Expression Of Ion Channel Subunits Research Articles

The Role of Ion Channels in Functional Gastrointestinal Disorders (FGID): Evidence of Channelopathies and Potential Avenues for Future Research and Therapeutic Targets.

Several gastrointestinal (GI) tract abnormalities, including visceral hypersensitivity, motility, and intestinal permeability alterations, have been implicated in functional GI disorders (FGIDs). Ion channels play a crucial role in all the functions mentioned above. Hormones and natural molecules modulate these channels and represent targets of drugs and bacterial toxins. Mutations and abnormal functional expression of ion channel subunits can lead to diseases called channelopathies. These channelopathies in gastroenterology are gaining a strong interest, and the evidence of co-relationships is increasing. In this review, we describe the correlation status between channelopathies and FGIDs. Different findings are available. Among others, mutations in the ABCC7/CFTR gene have been described as a cause of constipation and diarrhea. Mutations of the SCN5A gene are instead associated with irritable bowel syndrome. In contrast, mutations of the TRPV1 and TRPA genes of the transient receptor potential (TRP) superfamily manifest hypersensitivity and visceral pain in sensory nerves. Recently, mice and humans affected by Cantu syndrome (CS), which is associated with the mutations of the KCNJ8 and ABCC9 genes encoding for the Kir6.1 and SUR2 subunits, showed dysfunction of contractility throughout the intestine and death in the mice after the weaning on solid food. The discovery of a correlation between channelopathies and FIGD opens new avenues for discovering new direct drug targets for specific channelopathies, leading to significant implications for diagnosing and treating functional GI diseases.

Mineralocortidoid Receptor Mediates Uremic Serum‐Induced Increase in Endothelial Cell Dysfunction

Patients with chronic kidney disease (CKD) have a markedly increased incidence of cardiovascular disease (CVD) and mortality compared with the age‐matched general population. The high concentration of circulating uremic toxins in CKD patients leads to vascular inflammatory, thereby inducing endothelial dysfunction, which is associated with CVD development and progression. Plasma aldosterone levels are increased in CKD, and aldosterone has been found to increase vascular inflammation and fibrosis. The aim of our study was to analyze the influence of CKD in the expression of endothelial ion channels and its potential modification by mineralocorticoid receptor (MR) inhibition, which would consequently affect endothelial cell function. To that end we used primary human endothelial cells cultured in medium supplemented with pooled sera from either healthy or uremic patients undergoing dialysis. To obtain a complete profile of ion channel subunit expression in both cellular types, we performed high‐throughput qPCR of 92 ion channel genes using a custom‐designed Taqman low‐density array card. In addition, we characterized the influence of uremic serum on nitric oxide (NO) production and measured cortical stiffness using atomic force microscopy. Our data show that uremic serum potently increased MR expression. In addition, it induced remodeling of ion channel expression patterns, including increased expression of αENaC, a known transcriptional target of MR that promotes cortical stiffness and endothelial dysfunction. Exposure of endothelial cells to uremic serum significantly decreased NO production and produced stiffening of the endothelial cell cortex, which was prevented by inhibition of MR using spironolactone. Our data identifies MR as a possible mediator of uremia‐induced endothelial dysfunction and supports the proposed beneficial effects of MR inhibition during CKD.Support or Funding InformationThis work was supported by a grant from Ministerio de Economía e Innovación (MINECO, grant BFU2016‐78374‐R) to D.A.R.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multi-Scale Assessments of Cardiac Electrophysiology Reveal Regional Heterogeneity in Health and Disease.

The left and right ventricles of the four-chambered heart have distinct developmental origins and functions. Chamber-specific developmental programming underlies the differential gene expression of ion channel subunits regulating cardiac electrophysiology that persists into adulthood. Here, we discuss regional specific electrical responses to genetic mutations and cardiac stressors, their clinical correlations, and describe many of the multi-scale techniques commonly used to analyze electrophysiological regional heterogeneity.

