Novel Mechanisms of Trafficking Defect Caused by KCNQ1 Mutations Found in Long QT Syndrome

Long QT syndrome (LQTS) is a hereditary arrhythmia caused by mutations in genes for cardiac ion channels, including a potassium channel, KvLQT1. Inheritance of LQTS is usually autosomal-dominant, but autosomal-recessive inheritance can be observed in patients with LQTS accompanied by hearing loss. In this study, we investigated the functional alterations caused by KCNQ1 mutations, a deletion (delV595) and a frameshift (P631fs/19), which were identified in compound heterozygous state in two patients with autosomal-recessive LQTS not accompanied by hearing loss. Functional analyses showed that both mutations impaired cell surface expression due to trafficking defects. The mutations severely affected outward potassium currents without apparent dominant negative effects. It was found that delV595 impaired subunit binding, whereas P631fs/19 was retained in endoplasmic reticulum due to the newly added 19-amino acid sequence containing two retention motifs (R⁶³³GR and R⁶⁴⁶LR). This is the first report of novel mechanisms for trafficking abnormality of cardiac ion channels, providing us new insights into the molecular mechanisms of LQTS.     

Mutations in the K+-Channel KcsA Toward Kir Channels Alter Salt-Induced Clusterization and Blockade by Quaternary Alkylammonium Ions

Protein aggregation is a result of malfunction in protein folding, assembly, and transport, caused by protein mutation and/or changes in the cell environment, thus triggering many human diseases. We have shown that bacterial K⁺-channel KcsA, which acts as a representative model for ion channels, forms salt-induced large conductive complexes in a particular environment. In the present study, we investigated the effects of point mutations in the selectivity filter of KcsA on intrinsic stability, aggregation, and channel blocking behavior. First, we found that a low sodium chloride concentration in potassium-containing media induced fast transfer of single channels to a planar lipid bilayer. Second, increasing the sodium chloride concentration drastically increased the total channel current, indicating enhanced vesicle fusion and transfer of multiple channels to a planar lipid bilayer. However, such complexes exhibited high conductance as well as higher open probability compared to the unmodified KcsA behavior shown previously. Interestingly, the affinity of aggregated complexes for larger symmetric quaternary alkylammonium ions (QAs) was found to be much higher than that for tetraethylammonium, a classical blocker of the K⁺ channel. Based on these findings, we propose that mutant channel complexes exhibit larger pore dimensions, thus resembling more the topological properties of voltage-gated and inwardly rectifying K⁺ channels.     

Focus on TRP channels in cystic fibrosis

The Transient Receptor Potential (TRP) protein superfamily is a group of cation channels expressed in various cell types and involved in respiratory diseases such as cystic fibrosis (CF), the genetic disease caused by CF Transmembrane conductance Regulator (CFTR) mutations. In human airway epithelial cells, there is growing evidence for a functional link between CFTR and TRP channels. TRP channels contribute to transmitting extracellular signals into the cells and, in an indirect manner, to CFTR activity via a Ca²⁺ rise signaling. Indeed, mutated CFTR-epithelial cells are characterized by an increased Ca²⁺ influx and, on the opposite, by a decreased of magnesium influx, both being mediated by TRP channels. This increasing cellular Ca²⁺ triggers the activation of calcium-activated chloride channels (CaCC) or CFTR itself, via adenylyl cyclase, PKA and tyrosine kinases activation, but also leads to an exaltation of the inflammatory response. Another shortcoming in mutated CFTR-epithelial cells is a [Mg²⁺]_i decrease, associated with impaired TRPM7 functioning. This deregulation has to be taken into consideration in CF physiopathology, as Mg²⁺ is required for ATP hydrolysis and CFTR activity. The modulation of druggable TRP channels could supplement CF therapy either an anti-inflammatory drug or for CFTR potentiation, according to the balance between exacerbation and respite phases. The present paper focus on TRPA1, TRPC6, TRPM7, TRPV2, TRPV4, TRPV6 and ORAI 1, the proteins identified, for now, as dysfunctional channels, in CF cells.     

