Kv1.1 channels mediate network excitability and feed-forward inhibition in local amygdala circuits.

Kv1.1 containing potassium channels play crucial roles towards dampening neuronal excitability. Mice lacking Kv1.1 subunits (Kcna1-/-) display recurrent spontaneous seizures and often exhibit sudden unexpected death. Seizures in Kcna1-/- mice resemble those in well-characterized models of temporal lobe epilepsy known to involve limbic brain regions and spontaneous seizures result in enhanced cFos expression and neuronal death in the amygdala. Yet, the functional alterations leading to amygdala hyperexcitability have not been identified. In this study, we used Kcna1-/- mice to examine the contributions of Kv1.1 subunits to excitability in neuronal subtypes from basolateral (BLA) and central lateral (CeL) amygdala known to exhibit distinct firing patterns. We also analyzed synaptic transmission properties in an amygdala local circuit predicted to be involved in epilepsy-related comorbidities. Our data implicate Kv1.1 subunits in controlling spontaneous excitatory synaptic activity in BLA pyramidal neurons. In the CeL, Kv1.1 loss enhances intrinsic excitability and impairs inhibitory synaptic transmission, notably resulting in dysfunction of feed-forward inhibition, a critical mechanism for controlling spike timing. Overall, we find inhibitory control of CeL interneurons is reduced in Kcna1-/- mice suggesting that basal inhibitory network functioning is less able to prevent recurrent hyperexcitation related to seizures.

Kv1.1 subunits localize to cardiorespiratory brain networks in mice where their absence induces astrogliosis and microgliosis.

Cardiorespiratory collapse following a seizure is a suspected cause of sudden unexpected death in epilepsy (SUDEP), the leading cause of epilepsy-related mortality. In the commonly used Kcna1 gene knockout (Kcna1-/-) mouse model of SUDEP, cardiorespiratory profiling reveals an array of aberrant breathing patterns that could contribute to risk of seizure-related mortality. However, the brain structures mediating these respiratory abnormalities remain unknown. We hypothesize that Kv1.1 deficiency in respiratory control centers of the brain contribute to respiratory dysfunction in Kcna1-/- mice leading to increased SUDEP risk. Thus, in this study, we first used immunohistochemistry to map expression of Kv1.1 protein in cardiorespiratory brain regions of wild-type Kcna1+/+ (WT) mice. Next, GFAP and Iba1 immunostaining was used to test for the presence of astrogliosis and microgliosis, respectively, in the cardiorespiratory centers of Kcna1-/- mice, which could be indicative of seizure-related brain injury that could impair breathing. In WT mice, we detected Kv1.1 protein in all cardiorespiratory centers examined, including the basolateral amygdala, dorsal respiratory group, dorsal motor nucleus of vagus, nucleus ambiguus, ventral respiratory column, and pontine respiratory group, as well as chemosensory centers including the retrotrapezoid and median raphae nuclei. Extensive gliosis was observed in the same areas in Kcna1-/- mice suggesting that seizure-associated brain injury could contribute to respiratory abnormalities.

Kv1.1 potassium channel subunit deficiency alters ventricular arrhythmia susceptibility, contractility, and repolarization.

Epilepsy-associated Kv1.1 voltage-gated potassium channel subunits encoded by the Kcna1 gene have traditionally been considered absent in heart, but recent studies reveal they are expressed in cardiomyocytes where they could regulate intrinsic cardiac electrophysiology. Although Kv1.1 now has a demonstrated functional role in atria, its role in the ventricles has never been investigated. In this work, electrophysiological, histological, and gene expression approaches were used to explore the consequences of Kv1.1 deficiency in the ventricles of Kcna1 knockout (KO) mice at the organ, cellular, and molecular levels to determine whether the absence of Kv1.1 leads to ventricular dysfunction that increases the risk of premature or sudden death. When subjected to intracardiac pacing, KO mice showed normal baseline susceptibility to inducible ventricular arrhythmias (VA) but resistance to VA under conditions of sympathetic challenge with isoproterenol. Echocardiography revealed cardiac contractile dysfunction manifesting as decreased ejection fraction and fractional shortening. In whole-cell patch-clamp recordings, KO ventricular cardiomyocytes exhibited action potential prolongation indicative of impaired repolarization. Imaging, histological, and transcript analyses showed no evidence of structural or channel gene expression remodeling, suggesting that the observed deficits are likely electrogenic due to Kv1.1 deficiency. Immunoblots of patient heart samples detected the presence of Kv1.1 at relatively high levels, implying that Kv1.1 contributes to human cardiac electrophysiology. Taken together, this work describes an important functional role for Kv1.1 in ventricles where its absence causes repolarization and contractility deficits but reduced susceptibility to arrhythmia under conditions of sympathetic drive.