Reconstitution of synaptic Ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization

Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post-mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimer's) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimer's brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets. Disruption in the expression and function of synaptic ion channels is critical in the pathophysiology of human neurodegenerative diseases. We here show that synaptosomes purified from rodent and human frozen brain (control and Alzheimer disease) can be studied both biochemically and functionally. This method opens the way to directly testing the effects of novel drugs on native ion channels.

Dopaminergic Neurons Exhibit an Age-Dependent Decline in Electrophysiological Parameters in the MitoPark Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most diagnosed neurodegenerative disorder, and the classic motor symptoms of the disease are attributed to selective loss of dopaminergic neurons of the substantia nigra. The MitoPark mouse is a genetic model of PD that mimics many of the key characteristics of the disease and enables the study of progressive neurodegeneration in parkinsonism. Here we have identified functional deficits in the ion channel physiology of dopaminergic neurons from MitoPark mice that both precede and are concurrent with the time course of behavioral symptomatology. Because PD is a progressive disease with a long asymptomatic phase, identification of early functional adaptations could lay the groundwork to test therapeutic interventions that halt or reverse disease progression.

The role of Mineralocorticoid Receptor on Human Aortic Endothelial Ion Channel Expression Profile in Chronic Kidney Disease

Patients with chronic kidney disease (CKD) have a markedly increased incidence of cardiovascular events and cardiovascular disease (CVD) mortality compared with the age-matched general population. The high concentration of circulating uremic toxins in CDK patients may trigger vascular inflammatory responses, thereby inducing endothelial dysfunction, which is associated with CVD development and progression. In addition, plasma aldosterone levels are increased in CKD, and aldosterone has been found to increase vascular inflammation and fibrosis. The aim of our study was to analyze the influence of CKD in the expression of endothelial ion channels and its potential modification by mineralocorticoid receptor (MR) antagonism, which would consequently affect endothelial dysfunction. To that end we used human aortic endothelial cells (HAEC) cultured in medium supplemented with pooled sera from either healthy or uremic patients undergoing dialysis, as well as both groups in the presence of spironolactone. HAEC were found to express MR under the culture conditions used. To obtain a global portrait of ion channel expression in HAEC in the four groups tested, we performed high-throughput real-time polymerase chain reaction of 92 ion channel genes using a custom-designed Taqman low-density array card. We have obtained a profile of ion channel gene expression altered by uremic serum and the effect of spironolactone both on basal and altered expression of ion channel subunits.

Transcriptional Remodeling of Ion Channel Subunits by Flow Adaptation in Human Coronary Artery Endothelial Cells

Endothelial cells (ECs) are constantly exposed to blood flow-induced shear forces in the vessels and this is a major determinant of endothelial function. Ion channels have a major role in endothelial function and in the control of vascular tone. We hypothesized that shear force is a general regulator of ion channel expression, which will have profound effects on endothelial function. We examined this hypothesis using large-scale quantitative real-time RT-PCR. Human coronary artery ECs were exposed to two levels of flow-induced shear stress for 24 h, while control cells were grown under static conditions. The expression of ion channel subunits was compared between control and flow-adapted cells. We used primers against 55 ion channel and exchanger subunits and were able to detect 54 subunits. Five dyn/cm² of shear induced downregulation of 1 (NCX1) and upregulation of 18 subunits, including K_Ca2.2, K_Ca2.3, CX37, K_v1.5 and HCN2. Fifteen dyn/cm² of shear stress induced the expression of 30 ion channel subunits, including K_Ca2.3, K_Ca2.2, CX37, K_ir2.3 and K_Ca3.1. Our data demonstrate that substantial remodeling of endothelial ion channel subunit expression occurs with flow adaptation and suggest that altered ion channel expression may significantly contribute to vascular pathology associated with flow-induced alterations.

Anthelmintic resistance: markers for resistance, or susceptibility?