The Molecular Biology of Genetic-Based Epilepsies

Epilepsy is one of the most common neurological disorders characterized by abnormal electrical activity in the central nervous system. The clinical features of this disorder are recurrent seizures, difference in age onset, type, and frequency, leading to motor, sensory, cognitive, psychic, or autonomic disturbances. Since the discovery of the first monogenic gene mutation in 1995, it is proposed that genetic factor plays an important role in the mechanism of epilepsy. Genes discovered in idiopathic epilepsies encode for ion channel or neurotransmitter receptor proteins, whereas syndromes with epilepsy as a main feature are caused by genes that are involved in functions such as cortical development, mitochondrial function, and cell metabolism. The identification of these monogenic epilepsy-causing genes provides new insight into the pathogenesis of epilepsies. Although most of the identified gene mutations present a monogenic inheritance, most of idiopathic epilepsies are complex genetic diseases exhibiting a polygenic or oligogenic inheritance. This article reviews recent genetic and molecular progresses in exploring the pathogenesis of epilepsy, with special emphasis on monogenic epilepsy-causing genes, including voltage-gated channels (Na⁺, K⁺, Ca²⁺, Cl^?, and HCN), ligand-gated channels (nicotinic acetylcholine and GABA_A receptors), non-ion channel genes as well as the mitochondrial DNA genes. These progresses have improved our understanding of the complex neurological disorder.     

The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl^?) channels. The EAATs couple the transport of glutamate to the co-transport of three Na⁺ ions and one H⁺ ion into the cell, and the counter-transport of one K⁺ ion out of the cell. The EAAT Cl^? channel is activated by the binding of glutamate and Na⁺, but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, Glt_Ph, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl^? permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl^? permeation and the mechanisms that underlie their split personality.     

Erbliche Ionenkanalerkrankungen der Netzhaut

Retinal channelopathies are clinically and genetically heterogeneous, and are caused by mutations in genes for a variety of ion channels such as cyclic nucleotide-gated channels, voltage-gated potassium and calcium channels, an inwardly rectifying potassium channel, a calcium-dependent chloride channel and the TRPM1 channel. This broad spectrum of disease-associated ion channels is also reflected in the diversity of pathophysiological consequences. Mutations in retinal ion channels may affect phototransduction, thereby impairing the detection of light or interfere with the transmission of the stimulus from the photoreceptor to second-order neurons. Ion channels located in the retinal pigment epithelium, which supports normal retina function, can also be affected in some diseases.     

Multiple Discrete Transitions Underlie Voltage-Dependent Activation in CLC Cl/H Antiporters

Most mammalian chloride channels and transporters in the CLC family display pronounced voltage-dependent gating. Surprisingly, despite the complex nature of the gating process and the large contribution to it by the transport substrates, experimental investigations of the fast gating process usually produce canonical Boltzmann activation curves that correspond to a simple two-state activation. By using nonlinear capacitance measurements of two mutations in the ClC-5 transporter, here we are able to discriminate and visualize discrete transitions along the voltage-dependent activation pathway. The strong and specific dependence of these transitions on internal and external [Cl⁻] suggest that CLC gating involves voltage-dependent conformational changes as well as coordinated movement of transported substrates.     

Determinants of cation transport selectivity: Equilibrium binding and transport kinetics

ConclusionThe equilibrium ion-binding properties of ion channels and transporters can be difficult to discern from crystal structures alone, as proteins often adopt different lowest energy states depending on the ions bound. In cases where transport is slow, their inherent ion-binding preferences can be used to infer their transport preferences. However, in cases where transport is fast, the transport selectivity can hide their equilibrium preferences by accentuating the kinetics of ions hopping through a channel over its inherent ion-binding preferences. Thus, depending on the arrangement of ion-binding sites in a channel’s selectivity filter, one can achieve either selective or nonselective ion transport.The equilibrium K⁺ selectivity of some nonselective channels suggests a potential mechanism whereby they could evolve into a fast K⁺-selective channel. K⁺ channels and nonselective channels like CNG and HCN are related to one another in both sequence and structure, suggesting an evolutionary link between them. Swap experiments show that only a few mutations separate a nonselective channel from a K⁺-selective channel. One might imagine an evolutionary path between these channels in which the equilibrium preference for a K⁺ ion in a nonselective channel evolves into a K⁺-selective channel through these few mutations to create the selective ion queue. Alternatively, a slow single-ion channel with an equilibrium and transport preference for K⁺ ions could be transformed into a fast multi-ion channel through mutations that create a queue of K⁺-selective ion-binding sites, as is seen in most K⁺ channels studied to date.In the case of multi-ion selectivity filters, such as those found in K⁺ channels, the selectivity filter can be viewed as the active site that interacts with different queues of ions and water molecules. At least three properties emerge from multi-ion queues: (1) high conductance by reducing the affinity of multiple bound ions versus single ions; (2) high selectivity by allowing disfavored ions time to dissociate back into solution; and, consequently, (3) robust selectivity in an environment where ion concentrations can change. For transporters and carriers, the equilibrium preference and slow transport naturally create robust selectivity. In all these cases, equilibrium-based ion selectivity is achieved by slowing transport enough so that the disfavored ion is able to dissociate back into solution before transport takes place.     