Genetic ablation or pharmacological inhibition of Kv1.1 potassium channel subunits impairs atrial repolarization in mice.

Voltage-gated Kv1.1 potassium channel α-subunits, encoded by the Kcna1 gene, have traditionally been regarded as neural-specific with no expression or function in the heart. However, recent data revealed that Kv1.1 subunits are expressed in atria where they may have an overlooked role in controlling repolarization and arrhythmia susceptibility independent of the nervous system. To explore this concept in more detail and to identify functional and molecular effects of Kv1.1 channel impairment in the heart, atrial cardiomyocyte patch-clamp electrophysiology and gene expression analyses were performed using Kcna1 knockout ( Kcna1-/-) mice. Specifically, we hypothesized that Kv1.1 subunits contribute to outward repolarizing K+ currents in mouse atria and that their absence prolongs cardiac action potentials. In voltage-clamp experiments, dendrotoxin-K (DTX-K), a Kv1.1-specific inhibitor, significantly reduced peak outward K+ currents in wild-type (WT) atrial cells but not Kcna1-/- cells, demonstrating an important contribution by Kv1.1-containing channels to mouse atrial repolarizing currents. In current-clamp recordings, Kcna1-/- atrial myocytes exhibited significant action potential prolongation which was exacerbated in right atria, effects that were partially recapitulated in WT cells by application of DTX-K. Quantitative RT-PCR measurements showed mRNA expression remodeling in Kcna1-/- atria for several ion channel genes that contribute to the atrial action potential including the Kcna5, Kcnh2, and Kcnj2 potassium channel genes and the Scn5a sodium channel gene. This study demonstrates a previously undescribed heart-intrinsic role for Kv1.1 subunits in mediating atrial repolarization, thereby adding a new member to the already diverse collection of known K+ channels in the heart.

Ketogenic diet regulates the antioxidant catalase via the transcription factor PPARγ2.

We have previously found that the transcription factor PPARγ2 contributes to the mechanism of action of the ketogenic diet (KD), an established treatment for pediatric refractory epilepsy. Among the wide-array of genes regulated by PPARγ, previous studies have suggested that antioxidants such as catalase may have prominent roles in KD neuroprotective and antiseizure effects. Here, we tested the hypothesis that the KD increases catalase through activation of PPARγ2, and that this action is part of the mechanism of antiseizure efficacy of the KD. We determined catalase mRNA and protein expression in hippocampal tissue from epileptic Kcna1-/- mice, Pparγ2+/+ mice and Pparγ2-/- mice. We found that a KD increases hippocampal catalase expression in Kcna1-/- and Pparγ2+/+ mice, but not Pparγ2-/- mice. Next, we determined whether catalase contributes to KD seizure protection. We found that the KD reduces pentylenetetrazole (PTZ)-induced seizures; however, pretreatment with a catalase inhibitor occluded KD effects on PTZ seizures. These results suggest that the KD regulates catalase expression through PPARγ2 activation, and that catalase may contribute to the KD antiseizure efficacy.

The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet.

The ketogenic diet (KD) is used to treat refractory epilepsy, but the mechanisms underlying its neuroprotective effects remain unclear. Here, we show that the gut microbiota is altered by the KD and required for protection against acute electrically induced seizures and spontaneous tonic-clonic seizures in two mouse models. Mice treated with antibiotics or reared germ free are resistant to KD-mediated seizure protection. Enrichment of, and gnotobiotic co-colonization with, KD-associated Akkermansia and Parabacteroides restores seizure protection. Moreover, transplantation of the KD gut microbiota and treatment with Akkermansia and Parabacteroides each confer seizure protection to mice fed a control diet. Alterations in colonic lumenal, serum, and hippocampal metabolomic profiles correlate with seizure protection, including reductions in systemic gamma-glutamylated amino acids and elevated hippocampal GABA/glutamate levels. Bacterial cross-feeding decreases gamma-glutamyltranspeptidase activity, and inhibiting gamma-glutamylation promotes seizure protection in vivo. Overall, this study reveals that the gut microbiota modulates host metabolism and seizure susceptibility in mice.