The Consortium for Anthelmintic Resistance and Susceptibility (CARS) brings together researchers worldwide, with a focus of advancing knowledge of resistance and providing information on detection methods and treatment strategies. Advances in this field suggest mechanisms and features of resistance that are shared among different classes of anthelmintic. Benzimidazole resistance is characterized by specific amino acid substitutions in beta-tubulin. If present, these substitutions increase in frequency upon drug treatment and lead to treatment failure. In the laboratory, sequence substitutions in ion-channels can contribute to macrocyclic lactone resistance, but there is little evidence that they are significant in the field. Changes in gene expression are associated with resistance to several different classes of anthelmintic. Increased P-glycoprotein expression may prevent drug access to its site of action. Decreased expression of ion-channel subunits and the loss of specific receptors may remove the drug target. Tools for the identification and genetic analysis of parasitic nematodes and a new online database will help to coordinate research efforts in this area. Resistance may result from a loss of sensitivity as well as the appearance of resistance. A focus on the presence of anthelmintic susceptibility may be as important as the detection of resistance.

Ion Channel Subunit Expression Changes in Cardiac Purkinje Fibers

Purkinje fibers (PFs) play key roles in cardiac conduction and arrhythmogenesis. Congestive heart failure (CHF) causes well-characterized atrial and ventricular ion channel subunit expression changes, but effects on PF ion channel subunits are unknown. This study assessed changes in PF ion channel subunit expression (real-time PCR, immunoblot, immunohistochemistry), action potential properties, and conduction in dogs with ventricular tachypacing-induced CHF. CHF downregulated mRNA expression of subunits involved in action potential propagation (Nav1.5, by 56%; connexin [Cx]40, 66%; Cx43, 56%) and repolarization (Kv4.3, 43%, Kv3.4, 46%). No significant changes occurred in KChIP2, KvLQT1, ERG, or Kir3.1/3.4 mRNA. At the protein level, downregulation was seen for Nav1.5 (by 38%), Kv4.3 (42%), Kv3.4 (57%), Kir2.1 (26%), Cx40 (53%), and Cx43 (30%). Cx43 dephosphorylation was indicated by decreased larger molecular mass bands (pan-Cx43 antibody) and a 57% decrease in Ser368-phosphorylated Cx43 (phospho-specific antibody). Immunohistochemistry revealed reduced Cx40, Cx43, and phospho-Cx43 expression at intercalated disks. Action potential changes were consistent with observed decreases in ion channel subunits: CHF decreased phase 1 slope (by 56%), overshoot (by 32%), and phase 0 dV/dt(max) (by 35%). Impulse propagation was slowed in PF false tendons: conduction velocity decreased significantly from 2.2+/-0.1 m/s (control) to 1.5+/-0.1 m/s (CHF). His-Purkinje conduction also slowed in vivo, with HV interval increasing from 35.5+/-1.2 (control) to 49.3+/-3.4 ms (CHF). These results indicate important effects of CHF on PF ion channel subunit expression. Alterations in subunits governing conduction properties may be particularly important, because CHF-induced impairments in Purkinje tissue conduction, which this study is the first to describe, could contribute significantly to dyssynchronous ventricular activation, a major determinant of prognosis in CHF-patients.

Abstract 1517: Remodeling of Ion-Channel Expression and Cellular Electrophysiology of Cardiac Purkinje Fibers by Heart Failure

Purkinje fibers (PFs) play a key role in cardiac conduction and arrhythmogenesis. Heart failure (HF) causes extensive electrical remodeling. HF-induced changes in atrial and ventricular ion channel subunit expression have been well characterized, but little is known about HF-induced ion channel subunit remodeling and functional consequences in PFs. This study assessed ion channel subunit expression, action potential (AP) properties and conduction in cardiac PF false tendons from control and HF dogs. HF was induced by 2 wk ventricular tachypacing (240 bpm). Control and HF PFs were fast-frozen for ion channel subunit mRNA (RT-qPCR) and protein (Western Blot, immunohistochemistry) assessment. APs were studied with standard micro-electrodes. HF significantly downregulated mRNA expression of subunits involved in AP propagation (Nav1.5, by 56%**, **P<0.01; Cx40, by 66%**, Cx43, by 56%**), automaticity (HCN2, by 75%**; HCN4, by 78%**) and repolarization (Kv4.3, by 43%*, *P<0.05; minK, by 31%*). No significant changes occurred in KChIP2, KvLQT1, ERG, Kir2.1 or Kir3.1/3.4 mRNA. At the protein level, significant downregulation was seen for Nav1.5 (by 38%**), Kv4.3 (by 42%**), HCN4 (by 74%*), Cx40 (by 53%**) and Cx43 (by 30%**). Immunohistochemistry revealed reduced Cx40 and Cx43 expression at PF intercalated disks. AP analysis showed changes consistent with observed decreases in I to and I Na subunits: HF decreased phase 1 slope (by 56%**), AP overshoot (by 32%*) and dV/dt max (by 35%*). AP properties associated with unchanged subunits (eg, resting potential, overall AP duration) were unaltered. Because of consistently significant changes in subunits governing impulse propagation (Nav1.5, Cx40, Cx43), we examined HF effects on AP propagation in PF false tendons with dual microelectrodes: conduction velocity decreased from 2.2±0.1 m/s (control) to 1.5±0.1 m/s* (HF). We have characterized for the first time HF effects on ion-channel subunit expression in cardiac PFs, finding prominent alterations in a variety of important subunits that control AP repolarization and propagation. These changes in PF ion-channel subunits likely contribute to conduction disturbances and arrhythmogenesis in the failing heart.

