Density Of L-type Ca2 Research Articles

Inositol hexakisphosphate primes syndapin I/PACSIN 1 activation in endocytosis

Endocytosis is controlled by a well-orchestrated molecular machinery, where the individual players as well as their precise interactions are not fully understood. We now show that syndapin I/PACSIN 1 is expressed in pancreatic β cells and that its knockdown abrogates β cell endocytosis leading to disturbed plasma membrane protein homeostasis, as exemplified by an elevated density of L-type Ca2+ channels. Intriguingly, inositol hexakisphosphate (InsP6) activates casein kinase 2 (CK2) that phosphorylates syndapin I/PACSIN 1, thereby promoting interactions between syndapin I/PACSIN 1 and neural Wiskott–Aldrich syndrome protein (N-WASP) and driving β cell endocytosis. Dominant-negative interference with endogenous syndapin I/PACSIN 1 protein complexes, by overexpression of the syndapin I/PACSIN 1 SH3 domain, decreases InsP6-stimulated endocytosis. InsP6 thus promotes syndapin I/PACSIN 1 priming by CK2-dependent phosphorylation, which endows the syndapin I/PACSIN 1 SH3 domain with the capability to interact with the endocytic machinery and thereby initiate endocytosis, as exemplified in β cells.

Automated Action Potential Recording in Stem Cell-Derived Cardiomyocytes for Proarrhythmia Assessment

Development of in vitro toxicity assays based on the human stem cell-derived cardiomyocytes (SC-CMs) provides an opportunity to assess the specific cardiac safety end-points early in drug development. The cardiac action potential (AP) is an established biomarker for cardiac safety and efficacy in humans and a good detector of proarrhythmic events. However, its utility has been mainly limited by a low throughput of manual patch clamp and a restricted access to healthy human cardiomyocytes. The recent progress in automated electrophysiology combined with availability of human SC-CMs provides an exciting possibility to measure AP in human cells expressing endogenous cardiac ion channels.Electrophysiological properties of human SC-CMs were investigated with special focus on establishment of automated cell-based cardiac safety screens.For the first time assay protocols and procedures were established to measure and characterize both cardiac AP and the main underlying currents in human SC-CMs using automated electrophysiology platforms Patchliner(r) and Synchropatch(r)96 (Nanion Technologies GmbH). Employment of the higher throughput systems allowed capturing and evaluating AP and cardiac current profiles in cardiomyocyte cultures revealing specific heterogeneity. Outward potassium current mainly consisting of slow and rapid delayed rectifier and Ca2+-activated components as well as inward cardiac sodium current were recorded and characterized. Density of L-type Ca2+ current varied with the type and differentiation status of cardiomyocytes. The inward rectifier potassium current and a pacemaker current could also be measured. These results demonstrate that functional cardiac currents and appropriate AP present in human SC-CMs can be successfully recorded by automated electrophysiology systems.The automated cardiac AP measurements in human SC-CMs exemplify a novel screening method for early and sensitive detection of multiple ion channel effects in vitro completing the current core battery hERG assay and animal ECG measurements in pre-clinical safety assessment.

Quantification of repolarization reserve to understand interpatient variability in the response to proarrhythmic drugs: A computational analysis