Ketogenic diet-induced extension of longevity in epileptic Kcna1-null mice is influenced by gender and age at treatment onset.

Sudden unexpected death in epilepsy (SUDEP) is a leading cause of premature mortality in patients with epilepsy, and has been linked to multiple risk factors, including gender and early age at seizure onset. Despite the lack of a targeted therapy for SUDEP, it has recently been shown that a high-fat, low carbohydrate ketogenic diet (KD) enhances longevity in the epileptic Kcna1-null (KO) mouse, a validated model of SUDEP. Here, we asked whether the KD-driven prolongation of lifespan in KO mice is dependent on sex and/or age at treatment onset. We found that as KO mice aged, their daily seizure frequency steadily increased, but had early demise by postnatal day (PD) 46.9±0.8. In KO mice started on the KD at PD30, longevity was extended to a mean of PD69.8±1.7, accompanied with improved seizure control. Interestingly, while seizure control on the KD was similar between male and female mice, KD-fed female KO mice survived longer than their male counterparts. Further, epileptic mice initiated on the KD at PD25 had longer lifespans compared to those placed on the KD starting at PD35. Collectively, these data further support the notion that the KD can retard disease progression and sudden death in KO mice, but that this beneficial action is influenced by gender and age at the start of treatment.

Accumulation of rest deficiency precedes sudden death of epileptic Kv1.1 knockout mice, a model of sudden unexpected death in epilepsy.

OBJECTIVE: Chronic sleep deficiency is associated with early mortality. In the epileptic population, there is a higher prevalence of sleep disorders, and individuals with severe refractory epilepsy are at greater risk of premature mortality than the general population. Sudden unexpected death in epilepsy affects 1:1000 cases of epilepsy each year. Ketogenic diet (KD) treatment is one of the few effective options for refractory seizures. Despite KD reducing seizures and increasing longevity in Kv1.1 knockout (KO) mice, they still succumb to sudden death. This study aims to determine whether (1) the rest profiles of KO and KD-treated KO (KOKD) mice resemble each other as a function of either age or proximity to death and (2) the timing of death correlates with acute or chronic changes in rest. METHODS: Noninvasive actimetry was used to monitor rest throughout the lives of KO and wild-type (WT) littermates administered standard diet or KD. RESULTS: As KO mice age, rest is reduced (P < .0001). Rest is significantly improved in KDKO mice (P < .0001), resembling WT values at several ages. When age is removed as a variable and data are realigned to the day of death, the rest profiles of KO and KOKD groups worsen to similar degrees as a function of proximity to death. The amount of rest acutely is not sensitive to the timing of death, whereas chronic rest deficiency profiles (10-15 days prior to death) of both groups were indistinguishable. Chronic accumulation of rest deficiency over the final 15 days was associated with 75% of deaths. SIGNIFICANCE: Our data suggest that the accumulated rest deficiency is associated with sudden death in Kv1.1 KO mice. These data (1) support the proposed clinical hypothesis that chronic sleep deficiency may be associated with early mortality in epileptic patients and (2) warrant future preclinical and clinical studies on sleep monitoring in epileptic patients.

Kv1.1 channelopathy abolishes presynaptic spike width modulation by subthreshold somatic depolarization.

Although action potentials propagate along axons in an all-or-none manner, subthreshold membrane potential fluctuations at the soma affect neurotransmitter release from synaptic boutons. An important mechanism underlying analog-digital modulation is depolarization-mediated inactivation of presynaptic Kv1-family potassium channels, leading to action potential broadening and increased calcium influx. Previous studies have relied heavily on recordings from blebs formed after axon transection, which may exaggerate the passive propagation of somatic depolarization. We recorded instead from small boutons supplied by intact axons identified with scanning ion conductance microscopy in primary hippocampal cultures and asked how distinct potassium channels interact in determining the basal spike width and its modulation by subthreshold somatic depolarization. Pharmacological or genetic deletion of Kv1.1 broadened presynaptic spikes without preventing further prolongation by brief depolarizing somatic prepulses. A heterozygous mouse model of episodic ataxia type 1 harboring a dominant Kv1.1 mutation had a similar broadening effect on basal spike shape as deletion of Kv1.1; however, spike modulation by somatic prepulses was abolished. These results argue that the Kv1.1 subunit is not necessary for subthreshold modulation of spike width. However, a disease-associated mutant subunit prevents the interplay of analog and digital transmission, possibly by disrupting the normal stoichiometry of presynaptic potassium channels.

