Ion Channel Expression Levels Research Articles

Homeostatic regulation of neuronal function: importance of degeneracy and pleiotropy.

Neurons maintain their average firing rate and other properties within narrow bounds despite changing conditions. This homeostatic regulation is achieved using negative feedback to adjust ion channel expression levels. To understand how homeostatic regulation of excitability normally works and how it goes awry, one must consider the various ion channels involved as well as the other regulated properties impacted by adjusting those channels when regulating excitability. This raises issues of degeneracy and pleiotropy. Degeneracy refers to disparate solutions conveying equivalent function (e.g., different channel combinations yielding equivalent excitability). This many-to-one mapping contrasts the one-to-many mapping described by pleiotropy (e.g., one channel affecting multiple properties). Degeneracy facilitates homeostatic regulation by enabling a disturbance to be offset by compensatory changes in any one of several different channels or combinations thereof. Pleiotropy complicates homeostatic regulation because compensatory changes intended to regulate one property may inadvertently disrupt other properties. Co-regulating multiple properties by adjusting pleiotropic channels requires greater degeneracy than regulating one property in isolation and, by extension, can fail for additional reasons such as solutions for each property being incompatible with one another. Problems also arise if a perturbation is too strong and/or negative feedback is too weak, or because the set point is disturbed. Delineating feedback loops and their interactions provides valuable insight into how homeostatic regulation might fail. Insofar as different failure modes require distinct interventions to restore homeostasis, deeper understanding of homeostatic regulation and its pathological disruption may reveal more effective treatments for chronic neurological disorders like neuropathic pain and epilepsy.

High-level expression of voltage-gated sodium channels in thymidine-arrested human embryonic kidney cells.

The benefit of using heterologous mammalian cell lines such as human embryonic kidney (HEK293) cells for expressing voltage-gated ion channels includes efficient translation and processing of such large membrane proteins. Still, the biophysical assessment of these channels is hindered by their low expression and reduced localization on the surface of a cell. Having a higher expression level of ion channels is crucial for investigating mutations that cause a decrease in the current amplitude as well as for measuring tiny currents such as gating currents and gating pore currents that are in the range of 0.1 to 1% of the peak current. We aimed at improving the efficiency of ion channel expression and enhancing their localization in cell membranes by modifying the protocol of cell cycle arrest at the G1/S boundary using thymidine to direct the cells into increasing the expression machinery during viral transduction of mammalian voltage-gated sodium channels. The combination of cell arrest at the G1/S boundary and mammalian baculovirus system (BacMam) resulted in a 5-fold increase in the current density of different mammalian voltage-gated sodium channels in HEK293 cells compared to the current literature. By applying this modified protocol for expression, we succeeded to measure a pathogenic gating pore current in HEK293 cells of ∼0.5% of the central pore current induced by an autism-related mutation in the R2 gating charge in Domain II (R853Q) of Nav1.2, which is blocked in a voltage-dependent manner when the voltage sensors activate. These results show that our protocol is a universal one that can be applied on different voltage-gated ion channels and possibly other membrane proteins. This ultimately will pave the way to decipher the structure and function of ion channels and their association with ion channelopathies and other diseases.

Observation of topological action potentials in engineered tissues

The interface between two tissues can have very different bioelectrical properties compared to either tissue on its own. Here we show that an interface between non-excitable tissues can be electrically excitable because of an interaction between the currents passing through the gap junctions—electrically resistive intercellular connections—and the non-linear current–voltage dependence in the ion channels on either side of the interface. Our theory shows that this topologically robust excitability occurs over a far larger range of ion channel expression levels than can support excitability in the bulk. The corresponding interfacial action potentials can cause local elevations in calcium concentration, possibly providing a bioelectrical mechanism for interface sensing. The observed topological action potentials point to the possibility of other types of topological effect in electrophysiology and at other diffusively coupled interfaces. Interfaces between non-excitable tissues can be electrically excitable, suggesting a possible bioelectrical mechanism for interface sensing.