Genome-Wide Analysis of Potassium Transport-Related Genes in Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and Their Role in Abiotic Stress Responses

Potassium is the most abundant inorganic cation that constitutes up to 10% of the total plant dry weight and plays a prominent role in plant growth and development. Plants exhibit a complex but highly organized system of channels and transporters, which are involved in absorption and distribution of K⁺ from soil to different parts of plants. In this study, we explored the K⁺ transport system in chickpea genome and identified 36 genes encoding potassium channels and transporters. The identified genes were further classified on the basis of their domain structure and conserved motifs. It includes K⁺ transporters (23 genes: 2 HKTs, 6 KEAs, and 15 KUP/HAK/KTs) and K⁺ channels (13 genes: 8 Shakers and 5 TPKs). Chromosomal localization of these genes demonstrated that various K⁺ transporters and channels are randomly distributed across all the eight chromosomes. Comparative phylogenetic analysis of K⁺ transport system genes from Arabidopsis thaliana, Glycine max, Medicago truncatula, and Oryza sativa revealed their strong conservation in different plant species. Similarly, gene structure analysis displayed conservation of family-specific intron/exon organization in the K⁺ transport system genes. Evolutionary analysis of these genes suggested the segmental duplication as principal route of expansion for this family in chickpea. Several abiotic stress-related cis-regulatory elements were also identified in promoter regions suggesting their role in abiotic stress tolerance. Expression analysis of selected genes under drought, heat, osmotic, and salt stress demonstrated their differential expression in response to these stresses. This signifies the importance of these genes in the modulation of stress response in chickpea. Present study provides the first insight into K⁺ transport system in chickpea and can serve as a basis for their functional analysis.     

成骨不全及其分子机制

成骨不全作为罕见性遗传性结缔组织疾病,具有临床异质性与遗传异质性,迄今已经分为15个亚型.有常染色体显性遗传与常染色体隐性遗传两种遗传方式.常染色体显性遗传以Ⅰ型胶原蛋白结构基因COL1A1、COL1A2突变为主.非Ⅰ型胶原蛋白突变的常染色体隐性遗传的成骨不全患者数量少,但致病基因种类多,涉及到胶原合成后异常修饰,胶原蛋白分子伴侣及羧基端前肽剪切酶缺陷、成骨细胞与破骨细胞分化及转录因子异常、钙离子通道与Wnt信号通路分子等诸多方面.致病基因及其机制的研究,对于成骨不全的基因确诊及个体化药物治疗意义重大.     

CFTR structure and function: is there a role in the kidney?

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective protein processing that leads to changes in function and regulation of this chloride channel. Despite of the expression of CFTR in the kidney, patients with CF do not present major renal dysfunction, but it is known that both the urinary excretion of proteins and renal capacity to concentrate and dilute urine are altered in these patients. CFTR mRNA is expressed in all nephron segments of rat and human, and this abundance is more prominent in renal cortex and outer medulla renal areas. CFTR protein was detected in apical surface of both proximal and distal tubules of rat kidney but not in the outer medullary collecting ducts. Studies have demonstrated that CFTR does not only transport Cl⁻ but also ATP. ATP transport by CFTR could be involved in the control of other ion transporters such as Na⁺ (ENaC) and K⁺ (renal outer medullary potassium) channels, especially in TAL and CCD. In the kidney, CFTR also might be involved in the endocytosis of low-molecular-weight proteins by proximal tubules. This review is focused on the CFTR function and structure, its role in the renal physiology, and its modulation by hormones involved in the control of extracellular fluid volume.     

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness¹ and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them². This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia³. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males⁴. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.     

Presynaptic Glycine Receptors Influence Plasma Membrane Potential and Glutamate Release

Glycine is a classical inhibitory neurotransmitter however presynaptic glycine receptors have rather depolarizing action. Reasons for latter phenomenon are unknown. In the present paper we have investigated how glycine influences cytosolic chloride level monitored by fluorescent dye SPQ, membrane potential monitored by fluorescent dye DiSC3(5) and [¹⁴C]-glutamate release in synaptosomes. We estimated that cytosolic chloride concentration in synaptosomes was about 52 ± 1 mM. Glycine (1 mM) induced chloride efflux and caused slow plasma membrane depolarization. Chloride efflux was almost completely blocked by 100 μM strychnine whilst glycine-induced depolarization was only partially. We also showed that 1 mM glycine induced [¹⁴C]-glutamate release via a strychnine-insensitive pathway. Hence we have concluded that glycine was able to induce two independent effects in synaptosomes: (1) Chloride efflux with following depolarization. This efflux was sensitive to strychnine and thereby is probably conducted through glycine-gated ion channels. (2) Glutamate release seems to be mediated by glycine transporters.     

