present article, we describe clinical, genetic, and functional data from 17 patients/families whose electroclinical presentation was consistent with the diagnosis of BFNE. Sixteen different heterozygous mutations were found in KCNQ2, including 10 substitutions, three insertions/deletions and three large deletions. One substitution was found in KCNQ3. Most of these mutations were novel, except for four KCNQ2 substitutions that were shown to be recurrent. Electrophysiological studies in mammalian cells revealed that homomeric or heteromeric KCNQ2 and/or KCNQ3 channels carrying mutant subunits with newly found substitutions displayed reduced current densities. In addition, we describe, for the first time, that some mutations impair channel regulation by syntaxin-1A, highlighting a novel pathogenetic mechanism for KCNQ2-related epilepsies.

Pathogenic variants in KCNQ2 and KCNQ3, paralogous genes encoding Kv7.2 and Kv7.3 voltage-gated K+ channel subunits, are responsible for early-onset developmental/epileptic disorders characterized by heterogeneous clinical phenotypes ranging from benign familial neonatal epilepsy (BFNE) to early-onset developmental and epileptic encephalopathy (DEE). KCNQ2 variants account for the majority of pedigrees with BFNE and KCNQ3 variants are responsible for a much smaller subgroup, but the reasons for this imbalance remain unclear. Analysis of additional pedigrees is needed to further clarify the nature of this genetic heterogeneity and to improve prediction of pathogenicity for novel variants. We identified a BFNE family with two siblings and a parent affected. Exome sequencing on samples from both parents and siblings revealed a novel KCNQ3 variant (c.719T>G; p.M240R), segregating in the three affected individuals. The M240 residue is conserved among human Kv7.2-5 and lies between the two arginines (R5 and R6) closest to the intracellular side of the voltage-sensing S4 transmembrane segment. Whole cell patch-clamp recordings in Chinese hamster ovary (CHO) cells revealed that homomeric Kv7.3 M240R channels were not functional, whereas heteromeric channels incorporating Kv7.3 M240R mutant subunits with Kv7.2 and Kv7.3 displayed a depolarizing shift of about 10 mV in activation gating. Molecular modeling results suggested that the M240R substitution preferentially stabilized the resting state and possibly destabilized the activated state of the Kv7.3 subunits, a result consistent with functional data. Exposure to β-hydroxybutyrate (BHB), a ketone body generated during the ketogenic diet (KD), reversed channel dysfunction induced by the M240R variant. In conclusion, we describe the first missense loss-of-function (LoF) pathogenic variant within the S4 segment of Kv7.3 identified in patients with BFNE. Studied under conditions mimicking heterozygosity, the M240R variant mainly affects the voltage sensitivity, in contrast to previously analyzed BFNE Kv7.3 variants that reduce current density. Our pharmacological results provide a rationale for the use of KD in patients carrying LoF variants in Kv7.2 or Kv7.3 subunits.

Many idiopathic epilepsy syndromes have a characteristic age dependence, the underlying molecular mechanisms of which are largely unknown. Here we propose a mechanism that can explain that epileptic spells in benign familial neonatal-infantile seizures occur almost exclusively during the first days to months of life. Benign familial neonatal-infantile seizures are caused by mutations in the gene SCN2A encoding the voltage-gated Na(+) channel Na(V)1.2. We identified two novel SCN2A mutations causing benign familial neonatal-infantile seizures and analysed the functional consequences of these mutations in a neonatal and an adult splice variant of the human Na(+) channel Na(V)1.2 expressed heterologously in tsA201 cells together with beta1 and beta2 subunits. We found significant gating changes leading to a gain-of-function, such as an increased persistent Na(+) current, accelerated recovery from fast inactivation or altered voltage-dependence of steady-state activation. Those were restricted to the neonatal splice variant for one mutation, but more pronounced for the adult form for the other, suggesting that a differential developmental splicing does not provide a general explanation for seizure remission. We therefore analysed the developmental expression of Na(V)1.2 and of another voltage-gated Na(+) channel, Na(V)1.6, using immunohistochemistry and real-time reverse transcription-polymerase chain reaction in mouse brain slices. We found that Na(V)1.2 channels are expressed early in development at axon initial segments of principal neurons in the hippocampus and cortex, but their expression is diminished and they are gradually replaced as the dominant channel type by Na(V)1.6 during maturation. This finding provides a plausible explanation for the transient expression of seizures that occur due to a gain-of-function of mutant Na(V)1.2 channels.

