Physiologic gating properties of unitary cardiac L-type Ca channels

The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca²⁺ ions through sarcolemmal L-type Ca²⁺ channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca²⁺ ions permeating the channel. Unitary LCC currents were recorded with 2 mM external Ca²⁺ ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of −12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53 ± 0.02 ms at −30 mV to 1.08 ± 0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca²⁺ ion entry. Thus, the average amount of Ca²⁺ ions entering through an individual LCC opening (∼300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca²⁺ ion concentration. Moreover, they provide insight into local Ca²⁺-induced Ca²⁺ release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes.     

Adenylyl cyclase/cAMP-PKA-mediated phosphorylation of basal L-type Ca(2+) channels in mouse embryonic ventricular myocytes

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (I_Ca,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca²⁺ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5-E11.5) than late developmental stage (LDS, E16.5-E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, I_Ca,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca²⁺ influx via LTCCs on AC activity, leading to the inability to inhibit AC.     

Testosterone is a potent inhibitor of L-type Ca(2+) channels

Testosterone administration is beneficial in alleviating myocardial ischaemia in men with significant coronary artery disease (CAD), a condition which is associated with hypotestosteronaemia. Infusion of physiological concentrations of testosterone into coronary arteries at angiography results in rapid vasodilatation in patients with CAD. Whilst the cardiovascular benefits of testosterone have long been documented, the underlying mechanism(s) have not yet been revealed. Here, we have investigated whether testosterone might act like widely prescribed antihypertensive dihydropyridines, as an endogenous Ca(2+) channel antagonist. To do this, we used the whole-cell patch-clamp technique to record Ca(2+) currents from the A7r5 smooth muscle cell line and HEK 293 cells stably expressing either L- or T-type Ca(2+) channels. We demonstrate that testosterone directly inhibited both native and human recombinant vascular L-type Ca(2+) channels in a manner that was voltage-independent and, crucially, displayed an IC(50) value of 38 nM, a value within the physiological range. At higher (supraphysiological) concentrations both native and human recombinant T-type channels were also inhibited by testosterone. Our data indicate that testosterone acts like widely prescribed antihypertensive dihydropyridines to reduce Ca(2+) influx into vascular smooth muscle and so promote vasodilation. This effect is likely to account for its beneficial cardiovascular actions.     

The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes

We examined the effects of the mitochondrial Ca(2+)-activated K(+) (mitoBK(Ca)) channel activator NS 1619 on L-type Ca(2+) channels in rat ventricular myocytes. NS 1619 inhibited the Ca(2+) current in a dose-dependent manner. NS 1619 shifted the activation curve to more positive potentials, but did not have a significant effect on the inactivation curve. Pretreatment with inhibitors of membrane BK(Ca) channel, mitoBK(Ca) channel, protein kinase C, protein kinase A, and protein kinase G had little effect on the Ca(2+) current and did not alter the inhibitory effect of NS 1619 significantly. The application of additional NS 1619 in the presence of isoproterenol, a selective beta-adrenoreceptor agonist, reduced the Ca(2+) current to approximately the same level as a single application of NS 1619. In conclusion, our results suggest that NS 1619 inhibits the Ca(2+) current independent of the mitoBK(Ca) channel and protein kinases. Since NS 1619 is widely used to study mitoBK(Ca) channel function, it is essential to verify these unexpected effects of NS 1619 before experimental data can be interpreted accurately.     