"Repolarization reserve" is frequently invoked to explain why potentially proarrhythmic drugs cause, across a population, a range of changes to cardiac action potentials (APs). However, the mechanisms underlying this interindividual variability are not understood quantitatively. The purpose of this study was to perform a novel analysis of mathematical models of ventricular myocytes to quantify repolarization reserve and gain insight into the factors responsible for variability in the response to proarrhythmic drugs. In several models of human or canine ventricular myocytes, variability was simulated by randomizing model parameters and running repeated simulations. With each randomly generated set of parameters, APs before and after simulated 75% block of the rapid delayed rectifier current (I(Kr)) were calculated. Multivariable regression was performed to determine how much each model parameter attenuated or exacerbated the AP prolongation caused by the I(Kr)-blocking drug. Simulations with a human ventricular myocyte model suggest that drug response is influenced most strongly by (1) the density of I(Kr), (2) the density of slow delayed rectifier current I(Ks), (3) the voltage dependence of I(Kr) inactivation, (4) the density of L-type Ca2+ current, and (5) the kinetics of I(Ks) activation. The analysis also identified mechanisms underlying nonintuitive behavior, such as ionic currents that prolong baseline APs but decrease drug-induced AP prolongation. Finally, the simulations provided quantitative insight into conditions that aggravate the drug response, such as silent ion channel mutations and heart failure. These modeling results provide the first thorough quantification of repolarization reserve and improve our understanding of interindividual variability in adverse drug reactions.

Impaired Local Calcium Signaling in Primary Cultured Adult Rat Ventricular Myocytes

Although cultured adult cardiac myocytes have been adopted in studying protein functions in combination with cell-level genetic modifications, cellular alterations by culturing itself need to be clarified to understand real function of the protein genetically altered. We systematically compared contractile properties, calcium ion (Ca2+) signaling, transverse (t)-tubules, ryanodine receptor distributions between freshly isolated and two-days cultured adult rat ventricular myocytes. Density of t-tubules was remarkably decreased by culture. In cultured myocytes, cell shortenings were attenuated by ∼60% and relaxation was slowed. Consistently, magnitudes of action potential-induced Ca2+ transients were decreased to ∼50% and decays of the Ca2+ transients were retarded by culture. In cultured cells, density of L-type Ca2+ current was reduced to ∼40% and its inactivation was retarded. The latter is consistent with smaller Ca2+ transients in cultured group. However, sarcoplasmic reticulum Ca2+ contents were not different between two groups. To know the mechanism for smaller Ca2+ transient in cultured cells we examined Ca2+ sparks in these two groups of cells. The frequency of spontaneous Ca2+ sparks was significantly decreased by culturing. The amplitude, duration, and time-to-peak of individual Ca2+ sparks were not different between the two groups. Mean spark width was two-fold larger in cultured cells compared with freshly isolated cells. Quantitative analysis of immunofluorescence revealed shortening of longitudinal spacing between RyR2 clusters, and less dense and disorganized distributions of RyR2 clusters in cultured cells, which may be related to lower frequency of sparks in these cells. These results provide evidence on significant difference in Ca2+ sparks as well as excitation-contraction coupling in primary cultured adult ventricular myocytes. (This work was supported by National Research Foundation of Korea grants funded by the Ministry of Education, Science and Technolgy (2010-0000070).)

Understanding Pro-Arrhythmic Effects of Drugs using Computational Models and Parameter Sensitivity Analysis

Increased risk of ventricular arrhythmia is a dangerous side effect of many pharmacological agents. Often, drugs block the K+ channel responsible for rapid delayed rectifier current (IKr), leading to delayed of action potentials, prolongation of the QT interval, and increased arrhythmia risk. Some drugs, however, block IKr potently but are nonetheless safe. In addition, the effects of a drug on action potential morphology depend not just on the channel that is blocked, but also on the other channels present in the cell, a concept known as repolarization reserve. We have gained new insight into both phenomena through analysis of ventricular myocyte computational models with parameter randomization and multivariable regression. The most likely targets of a non-specific drug can be deduced from the relationship between action potential duration and drug concentration, if the data are compared to the parameter sensitivity analysis of an appropriate electrophysiological model. Simulations also provide insight into how the electrophysiological substrate of a ventricular myocyte affects the response of the cell to a drug that blocks IKr. Such a drug always prolongs action potential duration, but the effects can be either exacerbated or attenuated, depending on the characteristics of the other ion channels present. Specifically, simulations with a common human ventricular myocyte model suggest that the most important factors influencing the response to an IKr-blocking drug are: 1) the underlying density of IKr; 2) the density of slow delayed rectifier current IKs; 3) the voltage-dependence of IKr inactivation; 4) the density of L-type Ca2+ current, and 5) the kinetics of IKs activation. These simulations provide for a quantification of the important concept of reserve, and demonstrate how analysis of computational models can provide insight into the factors that influence adverse drug reactions.