Effect of sotagliflozin on ventricular arrhythmias in mice with myocardial infraction

BackgroundVentricular arrhythmias (VAs) after myocardial infarction (MI) are an important cause of death in patients. Inhibition of sodium-glucose ligand transporter type 2 (SGLT-2) has been shown to reduce cardiovascular mortality in myocardial infarction independent of glycemic sham and prevent ventricular arrhythmias, and it is unclear whether these effects can be further enhanced by additional SGLT-1 inhibition. We investigated the effects of chronic treatment with the dual SGLT-1 and 2 inhibitor sotagliflozin (Sto) on VAs and their underlying mechanisms after MI. MethodsMI was induced by ligation of the left anterior coronary artery in 6–8 weeks-old male mice, which were fed Sto at a dose of 30 mg/kg/day for 4 weeks after surgery. The effects were assessed by echocardiography, histopathology, Langendorff Heart Perfusion System, ambulatory electrocardiography, and Western blot. In vitro experiments were performed to assess the effect of Sto on cardiomyocytes subjected to hypoxic. ResultsCompared to the MI group, Sto treatment significantly improved cardiac function and reduced infarct size and fibrosis levels. Sto also decreased action potential duration (APD) and APD alternation thresholds. Ambulatory ECG and BURST stimulation showed that Sto reduced the incidence of VAs. In addition, Sto significantly improved the expression levels of ion channels and CX43 and ameliorated the abnormal expression of Ca2+ handling proteins after MI. Mechanistically, in vivo and in vitro experiments confirmed that TLR4/CaMKII activation was enhanced after MI, while Sto significantly inhibited the activation of the TLR4/CaMKII signaling pathway. ConclusionSto improved LV remodeling and reduced the incidence of VAs after MI, which was regulated by modulating the TLR4/CaMKII signaling pathway.

In Silico Analysis of Ion Channels and Their Correlation with Epithelial to Mesenchymal Transition in Breast Cancer.

Simple SummaryBreast cancer involves changes in the healthy cells of the breast resulting in rapid and abnormal division of cells that later spread to other parts of the body through the process of metastasis, which involves epithelial mesenchymal transition (EMT). Ion channels play a significant role in the switch from epithelial to mesenchymal transition through their contributions to cellular motility, cell volume regulation and cell cycle progression. Comprehensive computational analyses were performed to understand the role of ion channels in tumor/metastatic samples of breast cancer and their correlation with EMT.Uncontrolled growth of breast cells due to altered gene expression is a key feature of breast cancer. Alterations in the expression of ion channels lead to variations in cellular activities, thus contributing to attributes of cancer hallmarks. Changes in the expression levels of ion channels were observed as a consequence of EMT. Additionally, ion channels were reported in the activation of EMT and maintenance of a mesenchymal phenotype. Here, to identify altered ion channels in breast cancer patients, differential gene expression and weighted gene co-expression network analyses were performed using transcriptomic data. Protein–protein interactions network analysis was carried out to determine the ion channels interacting with hub EMT-related genes in breast cancer. Thirty-two ion channels were found interacting with twenty-six hub EMT-related genes. The identified ion channels were further correlated with EMT scores, indicating mesenchymal phenotype. Further, the pathway map was generated to represent a snapshot of deregulated cellular processes by altered ion channels and EMT-related genes. Kaplan–Meier five-year survival analysis and Cox regressions indicated the expression of CACNA1B, ANO6, TRPV3, VDAC1 and VDAC2 to be potentially associated with poor survival. Deregulated ion channels correlate with EMT-related genes and have a crucial role in breast cancer-associated tumorigenesis. Most likely, they are potential candidates for the determination of prognosis in patients with breast cancer.

Contractility assessment using hiPSC-CMs with alignment

Evaluation of cardiomyocyte contraction using hiPSC-CMs holds great promise to predict drug-induced heart failures in vitro. However, it has been difficult to detect the positive inotropic effects of drug using hiPSC-CMs. Due to the immature property, hiPSC-CMs do not show alignment. In this study, we evaluated whether hiPSC-CM with alignment can detect the drug-induced positive inotropic effects. We used hiPSC-CM (iCell Cardiomyocyte, FCDI), which were cultured using commercially available plates for alignment. After long-term culture, we found that the aligned culture improved the basal contraction velocity, which were measured by motion vector analysis (SI8000, Sony). To understand the molecular mechanisms of the contraction improvement, we performed next-gene sequencing analysis. Gene expression levels of ion channels and metabolism-related factors were up-regulated by alignment. In addition, we observed that isoproterenol enhanced contraction velocity in hiPSC-CMs with alignment in a dose-dependent manner. These results suggests that the aligned culture of hiPSC-CMs facilitates the maturation and the responses to isoproterenol.