Benign familial neonatal convulsions (BFNC) is an epileptic disorder caused by dominant mutations in the genes KCNQ2 and KCNQ3 encoding the K+ channels K(V)7.2 and K(V)7.3. We identified two novel KCNQ2 mutations in two BFNC families. One mutation predicted a truncated protein (S247X) that lacks the channel's pore region, the other resulted in the amino acid substitution S122L in the S2 segment of K(V)7.2. In comparison to wild-type (WT) K(V)7.2, functional analysis of S122L mutant channels in Xenopus oocytes revealed a significant positive shift and increased slope of the activation curve leading to significant current reduction in the subthreshold range of an action potential (75% reduction at -50 mV). Our results establish an important role of the K(V)7.2 S2 segment in voltage-dependent channel gating and demonstrate in a human disease that subthreshold voltages are likely to represent the physiologically relevant range for this K+ channel to regulate neuronal firing.

Heterozygous variants in KCNQ2 or, more rarely, KCNQ3 genes are responsible for early-onset developmental/epileptic disorders characterized by heterogeneous clinical presentation and course, genetic transmission, and prognosis. While familial forms mostly include benign epilepsies with seizures starting in the neonatal or early-infantile period, de novo variants in KCNQ2 or KCNQ3 have been described in sporadic cases of early-onset encephalopathy (EOEE) with pharmacoresistant seizures, various age-related pathological EEG patterns, and moderate/severe developmental impairment. All pathogenic variants in KCNQ2 or KCNQ3 occur in heterozygosity. The aim of this work was to report the clinical, molecular, and functional properties of a new KCNQ3 variant found in homozygous configuration in a 9-year-old girl with pharmacodependent neonatal-onset epilepsy and non-syndromic intellectual disability.

Benign Familial Neonatal Seizures (BFNS) is a rare, autosomal-dominant epilepsy of the newborn caused by mutations in K(v)7.2 (KCNQ2) or K(v)7.3 (KCNQ3) genes encoding for neuronal potassium (K(+)) channel subunits. In this study, we describe a sporadic case of BFNS; the affected child carried heterozygous missense mutations in both K(v)7.2 (D212G) and K(v)7.3 (P574S) alleles. Electrophysiological experiments revealed that the K(v)7.2 D212G substitution, neutralizing a unique negatively-charged residue in the voltage sensor of K(v)7.2 subunits, altered channel gating, leading to a marked destabilization of the open state, a result consistent with structural analysis of the K(v)7.2 subunit, suggesting a possible pathogenetic role for BFNS of this K(v)7.2 mutation. By contrast, no significant functional changes appeared to be prompted by the K(v)7.3 P574S substitution. Computational modelling experiments in CA1 pyramidal cells revealed that the gating changes introduced by the K(v)7.2 D212G increased cell firing frequency, thereby triggering the neuronal hyperexcitability which underlies the observed neonatal epileptic condition.

The voltage-gated potassium channels KV7.2 and KV7.3 (genes KCNQ2 and KCNQ3) constitute a major component of the M-current controlling the firing rate in many neurons. Mutations within these two channel subunits cause benign familial neonatal convulsions (BFNC). Here we identified a novel BFNC-causing mutation (E119G) in the S1-S2 region of KV7.2. Electrophysiological investigations in Xenopus oocytes using two-microelectrode voltage clamping revealed that the steady-state activation curves for E119G alone and its coexpressions with KV7.2 and/or KV7.3 wild-type (WT) channels were significantly shifted in the depolarizing direction compared to KV7.2 or KV7.2/KV7.3. These shifts reduced the relative current amplitudes for mutant channels particularly in the subthreshold range of an action potential (about 45% reduction at --50 mV for E119G compared to KV7.2, and 33% for E119G/KV7.3 compared to KV7.2/KV7.3 channels). Activation kinetics were significantly slowed for mutant channels. Our results indicate that small changes in channel gating at subthreshold voltages are sufficient to cause neonatal seizures and demonstrate the importance of the M-current for this voltage range. This was confirmed by a computer model predicting an increased burst duration for the mutation. On a molecular level, these results reveal a critical role in voltage sensing of the negatively charged E119 in S1-S2 of KV7.2, a region that-- according to molecular modelling - might interact with a positive charge in the S4 segment.