Atrial Remodeling and Atrial Fibrillation

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. It can occur at any age but is very rare in children and becomes extremely common in the elderly, with a prevalence approaching 20% in patients >85 years of age.1 AF is associated with a wide range of potential complications and contributes significantly to population morbidity and mortality. Present therapeutic approaches to AF have major limitations, including limited efficacy and significant adverse effect liability. These limitations have inspired substantial efforts to improve our understanding of the mechanisms underlying AF, with the premise that improved mechanistic insights will lead to innovative and improved therapeutic approaches.2 Our understanding of AF pathophysiology has advanced significantly over the past 10 to 15 years through an increased awareness of the role of “atrial remodeling.” Any persistent change in atrial structure or function constitutes atrial remodeling. Many forms of atrial remodeling promote the occurrence or maintenance of AF by acting on the fundamental arrhythmia mechanisms illustrated in Figure 1. Both rapid ectopic firing and reentry can maintain AF. Reentry requires a suitable vulnerable substrate, as well as a trigger that acts on the substrate to initiate reentry. Ectopic firing contributes to reentry by providing triggers for reentry induction. Atrial remodeling has the potential to increase the likelihood of ectopic or reentrant activity through a multitude of potential mechanisms. This article reviews the types of atrial remodeling, their underlying pathophysiology, the molecular basis of their occurrence, and finally, their potential therapeutic significance. Figure 1. General schema representing AF mechanisms and the role of remodeling. The mechanisms underlying AF are portrayed schematically in Figure 2. AF can be maintained by rapid focal firing, which may itself be regular but result in fibrillatory activity because of wave breakup in portions of the atrium that …

Expression of Kir2.1 and Kir6.2 transgenes under the control of the α-MHC promoter in the sinoatrial and atrioventricular nodes in transgenic mice

Kir2.1 and Kir6.2 are ion channel subunits partly responsible for the background inward rectifier and ATP-sensitive K + currents (I K1 and I KATP) in the heart. Very little is known about how the distribution of ion channel subunits is controlled. In this study, we have investigated the expression (at protein and mRNA levels) of GFP-tagged Kir2.1 and Kir6.2 transgenes under the control of the α-MHC promoter in the sinoatrial node (SAN), atrioventricular node (AVN), His bundle and working myocardium of transgenic mice. After dissection, serial 10-μm cryosections were cut. Histological staining was carried out to identify tissues, confocal microscopy was carried out to map the distribution of the GFP-tagged ion channel subunits and in situ hybridization was carried out to map the distribution of corresponding mRNAs. We demonstrate heterologous expression of the ion channel subunits in the working myocardium, but not necessarily in the SAN, AVN or His bundle; the distribution of the subunits does not correspond to the expected distribution of α-MHC. Both protein and mRNA expression does, however, correspond to the expected distributions of native Kir6.2 and Kir2.1 in the SAN, AVN, His bundle and working myocardium. The data demonstrate novel transcriptional and/or post-transcriptional control of ion channel subunit expression and raise important questions about the control of regional expression of ion channels.