Co-expression of calcium and hERG potassium channels reduces the incidence of proarrhythmic events.

Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. While there is considerable variability in cardiac ion channel expression levels between individuals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression datasets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance. Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, ICaL) and KCNH2 (rapid delayed rectifier K+ current, IKr), which was also observed in human adult heart tissue samples. In silico modelling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for proarrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human-induced pluripotent stem cell-derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of IKr. Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C-KCNH2 co-expression increases the robustness of cardiac electrical activity.

Abstract 247: Identification of the GABAA receptor in melanoma brain metastases patient tumors and demonstration that it is a viable drug target using benzodiazepine-derivatives

Abstract Primary cutaneous melanoma is often successfully treated in early stages in patients with regional disease. While distant metastatic disease, including brain metastasis, that develops in most stage IV melanoma patients, carries a particularly poor prognosis with median overall survival of only 4-5 months. Standard-of-care treatment of brain metastases includes surgery, radiation, and chemotherapy. The fact that melanoma is a radio-resistant cancer, significantly limits treatment options for melanoma that has metastasized to the brain. There is clearly a significant demand for new therapeutic approaches. We have conducted a whole transcriptomic analysis of melanoma brain metastases to provide insight into molecular changes that may contribute to metastasis as well as to identify potential therapeutic targets in the metastasized cancer. Total RNA was extracted from 29 formalin-fixed paraffin-embedded (FFPE) melanoma brain metastatic samples, libraries constructed and enriched for transcript fragments with coding regions. Libraries were subjected to Transcriptome Capture (TCap) targeting 21,415 genes, which represents more than 98% of the total RefSeq exome. Sequencing was performed on the Illumina HiSeq platform. Gene expression analysis of melanoma brain metastatic samples reveals high expression levels of ion channels, including subunits of the ligand-gated neurotransmitter GABAA receptors. We will present differential expression analysis between recent melanoma transcriptomic studies [1, 2] and melanoma brain metastases samples. More than 20% of FDA approved drugs target ion channels. We report that repurposing of one such class of drugs targeting GABAA receptors can impair melanoma cell viability in vitro and reduce tumor volume in vivo. GABAA receptors can serve as a potential therapeutic target for treatment brain metastasis.

Fluvastatin inhibits Rab5-mediated IKs internalization caused by chronic Ca2+-dependent PKC activation

Statins, in addition to their cholesterol lowering effects, can prevent isoprenylation of Rab GTPase proteins, a key protein family for the regulation of protein trafficking. Rab-GTPases have been shown to be involved in the control of membrane expression level of ion channels, including one of the major cardiac repolarizing channels, IKs. Decreased IKs function has been observed in a number of disease states and associated with increased propensity for arrhythmias, but the mechanism underlying IKs decrease remains elusive. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in variety of human diseases and have been suggested to acutely regulate IKs function. We hypothesize that chronic cPKC stimulation leads to Rab-mediated decrease in IKs membrane expression, and that can be prevented by statins. In this study we show that chronic cPKC stimulation caused a dramatic Rab5 GTPase-dependent decrease in plasma membrane localization of the IKs pore forming subunit KCNQ1, reducing IKs function. Our data indicates fluvastatin inhibition of Rab5 restores channel localization and function after cPKC-mediated channel internalization. Our results indicate a novel statin anti-arrhythmic effect that would be expected to inhibit pathological electrical remodeling in a number of disease states associated with high cPKC activation. Because Rab-GTPases are important regulators of membrane trafficking they may underlie other statin pleiotropic effects.