Benign familial epilepsies that present themselves in the first year of life include benign familial neonatal epilepsy (BFNE), benign familial neonatal-infantile epilepsy (BFNIE) and benign familial infantile epilepsy (BFIE). We used Sanger sequencing and targeted next-generation sequencing to detect gene mutations in a Chinese cohort of patients with these three disorders. A total of 79 families were collected, including 4 BFNE, 7 BFNIE, and 68 BFIE. Genetic testing led to the identification of gene mutations in 60 families (60 out of 79, 75.9%). A total of 42 families had PRRT2 mutations, 9 had KCNQ2 mutations, 8 had SCN2A mutations, and 1 had a GABRA6 mutation. In total three of four BFNE families were detected with KCNQ2 mutations. Mutations were detected in all BFNIE families, including 3 KCNQ2 mutations, 3 SCN2A mutations, and 1 PRRT2 mutation. Gene mutations were identified in 50 out of 68 BFIE families (73.5%), including 41 PRRT2 mutations (41 out of 68, 60.3%), 5 SCN2A mutations, 3 KCNQ2 mutations, and 1 GABRA6 mutation. Our results confirmed that mutations in KCNQ2, SCN2A, and PRRT2 are major genetic causes of benign familial epilepsy in the first year of life in the Chinese population. KCNQ2 is the major gene related to BFNE. PRRT2 is the main gene responsible for BFIE.

Epilepsies can be caused by specific genetic anomalies or by non-genetic factors, but in many cases the underlying cause is unknown. Mutations in the SCN1A and SCN2A genes are reported in childhood epilepsies; in particular SCN1A was found mutated in patients with Dravet syndrome and with generalized epilepsy with febrile seizures plus (GEFS+). In this paper we report a patient presenting with an atypical epileptic syndrome whose phenotype partially overlaps both Dravet syndrome and benign familial neonatal-infantile seizures (BFNIS). Array-CGH analysis suggested the presence of a mosaic duplication (about 12Mb) at the level of chromosome 2q23.3q24.3 involving SCN2A and SCN3A genes. Additional analyses (radiolabeled RFLP and quantitative PCR) confirmed the mosaicism of the duplication. We suggest that the array-CGH analysis is mandatory for children presenting with epilepsy and psycho-motor retardation even without dysmorphisms or other clinical features suggesting a specific genetic/epileptic syndrome. The analysis must nevertheless be performed taking into account the possibility of a mosaicism.

A benign prognosis has been claimed in benign familial infantile seizures (BFIS). However, few studies have assessed the long-term evolution of these patients. The objective of this study is to describe atypical courses and presentations in BFIS families with mutations in PRRT2 gene. We studied clinically affected individuals from five BFIS Spanish families. We found mutations in PRRT2 in all 5 families. A non-BFIS phenotype or an atypical BFIS course was found in 9/25 (36%) patients harbouring a PRRT2 mutation. Atypical features included neonatal onset, mild hemiparesis, learning difficulties or mental retardation, and recurrent seizures during adulthood. We also report a novel PRRT2 mutation (c.121_122delGT). In BFIS families an atypical phenotype was present in a high percentage of the patients. These findings expand the clinical spectrum of PRRT2 mutations including non-benign epileptic phenotypes.