Genetic perturbations suggest a role of the resting potential in regulating the expression of the ion channels of the KCNA and HCN families in octopus cells of the ventral cochlear nucleus.

Low-voltage-activated K+ (gKL) and hyperpolarization-activated mixed cation conductances (gh) mediate currents, IKL and Ih, through channels of the Kv1 (KCNA) and HCN families respectively and give auditory neurons the temporal precision required for signaling information about the onset, fine structure, and time of arrival of sounds. Being partially activated at rest, gKL and gh contribute to the resting potential and shape responses to even small subthreshold synaptic currents. Resting gKL and gh also affect the coupling of somatic depolarization with the generation of action potentials. To learn how these important conductances are regulated we have investigated how genetic perturbations affect their expression in octopus cells of the ventral cochlear nucleus (VCN). We report five new findings: First, the magnitude of gh and gKL varied over more than two-fold between wild type strains of mice. Second, average resting potentials are not different in different strains of mice even in the face of large differences in average gKL and gh. Third, IKL has two components, one being α-dendrotoxin (α-DTX)-sensitive and partially inactivating and the other being α-DTX-insensitive, tetraethylammonium (TEA)-sensitive, and non-inactivating. Fourth, the loss of Kv1.1 results in diminution of the α-DTX-sensitive IKL, and compensatory increased expression of an α-DTX-insensitive, tetraethylammonium (TEA)-sensitive IKL. Fifth, Ih and IKL are balanced at the resting potential in all wild type and mutant octopus cells even when resting potentials vary in individual cells over nearly 10 mV, indicating that the resting potential influences the expression of gh and gKL. The independence of resting potentials on gKL and gh shows that gKL and gh do not, over days or weeks, determine the resting potential but rather that the resting potential plays a role in regulating the magnitude of either or both gKL and gh.

Regulation of brain PPARgamma2 contributes to ketogenic diet anti-seizure efficacy.

The ketogenic diet (KD) is an effective therapy primarily used in pediatric patients whom are refractory to current anti-seizure medications. The mechanism of the KD is not completely understood, but is thought to involve anti-inflammatory and anti-oxidant processes. The nutritionally-regulated transcription factor peroxisome proliferator activated receptor gamma, PPARγ, regulates genes involved in anti-inflammatory and anti-oxidant pathways. Moreover, endogenous ligands of PPARγ include fatty acids suggesting a potential role in the effects of the KD. Here, we tested the hypothesis that PPARγ contributes to the anti-seizure efficacy of the KD. We found that the KD increased nuclear protein content of the PPARγ2 splice variant by 2-4 fold (P<0.05) in brain homogenates from wild-type (WT) and epileptic Kv1.1 knockout (KO) mice, while not affecting PPARγ1. The KD reduced the frequency of seizures in Kv1.1KO mice by ~70% (P<0.01). GW9662, a PPARγ antagonist, prevented KD-mediated changes in PPARγ2 expression and prevented the anti-seizure efficacy of the KD in Kv1.1KO mice. Further supporting the association of PPARγ2 in mediating KD actions, the KD significantly prolonged the latency to flurothyl-induced seizure in WT mice by ~20-35% (P<0.01), but was ineffective in PPARγ2KO mice and neuron-specific PPARγKO mice. Finally, administering the PPARγ agonist pioglitazone increased PPARγ2 expression by 2-fold (P<0.01) and reduced seizures in Kv1.1KO mice by ~80% (P<0.01). Our findings implicate brain PPARγ2 among the mechanisms by which the KD reduces seizures and strongly support the development of PPARγ2 as a therapeutic target for severe, refractory epilepsy.

Ketogenic diet treatment increases longevity in Kcna1-null mice, a model of sudden unexpected death in epilepsy.

Individuals with poorly controlled epilepsy have a higher risk for sudden unexpected death in epilepsy (SUDEP). With approximately one third of people with epilepsy not achieving adequate seizure control with current antiseizure drugs, there is a critical need to identify treatments that reduce risk factors for SUDEP. The Kcna1-null mutant mouse models risk factors and terminal events associated with SUDEP. In the current study, we demonstrate the progressive nature of epilepsy and sudden death in this model (mean age of mortality (± SEM), postnatal day [P] 42.8 ± 1.3) and tested the hypothesis that long-term treatment with the ketogenic diet (KD) will prolong the life of Kcna1-null mice. We found that the KD postpones disease progression by delaying the onset of severe seizures and increases the lifespan of these mutant mice by 47%. Future studies are needed to determine the mechanisms underlying the KD effects on longevity.