Neurotransmitter identity and electrophysiological phenotype are genetically coupled in midbrain dopaminergic neurons

Most neuronal types have a well-identified electrical phenotype. It is now admitted that a same phenotype can be produced using multiple biophysical solutions defined by ion channel expression levels. This argues that systems-level approaches are necessary to understand electrical phenotype genesis and stability. Midbrain dopaminergic (DA) neurons, although quite heterogeneous, exhibit a characteristic electrical phenotype. However, the quantitative genetic principles underlying this conserved phenotype remain unknown. Here we investigated the quantitative relationships between ion channels’ gene expression levels in midbrain DA neurons using single-cell microfluidic qPCR. Using multivariate mutual information analysis to decipher high-dimensional statistical dependences, we unravel co-varying gene modules that link neurotransmitter identity and electrical phenotype. We also identify new segregating gene modules underlying the diversity of this neuronal population. We propose that the newly identified genetic coupling between neurotransmitter identity and ion channels may play a homeostatic role in maintaining the electrophysiological phenotype of midbrain DA neurons.

SIGMAR1 Regulates Membrane Electrical Activity in Response to Extracellular Matrix Stimulation to Drive Cancer Cell Invasiveness.

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition.

Electrical/ Electrochemical Impedance Measurement for Biological Applications

As a principle of transduction, the impedance technique has been applied in the field of biological study ranging from microbiology to cancer cell/tissue studies. This paper summarizes the applications of electrical/electrochemical impedance spectroscopy that have been practiced in our research in the past several years, mainly including two distinct fields--the detection of foodborne bacterial pathogens and the study of oral cancer cells. Foodborne diseases caused by foodborne pathogens have been a serious threat to public health for decades and remain one of the major concerns of our society. The development of rapid, sensitive, and specific methods is central to implementing effective practice to ensure food safety and security. We have been using impedance technique as a means to detect and/or quantify foodborne pathogenic bacteria. This paper will particular introduce the recent significant development in this field, including the use of microfabricated microelectrodes, microfluidic chip-based devices, nanoparticles and integration with other techniques such as dielectrophoresis. Based on the fundamental impedance microbiology, which is a technique based on the measurement of changes in electrical impedance of a culture medium or a reaction solution resulting from the bacterial growth, we have added some new aspects to this subject that have made the impedance technique a more valuable technique for detection of foodborne bacterial pathogens. These aspects include the use of different electrode systems and the analysis of impedance components using equivalent circuits for better improvement on the detection systems. The advances in microfabrication technologies have launched the use of microfabricated microarray electrodes in impedance detection and the miniaturization of impedance microbiology into a chip format, which has shown great promising in rapid detection of bacterial growth. Further, the integration of impedance technique with biosensor technology has led to the development of impedance biosensors that is expending rapidly for bacteria detection in recent years. Impedance biosensors for bacteria detection are based on the analysis of electrical properties of bacterial cells when they are attached to or associated with the electrodes. Such impedance biosensor methods have substantially reduced the assay time down to 30 min~2 h compared with growth-based impedance methods. The dimensional compatibility of those microfabricated biosensors with the target bacterial cells has enabled them to detect the binding of bacterial cells on its surface without any amplification step. On the other hand, electrical impedance spectroscopy (EIS) enables direct sensing of cellular activities occurring on an electrode surface by measuring the induced capacitance and/or resistance changes by cells attached on the electrode. Particular, EIS is capable of distinguishing different types of cells based on the cellular activity-induced electrical signals. We have demonstrated the use of real-time impedance technique to study the cellular activities of oral cancer cells in a label-free manner, including cell proliferation, adhesion, spreading, and apoptosis induced by drug treatments. Furthermore, it is known that during transformation from non-cancer cells to caner cells, the cellular morphology, structure, and function are altered, which may includes, but are not limited to changes in the nuclei, composition of the cytoplasm, structure of the cell membrane, changes in the nuclei, composition of the cytoplasm, structure of the cell membrane, and expression levels of ion channels, etc. These changes may result in alterations in cancer cell properties that could be detected by the EIS well before these morphological features that normally used in detection can be seen or biomarkers are developed. We have explored the use of real-time impedance measurements to distinguish oral cancer cells from normal oral epithelial cells in label-free manner by studying the kinetics of cell spreading and attachment of the two cell types.

