Functional characterisation of missense variations in the Kir4.1 potassium channel (KCNJ10) associated with seizure susceptibility

Recent genetic linkage studies have identified an association between missense variations in the gene encoding the Kir4.1 potassium channel (KCNJ10) and seizure susceptibility phenotypes in both humans and mice. The results of this study demonstrate that these variations (T262S and R271C) do not produce any observable changes in channel function or in predicted channel structure. It is therefore unlikely that the seizure susceptibility phenotypes associated with these missense variations are caused by changes in the intrinsic functional properties of Kir4.1.     

Localization of the ATP-sensitive potassium channel subunit (Kir6. 1/uK(ATP)-1) in rat brain

The Kir6.1/uK(ATP)-1, subunit of ATP-sensitive K(+) channels (K(ATP)), was localized in adult rat brain by in situ hybridization and immunohistochemistry. The mRNA of this subunit was ubiquitously expressed in various neurons and nuclei of the adult rat brain. Interestingly, Kir6.1/uK(ATP)-1 mRNA was also expressed in glial cells. Kir6.1/uK(ATP)-1 protein staining gave a dispersed array of fine dots throughout all neurons and glial cells examined. Under electron microscope, the immunoreactive products were specifically restricted to the mitochondria. The present study indicates that this K(ATP) subunit is localized in the mitochondria and may play a fundamental role in vital brain function.     

Focal epileptic spasms, involving one leg, manifesting during the clinical course of west syndrome (WS)

OBJECTIVES: Epileptic spasms (ES) in West syndrome (WS) are classified as being of the generalized seizure type. However, evidence of a focal neocortical origin has been proposed based mainly on surgical WS series. We report herein two infants with WS, whose ES subsequently evolved into focal ES of the right lower extremity. METHODS: Medical records, electroencephalographic and neuroimaging findings were retrospectively analyzed. RESULTS: A 2 year-old Japanese girl developed ES at 3 months of age. Focal ES of the right leg manifested one month after successful ACTH therapy performed at 6 months of age. Brief jerking of the right leg, every few seconds, occurred immediately after awakening. A 7 month-old girl with symptomatic WS had developed focal seizures with postural signs at 2 months of age. The seizures gradually diminished and had been replaced by ES by 3 months of age. ES gradually evolved into focal ES of the right leg. A video-polygraphic study demonstrated the ES with marked predominance in the right leg corresponding to a left sided predominant paroxysmal biphasic slow wave complex. Brain MRI revealed no abnormal findings although interictal EEG demonstrated left centro-parieto-temporal localized spike foci. CONCLUSIONS: The focal ES of one lower limb, following treatment of ES in these two infants with WS, suggests the origin of the ES in the first case to be a neocortical focus involving the primary motor cortex representing the lower limb while that in the second case involves more widespread neocortical area with predominance in the same motor cortex as the first case.     

Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: phenotypic impact in retina.

The inwardly rectifying potassium channel Kir4.1 has been suggested to underlie the principal K(+) conductance of mammalian Müller cells and to participate in the generation of field potentials and regulation of extracellular K(+) in the retina. To further assess the role of Kir4.1 in the retina, we generated a mouse line with targeted disruption of the Kir4.1 gene (Kir4.1 -/-). Müller cells from Kir4.1 -/- mice were not labeled with an anti-Kir4.1 antibody, although they appeared morphologically normal when stained with an anti-glutamine synthetase antibody. In contrast, in Müller cells from wild-type littermate (Kir4.1 +/+) mice, Kir4.1 was present and localized to the proximal endfeet and perivascular processes. In situ whole-cell patch-clamp recordings showed a 10-fold increase in the input resistance and a large depolarization of Kir4.1 -/- Müller cells compared with Kir4.1 +/+ cells. The slow PIII response of the light-evoked electroretinogram (ERG), which is generated by K(+) fluxes through Müller cells, was totally absent in retinas from Kir4.1 -/- mice. The b-wave of the ERG, in contrast, was spared in the null mice. Overall, these results indicate that Kir4.1 is the principal K(+) channel subunit expressed in mouse Müller glial cells. The highly regulated localization and the functional properties of Kir4.1 in Müller cells suggest the involvement of this channel in the regulation of extracellular K(+) in the mouse retina.     

Reduced expression of the KATP channel subunit, Kir6.2, is associated with decreased expression of neuropeptide Y and agouti-related protein in the hypothalami of Zucker diabetic fatty rats