Orexin Receptor Antagonism Improves Sleep and Reduces Seizures in Kcna1-null Mice.

STUDY OBJECTIVE: Comorbid sleep disorders occur in approximately one-third of people with epilepsy. Seizures and sleep disorders have an interdependent relationship where the occurrence of one can exacerbate the other. Orexin, a wake-promoting neuropeptide, is associated with sleep disorder symptoms. Here, we tested the hypothesis that orexin dysregulation plays a role in the comorbid sleep disorder symptoms in the Kcna1-null mouse model of temporal lobe epilepsy. METHODS: Rest-activity was assessed using infrared beam actigraphy. Sleep architecture and seizures were assessed using continuous video-electroencephalography-electromyography recordings in Kcna1-null mice treated with vehicle or the dual orexin receptor antagonist, almorexant (100 mg/kg, intraperitoneally). Orexin levels in the lateral hypothalamus/perifornical region (LH/P) and hypothalamic pathology were assessed with immunohistochemistry and oxygen polarography. RESULTS: Kcna1-null mice have increased latency to rapid eye movement (REM) sleep onset, sleep fragmentation, and number of wake epochs. The numbers of REM and non-REM (NREM) sleep epochs are significantly reduced in Kcna1-null mice. Severe seizures propagate to the wake-promoting LH/P where injury is apparent (indicated by astrogliosis, blood-brain barrier permeability, and impaired mitochondrial function). The number of orexin-positive neurons is increased in the LH/P compared to wild-type LH/P. Treatment with a dual orexin receptor antagonist significantly increases the number and duration of NREM sleep epochs and reduces the latency to REM sleep onset. Further, almorexant treatment reduces the incidence of severe seizures and overall seizure burden. Interestingly, we report a significant positive correlation between latency to REM onset and seizure burden in Kcna1-null mice. CONCLUSION: Dual orexin receptor antagonists may be an effective sleeping aid in epilepsy, and warrants further study on their somnogenic and ant-seizure effects in other epilepsy models.

Auditory deficits of Kcna1 deletion are similar to those of a monaural hearing impairment.

Kv1.1 subunits of low voltage-activated (Kv) potassium channels are encoded by the Kcna1 gene and crucially determine the synaptic integration window to control the number and temporal precision of action potentials in the auditory brainstem of mammals and birds. Prior electrophysiological studies showed that auditory signaling is compromised in monaural as well as in binaural neurons of the auditory brainstem in Kv1.1 knockout mice (Kcna1(-/-)). Here we examine the behavioral effects of Kcna1 deletion on sensory tasks dependent on either binaural processing (detecting the movement of a sound source across the azimuth), monaural processing (detecting a gap in noise), as well as binaural summation of the acoustic startle reflex (ASR). Hearing thresholds measured by auditory brainstem responses (ABR) do not differ between genotypes, but our data show a much stronger performance of wild type mice (+/+) in each test during binaural hearing which was lost by temporarily inducing a unilateral hearing loss (through short term blocking of one ear) thus remarkably, leaving no significant difference between binaural and monaural hearing in Kcna1(-/-) mice. These data suggest that the behavioral effect of Kv1.1 deletion is primarily to impede binaural integration and thus to mimic monaural hearing.

The Kv1.1 null mouse, a model of sudden unexpected death in epilepsy (SUDEP).