Electrophysiological and morphological maturation of murine fetal cardiomyocytes during electrical stimulation in vitro.

The aim of this study was to investigate whether continuous electrical stimulation affects electrophysiological properties and cell morphology of fetal cardiomyocytes (FCMs) in culture. Fetal cardiomyocytes at day 14.5 post coitum were harvested from murine hearts and electrically stimulated for 6 days in culture using a custom-made stimulation chamber. Subsequently, action potentials of FCM were recorded with glass microelectrodes. Immunostainings of α-Actinin, connexin 43, and vinculin were performed. Expression of ion channel subunits Kcnd2, Slc8a1, Cacna1, Kcnh2, and Kcnb1 was analyzed by quantitative reverse-transcriptase polymerase chain reaction. Action potential duration to 50% and 90% repolarization (APD50 and APD90) of electrically stimulated FCMs were significantly decreased when compared to nonstimulated control FCM. Alignment of cells was significantly higher in stimulated FCM when compared to control FCM. The expression of connexin 43 was significantly increased in stimulated FCM when compared to control FCM. The ratio between cell length and cell width of the stimulated FCM was significantly higher than in control FCM. Kcnh2 and Kcnd2 were upregulated in stimulated FCM when compared to control FCM. Expression of Slc8a1, Cacna1c, and Kcnb1 was not different in stimulated and control FCMs. The decrease in APD50 observed after electrical stimulation of FCM in vitro corresponds to the electrophysiological maturation of FCM in vivo. Expression levels of ion channels suggest that some important but not all aspects of the complex process of electrophysiological maturation are promoted by electrical stimulation. Parallel alignment, increased connexin 43 expression, and elongation of FCM are signs of a morphological maturation induced by electrical stimulation.

Remodeling of ion channel expression may contribute to electrophysiological consequences caused by methamphetamine in vitro and in vivo

Methamphetamine (MA) is a psychostimulant. MA may induce numerous cardiotoxic effects, leading to cardiac arrhythmias, heart failure, eventually leading to sudden cardiac death. The deleterious effects of methamphetamine work in tandem to disrupt the coordinated electrical activity of the heart and have been associated with life-threatening cardiac arrhythmias. Remodeling of ion channels is an important mechanism of arrhythmia. Although arrhythmogenic remodeling involves alterations in ion channel expression, it is yet unknown whether MA induced electrical remodeling by affecting gene expression, and whether the changes in protein expression are paralleled by alterations in mRNA expression. Our study focused on the expression of ion channels which were correlated to the electrical remodeling caused by MA. We used RT-PCR and western blot to assess of the transcript and translate levels of ion channel subunits, including Ito: kv1.4, kv1.7, kv3.4, kv4.2; IK1: kir2.1, kir2.2, kir2.3, kir2.4; and ICa-l: Ca2+α1, Ca2+β, respectively. The reversible effect of these changes after MA withdrawal was also evaluated. MA caused decrease in mRNA and protein levels in all ion channel subunits in vitro and also in vivo, is at this work. The kv3.4 and all 4 subunits of Kir2.0 family showed significant decrease than the other genes. Most of the channel subunit expression started to reverse after MA withdrawal for 4weeks and significantly reverse in all of the channel subunits after MA withdrawal for 8weeks. We found that CACNA1C and Kir2.0 family showed lower recoverability than the others after MA withdrawal for 8weeks. The reduction of the ion channel expression levels may be the molecular mechanism that mediates the electrical remodeling caused by methamphetamine.

Electrophysiological Differences Between fshb- and lhb-Expressing Gonadotropes in Primary Culture