KCNQ/Kv 7 channels conduct voltage-dependent outward potassium currents that potently decrease neuronal excitability. Heterozygous inherited mutations in their principle subunits Kv 7.2/KCNQ2 and Kv 7.3/KCNQ3 cause benign familial neonatal epilepsy whereas patients with de novo heterozygous Kv 7.2 mutations are associated with early-onset epileptic encephalopathy and neurodevelopmental disorders characterized by intellectual disability, developmental delay and autism. However, the role of Kv 7.2-containing Kv 7 channels in behaviors especially autism-associated behaviors has not been described. Because pathogenic Kv 7.2 mutations in patients are typically heterozygous loss-of-function mutations, we investigated the contributions of Kv 7.2 to exploratory, social, repetitive and compulsive-like behaviors by behavioral phenotyping of both male and female KCNQ2+/- mice that were heterozygous null for the KCNQ2 gene. Compared with their wild-type littermates, male and female KCNQ2+/- mice displayed increased locomotor activity in their home cage during the light phase but not the dark phase and showed no difference in motor coordination, suggesting hyperactivity during the inactive light phase. In the dark phase, KCNQ2+/- group showed enhanced exploratory behaviors, and repetitive grooming but decreased sociability with sex differences in the degree of these behaviors. While male KCNQ2+/- mice displayed enhanced compulsive-like behavior and social dominance, female KCNQ2+/- mice did not. In addition to elevated seizure susceptibility, our findings together indicate that heterozygous loss of Kv 7.2 induces behavioral abnormalities including autism-associated behaviors such as reduced sociability and enhanced repetitive behaviors. Therefore, our study is the first to provide a tangible link between loss-of-function Kv 7.2 mutations and the behavioral comorbidities of Kv 7.2-associated epilepsy.

Neuronal Kv7/Potassium Voltage-Gated Channel Subfamily Q (KCNQ) potassium channels underlie M-current that potently suppresses repetitive and burst firing of action potentials (APs). They are mostly heterotetramers of Kv7.2 and Kv7.3 subunits in the hippocampus and cortex, the brain regions important for cognition and behavior. Underscoring their critical roles in inhibiting neuronal excitability, autosomal dominantly inherited mutations in Potassium Voltage-Gated Channel Subfamily Q Member 2 (KCNQ2) and Potassium Voltage-Gated Channel Subfamily Q Member 3 (KCNQ3) genes are associated with benign familial neonatal epilepsy (BFNE) in which most seizures spontaneously remit within months without cognitive deficits. De novo mutations in KCNQ2 also cause epileptic encephalopathy (EE), which is characterized by persistent seizures that are often drug refractory, neurodevelopmental delay, and intellectual disability. Heterozygous expression of EE variants of KCNQ2 is recently shown to induce spontaneous seizures and cognitive deficit in mice, although it is unclear whether this cognitive deficit is caused directly by Kv7 disruption or by persistent seizures in the developing brain as a consequence of Kv7 disruption. In this study, we examined the role of Kv7 channels in learning and memory by behavioral phenotyping of the KCNQ2+/- mice, which lack a single copy of KCNQ2 but dos not display spontaneous seizures. We found that both KCNQ2+/- and wild-type (WT) mice showed comparable nociception in the tail-flick assay and fear-induced learning and memory during a passive inhibitory avoidance (IA) test and contextual fear conditioning (CFC). Both genotypes displayed similar object location and recognition memory. These findings together provide evidence that heterozygous loss of KCNQ2 has minimal effects on learning or memory in mice in the absence of spontaneous seizures.

Background: Nav1.2 encoded by the SCN2A gene is a brain-expressed voltage-gated sodium channel known to be associated with neurodevelopment disorders ranging from benign familial neonatal infantile seizures (BFIS) to developmental and epileptic encephalopathy (DEE) and autism spectrum disorder. Interestingly, status epilepticus during slow sleep (ESES), which aggravates cognitive impairment, has been found in SCN2A-related epilepsy. However, the functional features and the relationship between SCN2A and ESES have not been researched. Method: We herein investigated the functional consequences of an unpublished de novo V911A and the other two published variants in patients with SCN2A-related disorder and ESES by whole-cell patch-clamp studies in transfected HEK293T cells. Results: The unpublished V911A and published K1933M variants detected in patients with DEE exhibited a profound gain-of-functional (GOF) change. Another published BFIS variant S863F significantly reduced current density as a loss-of-functional (LOF) change. The refractory epilepsy in the patient with V911A was controlled by using the precise treatment of oxcarbazepine (OXC) since the age of 3 months. ESES was found at 18 months during the seizure-free period. We finally chose an aggressive treatment for eliminating ESES by using methylprednisolone combined with levetiracetam and nitrazepam instead of the precise treatment of OXC. Conclusion: Both GOF and LOF variants in the SCN2A gene can lead to ESES among the phenotypes of DEE and BFIS. We should monitor the electroencephalogram regularly in the patients with SCN2A-related epilepsy even during their seizure-free period.

