A homozygous mutation in KCTD7 links neuronal ceroid lipofuscinosis to the ubiquitin-proteasome system

Neuronal ceroid lipofuscinosis (NCL) is a genetically heterogeneous group of lysosomal diseases that collectively compose the most common Mendelian form of childhood-onset neurodegeneration. It is estimated that ～8% of individuals diagnosed with NCL by conservative clinical and histopathologic criteria have been ruled out for mutations in the nine known NCL-associated genes, suggesting that additional genes remain unidentified. To further understand the genetic underpinnings of the NCLs, we performed whole-exome sequencing on DNA samples from a Mexican family affected by a molecularly undefined form of NCL characterized by infantile-onset progressive myoclonic epilepsy (PME), vision loss, cognitive and motor regression, premature death, and prominent NCL-type storage material. Using a recessive model to filter the identified variants, we found a single homozygous variant, c.550C>T in KCTD7, that causes a p.Arg184Cys missense change in potassium channel tetramerization domain-containing protein 7 (KCTD7) in the affected individuals. The mutation was predicted to be deleterious and was absent in over 6,000 controls. The identified variant altered the localization pattern of KCTD7 and abrogated interaction with cullin-3, a ubiquitin-ligase component and known KCTD7 interactor. Intriguingly, murine cerebellar cells derived from a juvenile NCL model (CLN3) showed enrichment of endogenous KCTD7. Whereas KCTD7 mutations have previously been linked to PME without lysosomal storage, this study clearly demonstrates that KCTD7 mutations also cause a rare, infantile-onset NCL subtype designated as CLN14.     

Up-regulation of Hyperpolarization-activated Cyclic Nucleotide-gated Channel 3 (HCN3) by Specific Interaction with K+ Channel Tetramerization Domain-containing Protein 3 (KCTD3)

Most ion channels consist of the principal ion-permeating core subunit(s) and accessory proteins that are assembled with the channel core. The biological functions of the latter proteins are diverse and include the regulation of the biophysical properties of the ion channel, its connection to signaling pathways and the control of its cell surface expression. There is recent evidence that native hyperpolarization-activated cyclic nucleotide-gated channel complexes (HCN1–4) also contain accessory subunits, among which TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) has been most extensively studied. Here, we identify KCTD3, a so far uncharacterized member of the potassium channel tetramerization-domain containing (KCTD) protein family as an HCN3-interacting protein. KCTD3 is widely expressed in brain and some non-neuronal tissues and colocalizes with HCN3 in specific regions of the brain including hypothalamus. Within the HCN channel family, KCTD3 specifically binds to HCN3 and leads to a profound up-regulation of cell surface expression and current density of this channel. HCN3 can also functionally interact with TRIP8b; however, we found no evidence for channel complexes containing both TRIP8b and KCTD3. The C terminus of HCN3 is crucially required for functional interaction with KCTD3. Replacement of the cytosolic C terminus of HCN2 by the corresponding domain of HCN3 renders HCN2 sensitive to regulation by KCTD3. The C-terminal-half of KCTD3 is sufficient for binding to HCN3. However, the complete protein including the N-terminal tetramerization domain is needed for HCN3 current up-regulation. Together, our experiments indicate that KCTD3 is an accessory subunit of native HCN3 complexes.     

KCTD10 interacts with proliferating cell nuclear antigen and its down‐regulation could inhibit cell proliferation

A novel gene (GenBank accession No. AF113208) named KCTD10 (potassium channel tetramerisation domain‐containing 10) was cloned from our 5300 EST database of human aorta cDNA library. Computational analysis showed that KCTD10 cDNA is 2,638 bp long, encoding 313 amino acids with a proliferating cell nuclear antigen binding motif, mapped to chromosome 12q24.11 with 7 exons, ubiquitously expressed in all 12 tested normal tissues and 7 of 8 tested tumor cell lines from MTN membranes by Northern blot. Nuclear localization of KCTD10 was observed in A549 cells. Yeast two‐hybrid analysis and immunoprecipitation assay showed that KCTD10 can interact with PCNA. In A549 cells, KCTD10 down‐regulation could inhibit cell proliferation, but its over‐expression could not influence cell proliferation. The results suggest that KCTD10 may be associated with DNA synthesis and cell proliferation. J. Cell. Biochem. 106: 409–413, 2009. © 2009 Wiley‐Liss, Inc.     