Rab11b GTPase Is Expressed In Ventricular Myocardium And Regulates Trafficking Of L-type Ca2+ Channels

Alterations in the density of L-type Ca2+ channels (LTCCs) have been implicated in several cardiac diseases; however, the mechanisms directing the trafficking of LTCCs have not been well defined. Nearly all steps of intracellular transport are regulated by Rab GTPases, a family of over 60 proteins with varying tissue distributions. The purpose of the present study was to determine if Rab11b, a protein localized to the endocytic recycling compartment, is expressed in the heart, and if it regulates membrane trafficking of LTCCs. RT-PCR using primers specific for Rab11b yielded a 280 base pair product from cDNA prepared from purified mouse ventricular myocytes and Western blot analysis using Rab11b-specific antibody detected a single band at 28 kDa in left ventricular lysate. To test whether Rab11b regulates trafficking of LTCCs, we expressed WT Rab11b or a dominant negative GDP-locked S25N mutant fused to GFP in HEK293 cells along with the pore forming Cav1.2 and auxiliary β2CN4 subunits. L-type currents were recorded in 10 mM Ba2+ (IBa,L) using the whole cell ruptured patch clamp technique. Expression of GFP-Rab11bS25N increased mean peak inward IBa,L from 42±3 pA/pF (n=14) in GFP-expressing control cells to 68±6 pA/pF (n=12), a 64.2% increase (p<0.005). Mean peak IBa,L of GFP-Rab11bWT cells (43±6 pA/pF, n=4) was not significantly different than the GFP control group. To determine whether Cav1.2 and Rab11b exist in a protein complex, HEK293 cells were transfected with HA-tagged Cav1.2, β2CN4, and GFP-Rab11bWT or S25N and lysates were used for immunoprecipitation with HA antibody. Interestingly, GFP-Rab11bS25N but not GFP-Rab11bWT associated with HA-Cav1.2, suggesting Rab11b preferentially interacts with Cav1.2 in its GDP-bound form. These data demonstrate that a Rab11b-dependent pathway is important for proper maintenance of LTCC density at the surface membrane.

Synaptic Amplification Versus Bistability in Motoneuron Dendritic Processing: A Top-Down Modeling Approach

Motoneurons have been shown to exhibit both bistable firing and synaptic amplification. Both of these behaviors have generally been attributed to a single mechanism-dendritic plateau potentials based on L-type Ca(2+) conductances. However, our recent discovery of a fast-amplification mode calls this into question. Here we examine the possibility that two mechanisms underlie these behaviors, one being a slow-mode bistability mechanism (i.e., the L-type Ca(2+)-conductance-based dendritic plateaus) and the other being a theoretical fast-mode amplification mechanism. A "top-down" motoneuron model that encapsulated these and other hypotheses was developed in which these mechanisms could be explored. The resulting final model simultaneously exhibits synaptic amplification, plateau potential formation, bistable firing patterns, and current-voltage (I-V) and frequency-current (F-I) hystereses. This model suggests that amplification and plateaus are mutually exclusive in the same dendrite/dendritic branch. Thus we predict that plateau generation does not occur in all dendritic branches. This could be readily accomplished by a large degree of variation in the density of L-type Ca(2+) channels believed to underlie plateau formation in these cells with the added benefit of spreading plateau onset over a wider voltage range, as is observed experimentally.