Synthesis and release of FSH and LH are differentially regulated by GnRH, but the mechanisms by which this regulation is achieved are not well understood. Teleost fish are powerful models for studying this differential regulation because they have distinct pituitary cells producing either FSH or LH. By using pituitary cultures from Atlantic cod (Gadus morhua), we were able to investigate and compare the electrophysiological properties of fshb- and lhb-expressing cells, identified by single-cell quantitative PCR after recording. Both cell types fired action potentials spontaneously. The relative number of excitable cells was dependent on reproductive season but varied in opposing directions according to season in the 2 cell types. Excitable and quiescent gonadotropes displayed different ion channel repertoires. The dynamics of outward currents and GnRH-induced membrane responses differed between fshb- and lhb-expressing cells, whereas GnRH-induced cytosolic Ca²⁺ responses were similar. Expression of Ca²⁺-activated K⁺ channels also differed with cell type and showed seasonal variation when measured in whole pituitary. The differential presence of these channels corresponds to the differences observed in membrane response to GnRH. We speculate that differences in ion channel expression levels may be involved in seasonal regulation of hormone secretion as well as the differential response to GnRH in LH- and FSH-producing gonadotropes, through differences in excitability and Ca²⁺ influx.

How conductance distributions are shaped by activity-dependent regulation rules

Neurons co-express a multitude of different ion channels that determine the electrophysiological behaviour of the cell. Recent experimental observations have revealed that the expression of different ion channels varies dramatically within neurons of a defined type, even when these neurons exhibit stereotyped electrical properties [1-5]. This variability has a structure visible in the correlations between different ion channel expression levels. Nonetheless,, the underlying mechanism that determines these correlations is unknown, nor is understood how these correlations can coexist with activity-dependent regulation mechanisms that are also known to exist in neurons [6]. We show that these observations are logical consequences of relatively simple control mechanisms that couple the expression levels of individual conductances to a cell-intrinsic readout of activity [7]. Crucially, these correlations are not visible when conductance space is searched to find combinations of conductances that give target behaviour. Therefore, activity-dependent regulation mechanisms constrain the solution space of potential combinations of membrane conductances to a characteristic subset. Furthermore, we show how the shape of the conductance distribution of a population of neurons is determined by the relative rates of expression of different conductances. Finally, degeneracy in the function of multiple ion channels means that regulation mechanisms can have widely-variable parameters yet remain stable; this is exemplified by tolerance to 'anti-homeostatic' regulation of a subset of conductances.

Pathophysiology of astroglial purinergic signalling

Astrocytes are fundamental for central nervous system (CNS) physiology and are the fulcrum of neurological diseases. Astroglial cells control development of the nervous system, regulate synaptogenesis, maturation, maintenance and plasticity of synapses and are central for nervous system homeostasis. Astroglial reactions determine progression and outcome of many neuropathologies and are critical for regeneration and remodelling of neural circuits following trauma, stroke, ischaemia or neurodegenerative disorders. They secrete multiple neurotransmitters and neurohormones to communicate with neurones, microglia and the vascular walls of capillaries. Signalling through release of ATP is the most widespread mean of communication between astrocytes and other types of neural cells. ATP serves as a fast excitatory neurotransmitter and has pronounced long-term (trophic) roles in cell proliferation, growth, and development. During pathology, ATP is released from damaged cells and acts both as a cytotoxic factor and a proinflammatory mediator, being a universal "danger" signal. In this review, we summarise contemporary knowledge on the role of purinergic receptors (P2Rs) in a variety of diseases in relation to changes of astrocytic functions and nucleotide signalling. We have focussed on the role of the ionotropic P2X and metabotropic P2YRs working alone or in concert to modify the release of neurotransmitters, to activate signalling cascades and to change the expression levels of ion channels and protein kinases. All these effects are of great importance for the initiation, progression and maintenance of astrogliosis-the conserved and ubiquitous glial defensive reaction to CNS pathologies. We highlighted specific aspects of reactive astrogliosis, especially with respect to the involvement of the P2X(7) and P2Y(1)R subtypes. Reactive astrogliosis exerts both beneficial and detrimental effects in a context-specific manner determined by distinct molecular signalling cascades. Understanding the role of purinergic signalling in astrocytes is critical to identifying new therapeutic principles to treat acute and chronic neurological diseases.