Understanding the molecular mechanisms underlying voltage-dependent gating in voltage-gated ion channels (VGICs) has been a major effort over the last decades. In recent years, changes in the gating process have emerged as common denominators for several genetically determined channelopathies affecting heart rhythm (arrhythmias), neuronal excitability (epilepsy, pain), or skeletal muscle contraction (periodic paralysis). Moreover, gating changes appear as the main molecular mechanism by which several natural toxins from a variety of species affect ion channel function. In this work, we describe the pathophysiological and pharmacological relevance of the gating process in voltage-gated K(+) channels encoded by the K(v)7 gene family. After reviewing the current knowledge on the molecular mechanisms and on the structural models of voltage-dependent gating in VGICs, we describe the physiological relevance of these channels, with particular emphasis on those formed by K(v)7.2-K(v)7.5 subunits having a well-established role in controlling neuronal excitability in humans. In fact, genetically determined alterations in K(v)7.2 and K(v)7.3 genes are responsible for benign familial neonatal convulsions, a rare seizure disorder affecting newborns, and the pharmacological activation of K(v)7.2/3 channels can exert antiepileptic activity in humans. Both mutation-triggered channel dysfunction and drug-induced channel activation can occur by impeding or facilitating, respectively, channel sensitivity to membrane voltage and can affect overlapping molecular sites within the voltage-sensing domain of these channels. Thus, understanding the molecular steps involved in voltage-sensing in K(v)7 channels will allow to better define the pathogenesis of rare human epilepsy, and to design innovative pharmacological strategies for the treatment of epilepsies and, possibly, other human diseases characterized by neuronal hyperexcitability.

Ribonucleotide reductase (RNR) is composed of two non-identical subunits, R1 and R2, and plays a crucial role in balancing the cellular dNTP pool, establishing it as an attractive cancer target. Herein, we report the discovery of a highly potent and selective small-molecule inhibitor, TAS1553, targeting protein-protein interaction between R1 and R2. TAS1553 is also expected to demonstrate superior selectivity because it does not directly target free radical or a substrate binding site. TAS1553 has shown antiproliferative activity in human cancer cell lines, dramatically reducing the intracellular dATP pool and causing DNA replication stress. Furthermore, we identified SLFN11 as a biomarker that predicts the cytotoxic effect of TAS1553. Oral administration of TAS1553 demonstrated robust antitumor efficacy against both hematological and solid cancer xenograft tumors and also provided a significant survival benefit in an acute myelogenous leukemia model. Our findings strongly support the evaluation of TAS1553 in clinical trials.

Deleterious variants in the voltage-gated sodium channel type 2 (Nav1.2) lead to a broad spectrum of phenotypes ranging from benign familial neonatal-infantile epilepsy (BFNIE), severe developmental and epileptic encephalopathy (DEE) and intellectual disability (ID) to autism spectrum disorders (ASD). Yet, the underlying mechanisms are still incompletely understood.