OBJECTIVE: Kv1.1 potassium channel null mouse (NULL) exhibits spontaneous seizure-related bradycardia, dies following seizure, and has been proposed as a model for vagus-mediated SUDEP. We characterized the cardiac events surrounding sudden unexpected death in epilepsy (SUDEP) in NULL during terminal asystole for comparison to patients with epilepsy who exhibit bradycardia and terminal or nonterminal asystole during/following seizure and explored the contribution of vagal-mediated bradycardia to SUDEP. METHODS: Electrocardiography (ECG) studies of 27 freely moving telemetered NULL mice was evaluated surrounding seizure-associated death. Chronic unilateral vagal section and, in a separate set of experiments, electrical stimulation of the cervical vagi in NULL and wild-type (WT) littermates assessed the role of the vagus nerve in seizure-related death. Seizure activity indicated by intense myogenic activity on the ECG recording correlated with visual and video recording. RESULTS: All NULL died following seizures, which were preceded by normal rhythm. Bradycardia followed seizure and led to slow ventricular escape rhythm (70-150 bpm) and asystole. The sequence from seizure to asystole was complete within approximately 3 min and was similar to that reported in individuals exhibiting ictal and postictal bradycardia/asystole. To address the singular role of vagus nerves in seizure-related asystole, cervical vagus nerves were stimulated in the absence of seizure. Heart rate was reduced 3 min to values similar to that following seizure but never produced asystole, suggesting activation of the vagi alone is insufficient for SUDEP. Nevertheless, unilateral chronic section of the vagus nerve increased survival time compared to nonsectioned NULL animals, supporting a role for the vagus nerve in seizure-associated death. SIGNIFICANCE: The Kv1.1 null mouse is a potential model for SUDEP in patients who experience ictal and postictal bradycardia. It offers the opportunity for evaluation of the combination of factors, in addition to vagal activation, necessary to produce a terminal asystole following seizure. It is notable that long-term studies that evaluate electroencephalography (EEG) and cardiorespiratory events surrounding nonfatal seizures may provide indices predictive of terminal seizure.

Targeting deficiencies in mitochondrial respiratory complex I and functional uncoupling exerts anti-seizure effects in a genetic model of temporal lobe epilepsy and in a model of acute temporal lobe seizures.

Mitochondria actively participate in neurotransmission by providing energy (ATP) and maintaining normative concentrations of reactive oxygen species (ROS) in both presynaptic and postsynaptic elements. In human and animal epilepsies, ATP-producing respiratory rates driven by mitochondrial respiratory complex (MRC) I are reduced, antioxidant systems are attenuated and oxidative damage is increased. We report that MRCI-driven respiration and functional uncoupling (an inducible antioxidant mechanism) are reduced and levels of H2O2 are elevated in mitochondria isolated from KO mice. Experimental impairment of MRCI in WT hippocampal slices via rotenone reduces paired-pulse ratios (PPRs) at mossy fiber-CA3 synapses (resembling KO PPRs), and exacerbates seizure-like events in vitro. Daily treatment with AATP [a combination therapy composed of ascorbic acid (AA), alpha-tocopherol (T), sodium pyruvate (P) designed to synergistically target mitochondrial impairments] improved mitochondrial functions, mossy fiber PPRs, and reduced seizure burden index (SBI) scores and seizure incidence in KO mice. AATP pretreatment reduced severity of KA-induced seizures resulting in 100% protection from the severe tonic-clonic seizures in WT mice. These data suggest that restoration of bioenergetic homeostasis in the brain may represent a viable anti-seizure target for temporal lobe epilepsy.

Evidence for presence and functional effects of Kv1.1 channels in ß-cells: general survey and results from mceph/mceph mice.

BACKGROUND: Voltage-dependent K(+) channels (Kv) mediate repolarisation of ß-cell action potentials, and thereby abrogate insulin secretion. The role of the Kv1.1 K(+) channel in this process is however unclear. We tested for presence of Kv1.1 in different species and tested for a functional role of Kv1.1 by assessing pancreatic islet function in BALB/cByJ (wild-type) and megencephaly (mceph/mceph) mice, the latter having a deletion in the Kv1.1 gene. METHODOLOGY/PRINCIPAL FINDINGS: Kv1.1 expression was detected in islets from wild-type mice, SD rats and humans, and expression of truncated Kv1.1 was detected in mceph/mceph islets. Full-length Kv1.1 protein was present in islets from wild-type mice, but, as expected, not in those from mceph/mceph mice. Kv1.1 expression was localized to the ß-cell population and also to α- and δ-cells, with evidence of over-expression of truncated Kv1.1 in mceph/mceph islets. Blood glucose, insulin content, and islet morphology were normal in mceph/mceph mice, but glucose-induced insulin release from batch-incubated islets was (moderately) higher than that from wild-type islets. Reciprocal blocking of Kv1.1 by dendrotoxin-K increased insulin secretion from wild-type but not mceph/mceph islets. Glucose-induced action potential duration, as well as firing frequency, was increased in mceph/mceph mouse ß-cells. This duration effect on action potential in ß-cells from mceph/mceph mice was mimicked by dendrotoxin-K in ß-cells from wild-type mice. Observations concerning the effects of both the mceph mutation, and of dendrotoxin-K, on glucose-induced insulin release were confirmed in pancreatic islets from Kv1.1 null mice. CONCLUSION/SIGNIFICANCE: Kv1.1 channels are expressed in the ß-cells of several species, and these channels can influence glucose-stimulated insulin release.

