Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.

Chemical inhibitors of the calcium entry channel TRPV6

Calcium entry channels in the plasma membrane are thought to play a major role in maintaining cellular Ca(2+) levels, crucial for growth and survival of normal and cancer cells. The calcium-selective channel TRPV6 is expressed in prostate, breast, and other cancer cells. Its expression coincides with cancer progression, suggesting that it drives cancer cell growth. However, no specific inhibitors for TRPV6 have been identified thus far.

Nerve excitability studies characterize Kv1.1 fast potassium channel dysfunction in patients with episodic ataxia type 1

Episodic ataxia type 1 is a neuronal channelopathy caused by mutations in the KCNA1 gene encoding the fast K(+) channel subunit K(v)1.1. Episodic ataxia type 1 presents with brief episodes of cerebellar dysfunction and persistent neuromyotonia and is associated with an increased incidence of epilepsy. In myelinated peripheral nerve, K(v)1.1 is highly expressed in the juxtaparanodal axon, where potassium channels limit the depolarizing afterpotential and the effects of depolarizing currents. Axonal excitability studies were performed on patients with genetically confirmed episodic ataxia type 1 to characterize the effects of K(v)1.1 dysfunction on motor axons in vivo. The median nerve was stimulated at the wrist and compound muscle action potentials were recorded from abductor pollicis brevis. Threshold tracking techniques were used to record strength-duration time constant, threshold electrotonus, current/threshold relationship and the recovery cycle. Recordings from 20 patients from eight kindreds with different KCNA1 point mutations were compared with those from 30 normal controls. All 20 patients had a history of episodic ataxia and 19 had neuromyotonia. All patients had similar, distinctive abnormalities: superexcitability was on average 100% higher in the patients than in controls (P < 0.00001) and, in threshold electrotonus, the increase in excitability due to a depolarizing current (20% of threshold) was 31% higher (P < 0.00001). Using these two parameters, the patients with episodic ataxia type 1 and controls could be clearly separated into two non-overlapping groups. Differences between the different KCNA1 mutations were not statistically significant. Studies of nerve excitability can identify K(v)1.1 dysfunction in patients with episodic ataxia type 1. The simple 15 min test may be useful in diagnosis, since it can differentiate patients with episodic ataxia type 1 from normal controls with high sensitivity and specificity.

In vivo assessment of HCN channel current (I(h)) in human motor axons

The "Trond" protocol of nerve excitability tests has been used widely to assess axonal function in peripheral nerve. In this study, the routine Trond protocol was expanded to refine assessment of cAMP-dependent, hyperpolarization-activated current (I(h)) activity. I(h) activity is generated by hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels in response to hyperpolarization. It limits activity-dependent hyperpolarization, contributes to neuronal automaticity, and is implicated in chronic pain states. Published data regarding I(h) activity in motor nerve are scant. We used additional strong, prolonged hyperpolarizing conditioning stimuli in the threshold electrotonus component of the Trond protocol to demonstrate the time-course of activation of I(h) in motor axons. Fifteen healthy volunteers were tested on four occasions during 1 week. I(h) action was revealed in the threshold electrotonus by the limiting and often reversal, after about 100 ms, of the threshold increase caused by strong hyperpolarizing currents. Statistical analysis by repeated-measures analysis of variance enabled confidence limits to be established for variation between subjects and within subjects. The results demonstrate that, of all the excitability parameters, those dependent on I(h) were the most characteristic of an individual, because variance between subjects was more than four times the variance within subjects. This study demonstrates a reliable method for in vivo assessment of I(h,) and also serves to document the normal variability in nerve excitability properties within subjects.