Chronic Probucol Treatment Decreases the Slow Component of the Delayed-Rectifier Potassium Current in CHO Cells Transfected With KCNQ1 and KCNE1

Indirect effects of drugs on ion channel expression levels on plasma membrane are focused as the cause of QT prolongation, and we explored the chronic effects of QT-prolonging drugs on the slow component of the delayed-rectifier potassium current (IKs). Chinese Hamster Ovary cells expressing IKs channels were constructed by transfecting KCNQ1/KCNE1 genes, and the IKs values were measured using IonWorks Quattro in the population patch-clamp mode. After 24 hours of treatment with IKs blockers (HMR1556, L-768673, or chromanol 293B) or human Ether-à-go-go related gene channel trafficking inhibitors (amiodarone,17-AAG, brefeldin A, pentamidine, thioridazine, or probucol), brefeldin A, pentamidine, and probucol decreased IKs. Probucol, which is a cholesterol-lowering drug and clinically reported to cause QT prolongation, potently inhibited the IKs in a concentration-dependent manner, with a half maximal inhibitory concentration of 149.1 nM. A reduction in the IKs by 1 μM of probucol was observed beginning 2 hours after treatment, and the current was reduced by about 80% at 24 hours. The activation and deactivation time constants of residual IKs currents became faster compared with that in the vehicle-treatment group. Acute application of probucol did not directly inhibit IKs channels at concentrations of up to 10 μM. Western blotting analysis indicated the reduction of multimeric complex of KCNQ1 proteins by probucol treatment but not monomeric form. These results suggest that chronic probucol treatment may contribute to QT prolongation in humans by decreasing the functional IKs channel complexes.

Ion Channel Remodeling in Failing and Non-Failing Hearts of Inherited DCM Mouse Model -Usefulness of Running Wheel in Detection of Heart Failure-

Dilated cardiomyopathy (DCM) is a progressive disease often associated with heart failure (HF) and sudden death (SD). We have been studying cause and mechanism of SD/lethal arrhythmia in DCM using a mouse model with a mutation in cardiac troponin T (ΔK210), which displays cardiac dilation and shows frequent death with t1/2 of ∼70 days. In experiments with such mouse models with progressive heart diseases, it is necessary to assess severity of HF in living mice. To do so we measured voluntary exercise using a running wheel (r=6cm). Wheel-running activity was basically similar between wild type (WT) and DCM mice (17.1±5.7 vs 17.5±6.9 x 103 rounds/day (rpd)) until 2 months old. After then, however, some DCM mice died suddenly while keeping their running activity and others started to lose running activity and died. DCM mice were divided into two groups; mice with normal (>15000 rpd) and decreased running activity (<2000 rpd), and heart weight/body weight ratio (HW/BW) and lung weight/body weight ratio (LW/BW) were determined as indicatives of cardiac dilation and congestive HF, respectively. There was a significant negative correlation between running activity and LW/BW ratio, indicating that running wheel is a useful for detecting congestive HF in mouse model. These results suggest that some of the DCM mice die suddenly without HF, probably with lethal arrhythmia, whereas others develop severe HF and die. We carried out real-time PCR analysis for BNP and ion channels in two groups. The difference and similarity in expression levels of ion channels between two groups will be discussed.

Electrical injury alters ion channel expression levels and electrophysiological properties in rabbit dorsal root ganglia neurons

The electrophysiological and morphological changes of nerve fibers induced by electrical injury have been widely addressed. However, the changes of ion channels in neurons after electrical shocks have not been systematically investigated yet. In this study, the sciatic nerves of rabbit were injured by 50 V 50 Hz, 110 V 50 Hz, and 220 V 50 Hz alternating current, respectively. One week later, the expression levels and electrophysiological changes of voltage-gated potassium (Kv) and sodium (Nav) channels in dorsal root ganglia (DRG) neurons were evaluated by RT-PCR, immunofluorescence staining and patch clamp technique. The Nav1.1 expression was decreased by 50 V injury. The Kv1.2, Kv1.4, Nav1.1 and Nav1.7 expression levels and Kv current densities were reduced after 110 V injury. Under the 220 V injury circumstance, Kv1.2, Nav1.1, Nav1.7 expression levels, Kv current densities and TTX-R Na + current densities were significantly decreased, while TTX-S Na + current densities increased. These findings suggest that the expression levels, subunit compositions, and electrophysiological properties of Kv and Nav channels are altered after electrical injury, and the severity of injury gets worse as injury voltage increases.