Dendrotoxin-κ suppresses tumor growth induced by human lung adenocarcinoma A549 cells in nude mice.

Voltage-gated K(+) (Kv) channels have been considered to be a regulator of membrane potential and neuronal excitability. Recently, accumulated evidence has indicated that several Kv channel subtypes contribute to the control of cell proliferation in various types of cells and are worth noting as potential emerging molecular targets of cancer therapy. In the present study, we investigated the effects of the Kv1.1-specific blocker, dendrotoxin-κ (DTX-κ, on tumor formation induced by the human lung adenocarcinoma cell line A549 in a xenograft model. Kv1.1 mRNA and protein was expressed in A549 cells and the blockade of Kv1.1 by DTX-κ, reduced tumor formation in nude mice. Furthermore, treatment with DTX-κ significantly increased protein expression of p21(Waf1/Cip1), p27(Kip1), and p15(INK4B) and significantly decreased protein expression of cyclin D3 in tumor tissues compared to the control. These results suggest that DTX-κ has anti-tumor effects in A549 cells through the pathway governing G1-S transition.

Kv1.1 potassium channel deficiency reveals brain-driven cardiac dysfunction as a candidate mechanism for sudden unexplained death in epilepsy.

Mice lacking Kv1.1 Shaker-like potassium channels encoded by the Kcna1 gene exhibit severe seizures and die prematurely. The channel is widely expressed in brain but only minimally, if at all, in mouse myocardium. To test whether Kv1.1-potassium deficiency could underlie primary neurogenic cardiac dysfunction, we performed simultaneous video EEG-ECG recordings and found that Kcna1-null mice display potentially malignant interictal cardiac abnormalities, including a fivefold increase in atrioventricular (AV) conduction blocks, as well as bradycardia and premature ventricular contractions. During seizures the occurrence of AV conduction blocks increased, predisposing Kv1.1-deficient mice to sudden unexplained death in epilepsy (SUDEP), which we recorded fortuitously in one animal. To determine whether the interictal AV conduction blocks were of cardiac or neural origin, we examined their response to selective pharmacological blockade of the autonomic nervous system. Simultaneous administration of atropine and propranolol to block parasympathetic and sympathetic branches, respectively, eliminated conduction blocks. When administered separately, only atropine ameliorated AV conduction blocks, indicating that excessive parasympathetic tone contributes to the neurocardiac defect. We found no changes in Kv1.1-deficient cardiac structure, but extensive Kv1.1 expression in juxtaparanodes of the wild-type vagus nerve, the primary source of parasympathetic input to the heart, suggesting a novel site of action leading to Kv1.1-associated cardiac bradyarrhythmias. Together, our data suggest that Kv1.1 deficiency leads to impaired neural control of cardiac rhythmicity due in part to aberrant parasympathetic neurotransmission, making Kcna1 a strong candidate gene for human SUDEP.

Cell-type-dependent molecular composition of the axon initial segment.

The exact site of initiation and shape of action potentials vary among different neuronal types. The reason for this variability is largely unknown, but the subunit composition, density and distribution of voltage-gated sodium (Nav) and potassium (Kv) channels within the axon initial segment (AIS) are likely to play a key role. Here, we asked how heterogeneous are the density and distribution of Nav and Kv channels within the AISs of a variety of excitatory and inhibitory neurons. Most of the studied cell types expressed a high density of Nav1.6, Kv1.1, and Kv1.2 subunits in their AIS, but the Nav1.1 subunit could only be detected in GABAergic interneurons. A proximo-distal gradient in the density of these subunits was observed within the AIS of certain nerve cells but not in others. For example, a gradual increase of the Nav1.6 subunit was observed in cortical layer 2/3 and hippocampal CA1 pyramidal cell (PC) AISs, whereas its density was rather uniform in layer 5 PC AISs. The Nav1.1 subunit was distributed evenly along the AIS of short-axon cells of the main olfactory bulb but was restricted to the proximal part of the AIS in cortical and cerebellar interneurons. Our results reveal a cell type-dependent expression of sodium and potassium channel subunits with varying densities along the proximo-distal axis of the AISs. This precise arrangement is likely to contribute to the diversity of firing properties observed among central neurons.