NO ARREST NEEDED TO PREVENT CARDIAC ARREST

Most vertebrates die within a few minutes when deprived of molecular oxygen(anoxia) because the heart and brain depend on a continuous supply of oxygen. However, some cold-blooded vertebrates are remarkably anoxia-tolerant and can survive for months without oxygen. One of the keys to surviving prolonged anoxia is being able to balance energy supply and demand. One way to achieve this is to reduce energy-consuming processes of cells to a level that can be supported by the decreased amount of energy available from anaerobic respiration. Since ion pumping is energetically costly, energy can potentially be conserved by reducing the density of cellular membrane ion transport channels in excitable tissues such as the heart. Matti Vornanen and Vesa Paajanen of the University of Joensuu, Finland, set out to test this `channel arrest' hypothesis in the crucian carp (Carassius carassius), an extremely anoxia-tolerant fish that spends a third of its life in anoxic water under the ice of shallow Finno-Scandinavian ponds.The oxygen content of the water in the ponds where crucian carp live is too low for the fish to produce energy aerobically for five and a half months throughout the winter, so carp need to conserve energy during winter. Vornanen and Paajenen reasoned that since L-type Ca2+ channels regulate the force of heart muscle contraction through changes in the amount of free intracellular Ca2+, these channels are good targets for channel arrest. They hypothesized that, if crucian carp use channel arrest to conserve energy during anoxia in winter, the number of dihydropyridine receptors(subunit of the L-type Ca2+ cardiac channel that triggers channel opening) and density of L-type Ca2+ current should both be decreased in winter-captured, anoxic carp compared with summer-captured,normoxic animals.Vornanen and Paajanen captured wild carp monthly throughout an entire year and measured the number of dihydropyridine receptors and the density of L-type Ca2+ current in the ventricles of the carp's hearts to see how these changed with the seasons. Unexpectedly, they found that the number of dihydropyridine receptors in winter fish was the same as in summer fish. Thus,cardiac L-type Ca2+ channels were not downregulated by seasonal anoxia in the natural environment, suggesting that carp do not use differential expression of the Ca2+ channel protein to save energy in ion pumping or in reduced cardiac contractility during anoxia. This finding, in conjunction with the team's earlier finding that inward rectifier K+ current density of crucian carps' heart cells is also unaffected by prolonged anoxia, led Vornanen and Paajanen to conclude that crucian carp do not use channel arrest as an energy-conserving mechanism in the heart during prolonged anoxia.However, Vornanen and Paajanen did observe a 6.1-fold reduction in Ca2+ current in winter compared with summer fish, indicating that low temperatures depress Ca2+ current. They suggest that when temperatures drop, the resulting decreased Ca2+ current may allow for sufficient savings in ATP-dependent ion pumping and reduced cardiac contractility to attain a balance between energy supply and demand, providing protection against anoxia.

NEUROPROTECTIVE EFFECT OF TREATMENT WITH CALCIUM ANTAGONISTS ON HYPERTENSIVE RETINA

The influence of hypertension and of treatment with the dihydropyridine-type Ca2+ antagonists lercanidipine, manidipine, nicardipine, and nimodipine and with non dihydropyridine-type vasodilator hydralazine on retinal nervous and glial fibrillary acidic protein (GFAP) immunoreactive astrocytes were investigated in male spontaneously hypertensive rats (SHR). Normotensive Wistar-Kyoto (WKY) were used as normotensive references group. Treatment of animals with oral equi-hypotensive doses of the above compounds started at 14 weeks of age and lasted for 12 weeks. Microanatomical analysis was extended to samples of frontal cortex and occipital cortex used as reference tissue. Different compounds investigated decreased to a similar extent systolic blood pressure values with the exception of nimodipine that in spite of the high dose used exerted a less pronounced hypotensive activity. Morphological changes including reduced thickness of retina and of inner plexiform, outer nuclear and layer of inner and outer segments plus outer limiting layer, and loss of ganglionic neurons were observed.GFAP-immunoreactive astrocyte hypertrophy was also found in control SHR. These phenomena were countered by treatment by treatment with dihydropyridine-type Ca2+ antagonists and to a lesser extent by hydralazine. The different Ca2+ antagonists tested exerted a similar protective effect on retinal, but not on brain neurons. The sensitivity of retina and cerebral cortex to anti-hypertensive treatment may be related to a different density of L-type Ca2+ channels in structures investigated or to kinetic reasons. The demonstration of a neuroprotective effect of Ca2+ antagonists on retina of SHR suggests that these compounds might protect to a some extent retina from hypertensive injury.