The link between obesity and diabetes is not fully understood but there is evidence to suggest that hypothalamic signalling pathways may be involved. The hypothalamic neuropeptides, pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and agouti-related protein (AGRP) are central to the regulation of food intake and have been implicated in glucose homeostasis. Therefore, the expression of these genes was quantified in hypothalami from diabetic Zucker fatty (ZDF) rats and nondiabetic Zucker fatty (ZF) rats at 6, 8, 10 and 14 weeks of age. Although both strains are obese, only ZDF rats develop pancreatic degeneration and diabetes over this time period. In both ZF and ZDF rats, POMC gene expression was decreased in obese versus lean rats at all ages. By contrast, although there was the expected increase in both NPY and AGRP expression in obese 14-week-old ZF rats, the expression of NPY and AGRP was decreased in 6-week-old obese ZDF rats with hyperinsulinaemia and in 14-week-old rats with the additional hyperglycaemia. Therefore, candidate genes involved in glucose, and insulin signalling pathways were examined in obese ZDF rats over this age range. We found that expression of the ATP-sensitive potassium (K(ATP)) channel component, Kir6.2, was decreased in obese ZDF rats and was lower compared to ZF rats in each age group tested. Furthermore, immunofluorescence analysis showed that Kir6.2 protein expression was reduced in the dorsomedial and ventromedial hypothalamic nuclei of 6-week-old prediabetic ZDF rats compared to ZF rats. The Kir6.2 immunofluorescence colocalised with NPY throughout the hypothalamus. The differences in Kir6.2 expression in ZF and ZDF rats mimic those of NPY and AGRP, which could infer that the changes occur in the same neurones. Overall, these data suggest that chronic changes in hypothalamic Kir6.2 expression may be associated with the development of hyperinsulinaemia and hyperglycaemia in ZDF rats.     

Morvan's syndrome associated with antibodies to multiple components of the voltage-gated potassium channel complex

We describe a patient presenting with a combination of muscle fasciculations, paresthesias, hyperhidrosis, as well as insomnia, agitation and confusion. He went on to develop psychosis and respiratory failure requiring intensive care. Electromyography confirmed the presence of neuromyotonia and CSF showed mild pleocytosis. Routine testing for voltage-gated potassium channel complex (VGKC-complex) antibodies was highly positive, confirming the clinical diagnosis of Morvan's syndrome. The patient improved after treatment with intravenous immunoglobulin and methylprednisolone. Further investigation of the antigenic targets using immunohistochemistry and cell-based assays revealed that he had autoantibodies targeting Lgi1, Caspr2 and Contactin-2/Tag-1, all proteins known to be complexed with VGKC in peripheral nerves and CNS. This is the first case of Morvan's syndrome from Cyprus and illustrates the clinical features as well as the emerging complexity of antigenic targets involved in the pathogenesis.     

Functional analysis of a novel potassium channel (KCNA1) mutation in hereditary myokymia

Myokymia is characterized by spontaneous, involuntary muscle fiber group contraction visible as vermiform movement of the overlying skin. Myokymia with episodic ataxia is a rare, autosomal dominant trait caused by mutations in KCNA1, encoding a voltage-gated potassium channel. In the present study, we report a family with four members affected with myokymia. Additional clinical features included motor delay initially diagnosed as cerebral palsy, worsening with febrile illness, persistent extensor plantar reflex, and absence of epilepsy or episodic ataxia. Mutation analysis revealed a novel c.676C>A substitution in the potassium channel gene KCNA1, resulting in a T226K nonconservative missense mutation in the Kv1.1 subunit in all affected individuals. Electrophysiological studies of the mutant channel expressed in Xenopus oocytes indicated a loss of function. Co-expression of WT and mutant cRNAs significantly reduced whole-oocyte current compared to expression of WT Kv1.1 alone.     

Laminin-induced aggregation of the inwardly rectifying potassium channel, Kir4.1, and the water-permeable channel, AQP4, via a dystroglycan-containing complex in astrocytes

Dystroglycan (DG) is part of a multiprotein complex that links the extracellular matrix to the actin cytoskeleton of muscle fibers and that is involved in aggregating acetylcholine receptors at the neuromuscular junction. This complex is also expressed in regions of the central nervous system where it is localized to both neuronal and glial cells. DG and the inwardly rectifying potassium channels, Kir4.1, are concentrated at the interface of astroglia and small blood vessels. These channels are involved in siphoning potassium released into the extracellular space after neuronal excitation. This raises the possibility that DG may be involved in targeting Kir4.1 channels to specific domains of astroglia. To address this question, we used mixed hippocampal cultures to investigate the distribution of DG, syntrophin, dystrobrevin, and Kir4.1 channels, as well as aquaporin-permeable water channels, AQP4. These proteins exhibit a similar distribution pattern and form aggregates in astrocytes cultured on laminin. Both DG and syntrophin colocalize with Kir4.1 channel aggregates in astrocytes. Similarly, DG colocalizes with AQP4 channel aggregates. Quantitative studies show a significant increase of Kir4.1 and AQP4 channel aggregates in astrocytes cultured in the presence of laminin when compared with those in the absence of laminin. These findings show that laminin has a role in Kir4.1 and AQP4 channel aggregation and suggest that this may be mediated via a dystroglycan-containing complex. This study reveals a novel functional role for DG in brain including K+ buffering and water homeostasis.     