双孔钾通道TREK-1 结构、分布、调控因素及其抗癫痫作用的研究进展

：钾离子通道是最大最复杂的离子通道家族,迄今为止在人类基因组中共克隆出了70 余种钾离子通道亚型,其中双孔钾离 子通道是近年来新发现的一类钾离子通道亚家族,它们在结构上与电压依赖性钾通道、钙激活钾通道,内向整流型钾通道等传统 的单孔钾离子通道差异很大。双孔钾离子通道,具有4 个跨膜片段,形成独特的2 个孔道结构域,主要介导背景钾电流。由于其介 导背景钾电流而参与并维持静息膜电位形成等重要生理作用而备受关注。近年来研究最多的双孔钾通道TREK-1 几乎表达于机 体的每一个细胞,可被细胞内酸度、膜牵张、多不饱和脂肪酸、温度、受体偶联第二信使系统调控,调节细胞兴奋性,参与一系列生 理、病理过程,与神经系统疾病如癫痫密切相关,本文就此做一综述。     

双孔钾通道TREK-1结构、分布、调控因素及其抗癫痫作用的研究进展

钾离子通道是最大最复杂的离子通道家族,迄今为止在人类基因组中共克隆出了70余种钾离子通道亚型,其中双孔钾离子通道是近年来新发现的一类钾离子通道亚家族,它们在结构上与电压依赖性钾通道、钙激活钾通道,内向整流型钾通道等传统的单孔钾离子通道差异很大。双孔钾离子通道,具有4个跨膜片段,形成独特的2个孔道结构域,主要介导背景钾电流。由于其介导背景钾电流而参与并维持静息膜电位形成等重要生理作用而备受关注。近年来研究最多的双孔钾通道TREK-1几乎表达于机体的每一个细胞,可被细胞内酸度、膜牵张、多不饱和脂肪酸、温度、受体偶联第二信使系统调控,调节细胞兴奋性,参与一系列生理、病理过程,与神经系统疾病如癫痫密切相关,本文就此做一综述。     

A Missense Mutation in KCTD17 Causes Autosomal Dominant Myoclonus-Dystonia

Myoclonus-dystonia (M-D) is a rare movement disorder characterized by a combination of non-epileptic myoclonic jerks and dystonia. SGCE mutations represent a major cause for familial M-D being responsible for 30%–50% of cases. After excluding SGCE mutations, we identified through a combination of linkage analysis and whole-exome sequencing KCTD17 c.434 G>A p.(Arg145His) as the only segregating variant in a dominant British pedigree with seven subjects affected by M-D. A subsequent screening in a cohort of M-D cases without mutations in SGCE revealed the same KCTD17 variant in a German family. The clinical presentation of the KCTD17-mutated cases was distinct from the phenotype usually observed in M-D due to SGCE mutations. All cases initially presented with mild myoclonus affecting the upper limbs. Dystonia showed a progressive course, with increasing severity of symptoms and spreading from the cranio-cervical region to other sites. KCTD17 is abundantly expressed in all brain regions with the highest expression in the putamen. Weighted gene co-expression network analysis, based on mRNA expression profile of brain samples from neuropathologically healthy individuals, showed that KCTD17 is part of a putamen gene network, which is significantly enriched for dystonia genes. Functional annotation of the network showed an over-representation of genes involved in post-synaptic dopaminergic transmission. Functional studies in mutation bearing fibroblasts demonstrated abnormalities in endoplasmic reticulum-dependent calcium signaling. In conclusion, we demonstrate that the KCTD17 c.434 G>A p.(Arg145His) mutation causes autosomal dominant M-D. Further functional studies are warranted to further characterize the nature of KCTD17 contribution to the molecular pathogenesis of M-D.     

PrPC interacts with potassium channel tetramerization domain containing 1 (KCTD1) protein through the PrP(51-136) region containing octapeptide repeats

To identify molecular interaction partners of the cellular prion protein (PrP(C)), we applied a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the potassium channel tetramerization domain containing 1 (KCTD1) as a PrP(C) interacting protein. Deletion mapping showed that PrP(C) specifically binds KCTD1 through the unstructured PrP(51-136) region. We further confirmed the interaction between PrP(C) and KCDT1 protein by co-immunoprecipitation in vivo and by a biosensor assay in vitro. Interestingly, the binding of an insertion mutant PrP(8OR) to KCTD1 is higher than that of wild-type PrP(C), suggesting an important role for an unstructured region harboring octapeptide repeats in the KCTD1-PrP(C) interaction. Our results identify a novel PrP(C)-interacting protein and suggest a new approach to investigating the unidentified physiological cellular function of PrP(C).     

The KCTD family of proteins: structure,function, disease relevance

The family of potassium channel tetramerizationdomain (KCTD) proteins consists of 26 members with mostly unknown functions. The name of the protein family is due to the sequence similarity between the conserved N-terminal region of KCTD proteins and the tetramerization domain in some voltage-gated potassium channels. Dozens of publications suggest that KCTD proteins have roles in various biological processes and diseases. In this review, we summarize the character of Bric-a-brack,Tram-track, Broad complex(BTB) of KCTD proteins, their roles in the ubiquitination pathway, and the roles of KCTD mutants in diseases. Furthermore, we review potential downstream signaling pathways and discuss future studies that should be performed.     