L-type Ca2+ channels in fetal and adult ovine cerebral arteries.

Recently, we reported that, whereas in cerebral arteries of the adult a majority of norepinephrine (NE)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) comes from release of the sarcoplasmic reticulum (SR) Ca2+ stores, in the fetus the SR Ca2+ stores are relatively small, and NE-induced increase in [Ca2+]i results mainly from activation of plasma membrane L-type Ca2+ channels (20). In an effort to establish further the role of L-type Ca2+ channels in the developing cerebral arteries, we tested the hypothesis that, in the fetus, increased reliance on plasmalemmal L-type Ca2+ channels is mediated, in part, by increased L-type Ca2+ channel density. We used 3H-labeled (+)isopropyl-4-(2,1,3-benzoxadiazol-4-y1)-1,4-dihydro-(2,6-dimethyl-5-methoxycarbonyl)pyridine-3-carboxylate (PN200-110, isradipine) to measure L-type Ca2+ channel density (Bmax) in the cerebral arteries, common carotid artery (CCA), and descending aortae of fetal (approximately 140 gestation days), newborn (7-10 days), and adult sheep. In the cerebral and common carotid arteries, B(max) values (fmol/mg protein) of fetuses and newborns were significantly greater than those of adults. Western immunoblotting assay also revealed that the density of L-type Ca2+ channel protein in the cerebral arteries and CCA was about twofold greater in the fetus than the adult. Finally, compared with the adult, fetal cerebral arteries demonstrated a significantly greater maximum tension and [Ca2+]i in response to stimulation with the L-type Ca2+ channel agonist Bay K 8644. In addition, Bay K 8644-stimulated fetal vessels demonstrated a maximal tension and [Ca2+]i similar to that observed in response to stimulation with 10(-4) NE. These results support the idea that fetal cerebrovascular smooth muscle relies more on extracellular Ca2+ and L-type Ca2+ channels for contraction than does the adult and that this increased reliance is mediated, in part, by greater L-type Ca2+ channel density. This may have important implications in the regulation of cerebral blood flow in the developing organism.

Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor.

Excitation-contraction coupling in skeletal muscle involves a voltage sensor in the plasma membrane which, in response to depolarization, causes an intracellular calcium-release channel to open. The skeletal isoform of the ryanodine receptor (RyR-1) functions as the Ca2+-release channel and the dihydropyridine receptor (DHPR) functions as the voltage sensor and also as an L-type Ca2+ channel. Here we examine the possibility that there is a retrograde signal from RyR-1 to the DHPR, using myotubes from mice homozygous for a disrupted RyR-1 gene (dyspedic mice). As expected, we find that there is no excitation-contraction coupling in dyspedic myotubes, but we also find that they have a roughly 30-fold reduction in L-type Ca2+-current density. Injection of dyspedic myotubes with RyR-1 complementary DNA restores excitation-contraction coupling and causes the density of L-type Ca2+ current to rise towards normal. Despite the differences in Ca2+-current magnitude, measurements of charge movement indicate that the density of DHPRs is similar in dyspedic and RyR-1-expressing myotubes. Our results support the possibility of a retrograde signal by which RyR-1 enhances the function of DHPRs as Ca2+ channels.

Altered functions of ion channels in diabetic beta cells.