Neonatal jaundice is prevalent among newborns and can lead to severe neurological deficits, particularly sensorimotor dysfunction. Previous studies have shown that bilirubin (BIL) enhances the intrinsic excitability of central neurons and this can potentially contribute to their overexcitation, Ca2+ overload, and neurotoxicity. However, the cellular mechanisms underlying elevated neuronal excitability remain unknown. By performing patch-clamp recordings from neonatal neurons in the rat medial vestibular nucleus (MVN), a crucial relay station for locomotor and balance control, we found that BIL (3 μM) drastically increases the spontaneous firing rates by upregulating the current-mediated voltage-gated sodium channels (VGSCs), while shifting their voltage-dependent activation toward more hyperpolarized potentials. Immunofluorescence labeling and western immunoblotting with an anti-NaV1.1 antibody, revealed that BIL elevates the expression of VGSCs by promoting their recruitment to the membrane. Furthermore, we found that this VGSC-trafficking process is Ca2+ dependent because preloading MVN neurons with the Ca2+ buffer BAPTA-AM, or exocytosis inhibitor TAT-NSF700, prevents the effects of BIL, indicating the upregulated activity and density of functional VGSCs as the core mechanism accountable for the BIL-induced overexcitation of neonatal neurons. Most importantly, rectification of such overexcitation with a low dose of VGSC blocker lidocaine significantly attenuates BIL-induced cell death. We suggest that this enhancement of VGSC currents directly contributes to the vulnerability of neonatal brain to hyperbilirubinemia, implicating the activity and trafficking of NaV1.1 channels as a potential target for neuroprotection in cases of severe jaundice.

Tuberous sclerosis complex (TSC) is a rare disorder of tissue growth and differentiation, characterized by benign hamartomas in the brain and other organs. Up to 90% of TSC patients develop epilepsy and 50% become medically intractable requiring resective surgery. The surgical outcome of TSC patients depends on the accurate identification of the epileptogenic zone consisting of tubers and the surrounding epileptogenic tissue. There is conflicting evidence whether the epileptogenic zone is in the tuber itself or in abnormally developed surrounding cortex. Here, we report the localization of the epileptiform activity among the many cortical tubers in a 4-year-old patient with TSC-related refractory epilepsy undergoing magnetoencephalography (MEG), electroencephalography (EEG), and diffusion tensor imaging (DTI). For MEG, we used a prototype system that offers higher spatial resolution and sensitivity compared to the conventional adult systems. The generators of interictal activity were localized using both EEG and MEG with equivalent current dipole (ECD) and minimum norm estimation (MNE) methods according to the current clinical standards. For DTI, we calculated four diffusion scalar parameters for the fibers passing through four ROIs defined: (i) at a large cortical tuber identified at the right quadrant, (ii) at the normal appearing tissue contralateral to the tuber, (iii) at the cluster formed by ECDs fitted at the peak of interictal spikes, and (iv) at the normal appearing tissue contralateral to the cluster. ECDs were consistently clustered at the vicinity of the large calcified cortical tuber. MNE and ECDs indicated epileptiform activity in the same areas. DTI analysis showed differences between the scalar values of the tracks passing through the tuber and the ECD cluster. In this illustrative case, we provide evidence from different neuroimaging modalities, which support the view that epileptiform activity may derive from abnormally developed tissue surrounding the tuber rather than the tuber itself.

Recessive variants in RARS2, a nuclear gene encoding a mitochondrial protein, were initially reported in pontocerebellar hypoplasia. Subsequently, a recessive RARS2 early-infantile (<12 weeks) developmental and epileptic encephalopathy was described with hypoglycaemia and lactic acidosis. Here, we describe two unrelated patients with a novel RARS2 phenotype and reanalyse the published RARS2 epilepsy phenotypes and variants. Our novel cases had infantile-onset myoclonic developmental and epileptic encephalopathy, presenting with a progressive movement disorder from 9 months on a background of normal development. Development plateaued and regressed thereafter, with mild to profound impairment. Multiple drug-resistant generalized and focal seizures occurred with episodes of non-convulsive status epilepticus. Seizure types included absence, atonic, myoclonic, and focal seizures. Electroencephalograms showed diffuse slowing, multifocal, and generalised spike-wave activity, activated by sleep. Both patients had compound heterozygous RARS2 variants with likely impact on splicing and transcription. Remarkably, of the now 52 RARS2 variants reported in 54 patients, our reanalysis found that 44 (85%) have been shown to or are predicted to affect splicing or gene expression leading to protein truncation or nonsense-mediated decay. We expand the RARS2 phenotypic spectrum to include infantile encephalopathy and suggest this gene is enriched for pathogenic variants that disrupt splicing.

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function.