Membrane Potential-regulated Transcription of the Resting K+ Conductance TASK-3 via the Calcineurin Pathway

The 2P domain K(+) channel TASK-3 is highly expressed in cerebellar granule neurons where it has been proposed to underlie the K(+) leak conductance, IKso. In a previous work we showed that expression of TASK-3 increases in cerebellar granule neurons as they mature in culture. Here we show that within the cerebellum, levels of TASK-3 mRNA increase as granule neurons migrate to their adult positions and receive excitatory mossy fiber input. To understand the mechanism of this increase in TASK-3 expression we used an in vitro model culturing the neurons in either depolarizing conditions mimicking neuronal activity (25K, 25 mm KCl) or in conditions which approach deafferentation (5K, 5 mm KCl). An important increase in TASK-3 mRNA is uniquely observed in 25K and is specific since other background K(+) channel levels remain unchanged or decrease. The rise in TASK-3 mRNA leads to an increase in TASK-3 protein and the IKso conductance resulting in hyperpolarization. Blocking L-type calcium channels or their downstream effector calcineurin, abrogates TASK-3 expression and IKso, leading to hyperexcitability. This is the first study demonstrating that depolarization-induced Ca(2+) entry can directly regulate cellular excitability by dynamically regulating the transcription of a resting K(+) conductance. The appearance of this conductance may play an important role in the transition of depolarized immature neurons to their mature hyperpolarized state during neuronal development.

Kv1.5 Is an Important Component of Repolarizing K + Current in Canine Atrial Myocytes

Although the canine atrium has proven useful in several experimental models of atrial fibrillation and for studying the effects of rapid atrial pacing on atrial electrical remodeling, it may not fully represent the human condition because of reported differences in functional ionic currents and ion channel subunit expression. In this study, we reassessed the molecular components underlying one current, the ultrarapid delayed rectifier current in canine atrium [IKur(d)], by evaluating the mRNA, protein, immunofluorescence, and currents of the candidate channels. Using reverse transcriptase-polymerase chain reaction, we found that Kv1.5 mRNA was expressed in canine atrium whereas message for Kv3.1 was not detected. Western analysis on cytosolic and membrane fractions of canine tissues, using selective antibodies, showed that Kv3.1 was only detectable in the brain preparations, whereas Kv1.5 was expressed at high levels in both atrial and ventricular membrane fractions. Confocal imaging performed on isolated canine atrial myocytes clearly demonstrated the presence of Kv1.5 immunostaining, whereas that of Kv3.1 was equivocal. Voltage- and current-clamp studies showed that 0.5 mmol/L tetraethylammonium had variable effects on sustained K+ currents, whereas a compound with demonstrated selectivity for hKv1.5 versus Kv3.1, hERG or the sodium channel, fully suppressed canine atrial IKur tail currents and depressed sustained outward K+ current. This agent also increased action potential plateau potentials and action potential duration at 20% and 50% repolarization. These results suggest that in canine atria, as in other species including human, Kv1.5 protein is highly expressed and contributes to IKur.

The endocrine system, vertigo and balance.

Steroid, amine and peptide hormones affect the peripheral vestibular system. Vasopressin hypersensitivity of the endolymphatic sac may be implicated in the pathogenesis of Meniere's disease. Specific vasopressin antagonists will help define the role of vasopressin in Meniere's disease. The modulation of central vestibular pathways by neuroactive steroids may involve effects on gamma-aminobutyric acid-ergic and glutaminergic pathways. The vestibular nuclei also express enzymes that are important in the synthesis of steroids and the modulation of their activity. Steroids mediate both facilitatory and deleterious effects of stress on vestibular compensation. The quality and quantity of stressor that determines the pattern of hormonal output, may be important. Clinical observation suggests that episodic ataxia type 2, a P/Q calcium channelopathy, may be phenotypically modulated by endocrine fluctuations. Steroid hormones may affect the episodic ataxia type 2 phenotype by modulation of voltage-gated calcium channel activity via second messenger systems and ion channel subunit expression. Despite evidence to support the link, the role of the endocrine system in vestibular function and disease is as yet virtually unexplored.