Calcium channels and channelopathies of the central nervous system

Several inherited human neurological disorders can be caused by mutations in genes encoding Ca²⁺ channel subunits. This review deals with known human and mouse calcium channelopathies of the central nervous system (CNS). The human diseases comprise: 1) a recessive retinal disorder, X-linked congenital stationary night blindness, associated with mutations in the CACNA1F gene, encoding α₁1.4 subunits of L-type channels; and 2) a group of rare allelic autosomal dominant human neurological disorders including familial hemiplegic migraine, episodic ataxia type 2, and spinocerebellar ataxia type 6, all associated with mutations in the CACNA1A gene, encoding α₁2.1 subunits of P/Q-type calcium channels. Mutations at the mouse orthologue of the CACNA1A gene cause a group of recessive neurological disorders, including the tottering, leaner, and rocker phenotypes with ataxia and absence epilepsy, and the rolling Nagoya phenotype with ataxia without seizures. Two other spontaneous mouse mutants with ataxia and absence epilepsy, lethargic and stargazer, have mutations in genes encoding a calcium channel auxiliary β subunit and a putative calcium channel auxiliary γ subunit. For each channelopathy, the review describes disease phenotype, channel genotype, and known functional consequences of the pathological mutations; in some cases, it also describes working hypothesis and/or speculations addressing the challenging question of how the alterations in channel function lead to selective cellular dysfunction and disease.     

TRP channels in disease

“Transient receptor potential” cation channels (TRP channels) play a unique role as cell sensors, are involved in a plethora of Ca²⁺-mediated cell functions, and play a role as “gate-keepers” in many homeostatic processes such as Ca²⁺ and Mg²⁺ reabsorption. The variety of functions to which TRP channels contribute and the polymodal character of their activation predict that failures in correct channel gating or permeation will likely contribute to complex pathophysiological mechanisms. Dysfunctions of TRPs cause human diseases but are also involved in a complex manner to contribute and determine the progress of several diseases. Contributions to this special issue discuss channelopathias for which mutations in TRP channels that induce “loss-“ or “gain-of-function” of the channel and can be considered “disease-causing” have been identified. The role of TRPs will be further elucidated in complex diseases of the intestinal, renal, urogenital, respiratory, and cardiovascular systems. Finally, the role of TRPs will be discussed in neuronal diseases and neurodegenerative disorders.     

Mutational Consequences of Aberrant Ion Channels in Neurological Disorders

Neurological channelopathies are attributed to aberrant ion channels affecting CNS, PNS, cardiac, and skeletal muscles. To maintain the homeostasis of excitable tissues, functional ion channels are necessary to rely electrical signals, whereas any malfunctioning serves as an intrinsic factor to develop neurological channelopathies. Molecular basis of these disease is studied based on genetic and biophysical approaches, e.g., loci positional cloning, whereas pathogenesis and bio-behavioral analysis revealed the dependency on genetic mutations and inter-current triggering factors. Although electrophysiological studies revealed the possible mechanisms of diseases, analytical study of ion channels remained unsettled and therefore underlying mechanism in channelopathies is necessary for better clinical application. Herein, we demonstrated (i) structural and functional role of various ion channels (Na⁺, K⁺, Ca²⁺,Cl^?), (ii) pathophysiology involved in the onset of their associated channelopathies, and (iii) comparative sequence and phylogenetic analysis of diversified sodium, potassium, calcium, and chloride ion channel subtypes.     

Nonsynaptic glycine release is involved in the early KCC2 expression

The cation‐chloride co‐transporters are important regulators of the cellular Cl^‐ homeostasis. Among them the Na⁺‐K⁺?2Cl^? co‐transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K⁺‐Cl^? co‐transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride‐mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na⁺ action potentials but was mimicked by a Ca²⁺‐dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume‐sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR‐activation is a necessary developmental process for the expression of functional spinal motor networks. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 764–779, 2016     

Ausgewählte Ionenkanalerkrankungen des peripheren Nervensystems

Ion channel disorders affecting the peripheral nervous system can manifest as altered pain perception or neuropathy. Several of these diseases are caused by mutations of the SCN9A gene, which encodes the voltage-gated sodium channel Na_v1.7. Whereas SCN9A gain of function mutations cause inherited erythromelalgia and other syndromes that are characterized by paroxysmal episodes of severe pain, loss of SCN9A function underlies congenital inability to experience pain. Together with the discovery of TRPV4 (“transient receptor potential channel 4”) mutations in scapuloperoneal spinal muscular atrophy and Charcot–Marie–Tooth disease type 2C, these data illustrate the central function of ion channels in diseases affecting not only the central but also the peripheral nervous system.