Expression, purification, and secondary structure characterization of recombinant KCTD1

Potassium channel tetramerization domain containing 1 (KCTD1) contains a BTB domain, which can facilitate protein-protein interactions that may be involved in the regulation of signaling pathways. Here we describe an expression and purification system that can provide a significant amount of recombinant KCTD1 from Escherichia coli. The cDNA encoding human KCTD1 was amplified and cloned into the expression vector pET-30a(+). The recombinant protein was expressed in E. coli BL21(DE3) cells and subsequently purified using affinity chromatography. To confirm that KCTD1 was correctly expressed and folded, the molecular weight and conformation were analyzed using mass spectroscopy, Western blot, and circular dichroism. Optimizing KCTD1 expression and investigating its secondary structure will provide valuable information for future structural and functional studies of KCTD1 and KCTD family proteins.     

A novel PDIP1-related protein, KCTD10, that interacts with proliferating cell nuclear antigen and DNA polymerase delta

Rat potassium channel tetramerisation domain-containing 10 (KCTD10) gene was cloned and identified as a novel member of polymerase delta-interacting protein 1 (PDIP1) gene family. Rat KCTD10 is highly expressed in lung and moderately expressed in heart and testis. KCTD10 shares significant similarity in amino acid sequence to PDIP1 and can interact with the small subunit of DNA polymerase delta and PCNA as PDIP1 does. Like PDIP1, the expression of KCTD10 gene can be induced by TNF-alpha in NIH3T3 cells.     

A New TASK for Dipeptidyl Peptidase-like Protein 6

Dipeptidyl Peptidase-like Protein 6 (DPP6) is widely expressed in the brain where it co-assembles with Kv4 channels and KChIP auxiliary subunits to regulate the amplitude and functional properties of the somatodendritic A-current, I_SA. Here we show that in cerebellar granule (CG) cells DPP6 also regulates resting membrane potential and input resistance by increasing the amplitude of the I_K(SO) resting membrane current. Pharmacological analysis shows that DPP6 acts through the control of a channel with properties matching the K_2P channel TASK-3. Heterologous expression and co-immunoprecipitation shows that DPP6 co-expression with TASK-3 results in the formation of a protein complex that enhances resting membrane potassium conductance. The co-regulation of resting and voltage-gated channels by DPP6 produces coordinate shifts in resting membrane potential and A-current gating that optimize the sensitivity of I_SA inactivation gating to subthreshold fluctuations in resting membrane potential.     

Regulation of recombinant human brain tandem P domain K+ channels by hypoxia: a role for O2 in the control of neuronal excitability?

The tandem P domain potassium channels, TREK1 and TASK1, are expressed throughout the brain but expression patterns do not significantly overlap. Since normal pO₂ in central nervous tissue is as low as 20 mmHg and can decrease even further in ischemic disease, it is important that the behaviour of human brain ion channels is studied under conditions of acute and chronic hypoxia. This is especially true for brain-expressed tandem P-domain channels principally because they are important contributors to neuronal resting membrane potential and excitability. Here, we discuss some recent data derived from two recombinant tandem P-domain potassium channels, hTREK1 and hTASK1. Hypoxia represents a potent inhibitory influence on both channel types and occludes the activation by arachidonic acid, intracellular acidosis and membrane deformation of TREK1. This casts doubt on the idea that TREK1 activation during brain ischemia might facilitate neuroprotection via hyperpolarising neurons in which it is expressed. Interestingly, hypoxia is unable to regulate alkalotic inhibition of TREK1 suggesting that this channel may be more intimately involved in control of excitability during physiological or pathological alkalosis.     

Genetic basis in epilepsies caused by malformations of cortical development and in those with structurally normal brain

Epilepsy is the most common neurological disorder affecting young people. The etiologies are multiple and most cases are sporadic. However, some rare families with Mendelian inheritance have provided evidence of genes’ important role in epilepsy. Two important but apparently different groups of disorders have been extensively studied: epilepsies associated with malformations of cortical development (MCDs) and epilepsies associated with a structurally normal brain (or with minimal abnormalities only). This review is focused on clinical and molecular aspects of focal cortical dysplasia, polymicrogyria, periventricular nodular heterotopia, subcortical band heterotopia, lissencephaly and schizencephaly as examples of MCDs. Juvenile myoclonic epilepsy, childhood absence epilepsy, some familial forms of focal epilepsy and epilepsies associated with febrile seizures are discussed as examples of epileptic conditions in (apparently) structurally normal brains.