Dissonance as a resource for probing the qubit depolarizing channel

Quantum channel identification, a standard problem in quantum metrology, is the task of estimating parameter(s) of a quantum channel. We investigate dissonance (quantum discord in the absence of entanglement) as an aid to quantum channel identification and find evidence for dissonance as a resource for quantum information processing. We consider the specific case of dissonant Bell-diagonal probes of the qubit depolarizing channel, using quantum Fisher information as a measure of statistical information extracted by the probe. In this setting dissonant quantum probes yield more statistical information about the depolarizing probability than do corresponding probes without dissonance and greater dissonance yields greater information. This effect only operates consistently when we control for classical correlation between the probe and its ancilla and the joint and marginal purities of the ancilla and probe.

Block of the hERG channel by bupivacaine: Electrophysiological and modeling insights towards stereochemical optimization

The hERG voltage-gated potassium channel mediates the cardiac I(Kr) current, which is crucial for the duration of the cardiac action potential. Undesired block of the channel by certain drugs may prolong the QT interval and increase the risk of malignant ventricular arrhythmias. Although the molecular determinants of hERG block have been intensively studied, not much is known about its stereoselectivity. Levo-(S)-bupivacaine was the first drug reported to have a higher affinity to block hERG than its enantiomer. This study strives to understand the principles underlying the stereoselectivity of bupivacaine block with the help of mutagenesis analyses and molecular modeling simulations. Electrophysiological measurements of mutated hERG channels allowed for the identification of residues involved in bupivacaine binding and stereoselectivity. Docking and molecular mechanics simulations for both enantiomers of bupivacaine and terfenadine (a non-stereoselective blocker) were performed inside an open-state model of the hERG channel. The predicted binding modes enabled a clear depiction of ligand-protein interactions. Estimated binding affinities for both enantiomers were consistent with electrophysiological measurements. A similar computational procedure was applied to bupivacaine enantiomers towards two mutated hERG channels (Tyr652Ala and Phe656Ala). This study confirmed, at the molecular level, that bupivacaine stereoselectively binds the hERG channel. These results help to lay the foundation for structural guidelines to optimize the cardiotoxic profile of drug candidates in silico.

Patient with syncope and LQTS carrying a mutation in the PAS domain of the hERG1 channel

We report the case of a woman with syncope and persistently prolonged QTc interval. Screening of congenital long QT syndrome (LQTS) genes revealed that she was a heterozygous carrier of a novel KCNH2 mutation, c.G238C. Electrophysiological and biochemical characterizations unveiled the pathogenicity of this new mutation, displaying a 2-fold reduction in protein expression and current density due to a maturation/trafficking-deficient mechanism. The patient's phenotype can be fully explained by this observation. This study illustrates the importance of performing genetic analyses and mutation characterization when there is a suspicion of congenital LQTS. Identifying mutations in the PAS domain or other domains of the hERG1 channel and understanding their effect may provide more focused and mutation-specific risk assessment in this population.

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Identification of a novel loss-of-function calcium channel gene mutation in short QT syndrome (SQTS6)

Short QT syndrome (SQTS) is a genetically determined ion-channel disorder, which may cause malignant tachyarrhythmias and sudden cardiac death. Thus far, mutations in five different genes encoding potassium and calcium channel subunits have been reported. We present, for the first time, a novel loss-of-function mutation coding for an L-type calcium channel subunit.

Regulation of the cardiac sodium channel Nav1.5 by utrophin in dystrophin-deficient mice

Duchenne muscular dystrophy (DMD) is a severe striated muscle disease due to the absence of dystrophin. Dystrophin deficiency results in dysfunctional sodium channels and conduction abnormalities in hearts of mdx mice. Disease progression in the mdx mouse only modestly reflects that of DMD patients, possibly due to utrophin up-regulation. Here, we investigated mice deficient in both dystrophin and utrophin [double knockout (DKO)] to assess the role of utrophin in the regulation of the cardiac sodium channel (Na(v)1.5) in mdx mice.