Selective impairment of glucose-induced insulin secretion and hyper-responsiveness to arginine are known features of GK rats, a genetic model of NIDDM. We focus on the ionic mechanism underlying these phenomena using patch-clamp techniques. Pancreatic islets were isolated from male GK rats and age-matched control Wistar rats and were subjected to dispersion and culture. Single channel recordings of KATP channels were performed using either on-cell mode or inside-out patch mode. Ca2+ channel currents were recorded under conventional whole-cell mode. In GK beta cells, ATP sensitivity of KATP channels itself was not altered, although glucose-induced closure of KATP channels was severely impaired. Among substrates for fuel metabolism, only dehydroxyacetone (DHA) reproduced this anomaly. On the other hand, current densities of L-type Ca2+ channels were increased in GK beta cells. Since DHA is a known substrate for glycerol phosphate shuttle, current data suggest that major metabolic deficit of GK beta cells resides in this shuttle. On the other hand, increased L-type Ca2+ channel activities might be an ionic basis for augmented insulin response to nonglucose depolarizing stimuli in GK beta cells.

Regulation of the L-type calcium channel alpha-1 subunit by chronic depolarization in the neuron-like PC12 and aortic smooth muscle A7r5 cell lines.

The regulation of L-type voltage-dependent Ca2+ channels by chronic depolarization was studied in the aortic smooth muscle A7r5 and neuron-type PC12 cell lines, by probing the expression and the functional state of their constitutive alpha-1 subunits. PC12 cells showed, after prolonged exposure to a high-K+ depolarizing solution, a 25% reduction of the functional Ca2+ channel density which was accompanied by a decrease of the alpha-1 subunit mRNA expression. In A7r5 cells submitted to a similar protocol of depolarization, 45Ca2+ uptake measurements revealed a fall in the functional activity of L-type Ca2+ channels which was not related to a modulation of their mRNA expression, but arose from a long-term voltage-dependent channel inactivation. Accordingly, the lag time and the mechanisms of recovery were different in the two cell types. In PC12 cells, when restoring physiological culture conditions, de novo synthesis of alpha-1 subunits allowed the recovery of the original density of L-type Ca2+ channels at the membrane surface. As for the A7r5 cells, we showed that after chronic depolarization, the complete restoration of the resting membrane potential and the related Ca2+ channel activity required a 2-day incubation in physiological medium and could probably be related to a normalization of the increased intracellular Ca2+ concentration. In contrast, it is noteworthy that, in PC12 cells, the only transient increase of intracellular Ca2+ content in the first hours of depolarization could account for the long-term down-regulation of L-type Ca2+ channels.

Metabolic inhibition of ICa,L and IK differs in feline left ventricular hypertrophy

To determine the intrinsic responsiveness of hypertrophied myocardium, electrophysiological properties of endocardial myocytes enzymatically dissociated from normal and hypertrophied feline left ventricle (LV) were compared during metabolic inhibition by 1 mM CN-. Chronic pressure overload was induced under surgical anesthesia. A single-pipette, whole cell clamp method was used to record action potential and membrane currents. Before CN-, action potential duration (APD) values at 90% repolarization (APD90) and at 0 mV (APD0mV) were significantly longer in hypertrophied cells. The current density of L-type Ca2+ currents (ICa,L) was not significantly different, whereas the time constant of the slow component (tau s) of ICa,L inactivation was significantly longer in hypertrophied cells. The current density of delayed rectifier K+ current (IK) was significantly smaller, the fast component (tau f) and tau s of IK activation were delayed, and those of IK deactivation were enhanced in hypertrophied cells. During exposure to CN-, APD shortened significantly more in hypertrophied cells; amplitude of ICa,L decreased, and the tau f and tau s of ICa,L inactivation shortened only in hypertrophied cells. However, IK showed no significant differences in changes in amplitude or kinetics during CN- exposure between normal and hypertrophied cells. Thus enhanced APD responsiveness to CN- is an intrinsic property of hypertrophied LV cells and ICa,L appears to be particularly affected by metabolic perturbation in such cells.