Loss of the potassium channel beta-subunit gene, KCNAB2, is associated with epilepsy in patients with 1p36 deletion syndrome

PURPOSE: Clinical features associated with chromosome 1p36 deletion include characteristic craniofacial abnormalities, mental retardation, and epilepsy. The presence and severity of specific phenotypic features are likely to be correlated with loss of a distinct complement of genes in each patient. We hypothesize that hemizygous deletion of one, or a few, critical gene(s) controlling neuronal excitability is associated with the epilepsy phenotype. Because ion channels are important determinants of seizure susceptibility and the voltage-gated K(+) channel beta-subunit gene, KCNAB2, has been localized to 1p36, we propose that deletion of this gene may be associated with the epilepsy phenotype. METHODS: Twenty-four patients were evaluated by fluorescence in situ hybridization with a probe containing KCNAB2. Clinical details were obtained by neurologic examination and EEG. RESULTS: Nine patients are deleted for the KCNAB2 locus, and eight (89%) of these have epilepsy or epileptiform activity on EEG. The majority of patients have a severe seizure phenotype, including infantile spasms. In contrast, of those not deleted for KCNAB2, only 27% have chronic seizures, and none had infantile spasms. CONCLUSIONS: Lack of the beta subunit would be predicted to reduce K(+) channel-mediated membrane repolarization and increase neuronal excitability, suggesting a possible relation between loss of this gene and the development of seizures. Because some patients with seizures were not deleted for KCNAB2, there may be additional genes within 1p36 that contribute to epilepsy in this syndrome. Hemizygosity of this gene in a majority of monosomy 1p36 syndrome patients with epilepsy suggests that haploinsufficiency for KCNAB2 is a significant risk factor for epilepsy.     

Infantile spasms in a patient with septo-optic dysplasia, partial agenesis of the corpus callosum and an interhemispheric cyst.

An uncommon association of infantile spasms, septo-optic dysplasia, partial agenesis of the corpus callosum and an interhemispheric cyst is described in a child. The combination of these findings has not, to our knowledge, been previously reported.     

The underlying etiology of infantile spasms (West syndrome): Information from the United Kingdom Infantile Spasms Study (UKISS) on contemporary causes and their classification2

Purpose: To examine the underlying etiology of infantile spasms from the United Kingdom Infantile Spasms Study (UKISS), using the pediatric adaptation of ICD 10. Methods: Infants were enrolled in a randomized controlled trial or a parallel epidemiologic study. Etiological information included history, examination, and investigations. The infants were classified as proven etiology, if a neurologic disease was identified; as no identified etiology, if no neurologic disease was identified; and as not fully investigated, if a major piece of information was missing. Proven etiology was subclassified using the pediatric adaptation of ICD 10. The results were then examined to identify further methods of classification. Results: Of 207 infants, 127 (61%) had proven etiology, 68 (33%) had no identified etiology, and 12 (6%) were not fully investigated. Etiologies were prenatal in 63, perinatal in 38, postnatal in 8, and 18 other. The most common etiologies were: hypoxic–ischemic encephalopathy (HIE) 21 (10%), chromosomal 16 (8%), malformations 16 (8%), stroke 16 (8%), tuberous sclerosis complex (TSC) 15 (7%), and periventricular leukomalacia or hemorrhage 11 (5%). The remaining 32 etiologies were all individually uncommon. Response to treatment is given for individual etiologies. Discussion: Our method of classification allows the reporting of results by individual diseases, disease groups, or categories and is structured and clear. It avoids the use of poorly defined terms such as symptomatic and cryptogenic. It can adapt to new neurologic diseases, such as gene defects, and can be used for comparison of different groups of infants, thereby aiding meta‐analysis.     

MERRF/MELAS overlap syndrome associated with 3243 tRNALeu(UUR) mutation of mitochondrial DNA

We report a case of myoclonus epilepsy associated with ragged-red fibers (MERRF)/mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episode (MELAS) overlap syndrome with hearing loss, external ophthalmoplegia, and myoclonus epilepsy in addition to stroke-like episode and diabetes mellitus. Pathologically, there was degeneration in the dentate nuclei, substantia nigra, red nucleus, and subthalamic nucleus which has been reported as characteristic of MERRF, as well as necrotic lesions of various stages in the cerebral cortex, characteristic of MELAS. The gene study disclosed 3243 mutation in the tRNA^Leu(UUR) gene of mitochondrial DNA. This case is the first neuropathological report of MERRF/MELAS overlap syndrome verified by gene analyses.     

Sporadic MERRF/MELAS overlap syndrome associated with the 3243 tRNALeu(UUR) mutation of mitochondrial DNA

We studied a patient with a mitochondrial encephalomyopathy characterized by the presence of all the cardinal features of both myoclonic epilepsy and ragged-red fibers (MERRF) and mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) syndromes. Muscle biopsy showed ragged-red fibers (RRF). Some RRF were cytochrome c oxidase (COX)-negative, while some others stained positive for COX. Muscle biochemistry revealed defects of complexes I and IV of the respiratory chain. Both muscle and blood mitochondrial DNA from the patient showed the presence of the mutation at nucleotide position 3243 in the tRNA^Leu(UUR) gene and the absence of point mutations related to MERRF syndrome. The proportions of mutant mtDNA were 70% in muscle and 30% in blood. The mutation was absent in blood from all maternal relatives, in hair follicles from the mother, and in muscle from one sister of the proband. Therefore, there was no evidence of maternal inheritance. © 1996 John Wiley & Sons, Inc.