Cardiac Sodium Channel Nav1.5 Mechanosensitivity is Inhibited by Ranolazine

The cardiac action potential (AP) is initiated by the depolarizing inward sodium current (I(Na)). The pore-forming subunit of the cardiac sodium channel, Na(v)1.5, is the main ion channel that conducts I(Na) in cardiac cells. Despite the large number of studies investigating Na(v)1.5, year after year, we are still learning new aspects regarding its roles in normal cardiac function and in diseased states. The clinical relevance of this channel cannot be understated. The cardiac I(Na) is the target of the class 1 anti-arrhythmic drugs(1), which are nowadays less frequently prescribed because of their well-documented pro-arrhythmic properties(2). In addition, since the first description in 1995 by Keating's group(3) of mutations in patients suffering from congenital long QT syndrome (LQTS) type 3, several hundred genetic variants in SCN5A, the gene coding for Na(v)1.5, have been reported and investigated(4). Interestingly, many of these genetic variants have been found in patients with diverse cardiac manifestations(5) such as congenital LQTS type 3, Brugada syndrome, conduction disorders, and more recently, atrial fibrillation and dilated cardiomyopathy. This impressive list underlines the importance of Na(v)1.5 in cardiac pathologies and raises the question about possible unknown roles and regulatory mechanisms of this channel in cardiac cells. Recent studies have provided experimental evidence that the function of Na(v)1.5, among many other described regulatory mechanisms(6), is also modulated by the mechanical stretch of the membrane in which it is embedded(7), thus suggesting that Na(v)1.5, like other ion channels, is "mechanosensitive". What does this mean? (SELECT FULL TEXT TO CONTINUE).

Stereoselective Inhibition of the hERG1 Potassium Channel

A growing number of drugs have been shown to prolong cardiac repolarization, predisposing individuals to life-threatening ventricular arrhythmias known as Torsades de Pointes. Most of these drugs are known to interfere with the human ether à-gogo related gene 1 (hERG1) channel, whose current is one of the main determinants of action potential duration. Prolonged repolarization is reflected by lengthening of the QT interval of the electrocardiogram, as seen in the suitably named drug-induced long QT syndrome. Chirality (presence of an asymmetric atom) is a common feature of marketed drugs, which can therefore exist in at least two enantiomers with distinct three-dimensional structures and possibly distinct biological fates. Both the pharmacokinetic and pharmacodynamic properties can differ between enantiomers, as well as also between individuals who take the drug due to metabolic polymorphisms. Despite the large number of reports about drugs reducing the hERG1 current, potential stereoselective contributions have only been scarcely investigated. In this review, we present a non-exhaustive list of clinically important molecules which display chiral toxicity that may be related to hERG1-blocking properties. We particularly focus on methadone cardiotoxicity, which illustrates the importance of the stereoselective effect of drug chirality as well as individual variations resulting from pharmacogenetics. Furthermore, it seems likely that, during drug development, consideration of chirality in lead optimization and systematic assessment of the hERG1 current block with all enantiomers could contribute to the reduction of the risk of drug-induced LQTS.

A 20-channel receive-only mouse array coil for a 3 T clinical MRI system

A 20-channel phased-array coil for MRI of mice has been designed, constructed, and validated with bench measurements and high-resolution accelerated imaging. The technical challenges of designing a small, high density array have been overcome using individual small-diameter coil elements arranged on a cylinder in a hexagonal overlapping design with adjacent low impedance preamplifiers to further decouple the array elements. Signal-to-noise ratio (SNR) and noise amplification in accelerated imaging were simulated and quantitatively evaluated in phantoms and in vivo mouse images. Comparison between the 20-channel mouse array and a length-matched quadrature driven small animal birdcage coil showed an SNR increase at the periphery and in the center of the phantom of 3- and 1.3-fold, respectively. Comparison with a shorter but SNR-optimized birdcage coil (aspect ratio 1:1 and only half mouse coverage) showed an SNR gain of twofold at the edge of the phantom and similar SNR in the center. G-factor measurements indicate that the coil is well suited to acquire highly accelerated images.