Neuroprotective effects of ginseng saponins against L-type Ca2+ channel-mediated cell death in rat cortical neurons

In the present study, we have examined any possible involvement of L-type Ca²⁺ channels in ginseng-mediated neuroprotective actions. Exposure to a 50 mM KCl (high-K) produced neuronal cell death, which was blocked by a selective L-type Ca²⁺ channel blocker in cultured cortical neurons. When cultured cells were co-treated with ginseng total saponin (GTS) and high-K, GTS reduced high-K-induced neuronal death. Using Ca²⁺ imaging techniques, we found that GTS inhibited high-K-mediated acute and long-term [Ca²⁺]_i changes. These GTS-mediated [Ca²⁺]_i changes were diminished by nifedipine. Furthermore, GTS-mediated effects were also diminished by a saturating concentration of Bay K (10 μM). After confirming the protective effect of GTS using a TUNEL assay, we found that ginsenosides Rf and Rg₃ are active components in ginseng-mediated neuroprotection. These results suggest that inhibition of L-type Ca²⁺ channels by ginseng could be one of the mechanisms for ginseng-mediated neuroprotection in cultured rat cortical neurons.     

Centrin is essential for the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels

Voltage-gated Ca(2+) channels play a critical role in controlling Ca(2+) entry in various cells. Ciliary reversal in Paramecium depends on the Ca(2+) influx through voltage-gated Ca(2+) channels on the ciliary membrane. One of the voltage-gated Ca(2+) channel mutants in Paramecium caudatum, cnrC, neither produces Ca(2+) action potentials nor responds to any depolarizing stimuli. Here, we report that the cnrC(+) gene product is P. caudatum centrin (Pccentrin1p), a member of the Ca(2+)-binding EF-hand protein superfamily. The Pccentrin1p gene of cnrC was found to contain a single-base deletion, a mutation that caused the loss of the fourth EF-hand of Pccentrin1p. Moreover, the wild-type Ca(2+) channel function was impaired by Pccentrin1p gene silencing, leading to the loss of current-evoked Ca(2+) action potentials and stimulated ciliary reversal. These results demonstrate that Pccentrin1p is indispensable for the activity of the voltage-gated Ca(2+) channels that control ciliary reversal in Paramecium.     

The protein kinase inhibitor, staurosporine, inhibits L-type Ca2+ current in rabbit atrial myocytes

A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca(2+) channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I-V relationship of LTCC (from -16.8+/-2.55 to -3.74+/-1.22pApF(-1) at 0 mV). The level of L-type Ca(2+) current (I(Ca,L)) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I(Ca,L) in a dose-dependent manner with a K(d) value of 61.98+/-6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 microM) and chelerythrine (3 microM), and PKA inhibitor, PKA-IP (5 microM), did not show any inhibitory effect on I(Ca,L). Additional application of ST in the presence of isoproterenol (1 microM), a selective beta-adrenoreceptor agonist, reduced peak I(Ca,L) (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.     

Regulation of L-type Ca2+ channels in the heart: Overview of recent advances

Regulation of L-type Ca²⁺ channels is complex, because many factors, such as phosphorylation, divalent cations, and proteins, specified or unspecified, have been shown to affect the channel activities. An additional complication is that these factors interact with one another to achieve final outcomes. Recent molecular technologies have helped to shed light on the mechanisms governing the activity of L-type Ca²⁺ channels. In this review article, three major topics concerning regulation of L-type Ca²⁺ channels in the heart are discussed, i.e. c-AMP dependent channel phosphorylation, role of magnesium (Mg²⁺), and the phenomenon of channel run-down.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca2+ sparks are the elementary events of intracellular Ca2+ release from the sar-coplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca2+ channel activation contributes to the genesis of spontaneous Ca2+ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca2+ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI2 which inhibits spontaneous L-type Ca2+ channel activation, the rate of occurrence of spontaneous Ca2+ sparks was halved from 4.20 to 2.04 events/(100 μm·s), with temporal and spatial properties of individual Ca2+ sparks unchanged. Analysis of the Cd2+-sensitive spark production revealed an open probability of-10-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca2+ channels in resting heart cells contribute significantly to the production of spontaneous Ca2+ sparks.     

Suppression of L-type Ca current by fluid pressure in rat ventricular myocytes: Possible role of Cl–OH exchange

The application of fluid pressure (FP) in ventricular myocytes using pressurized fluid flow inhibits L-type Ca²⁺ current (I_Ca), with approximately 80% of this effect coming through the enhancement of Ca²⁺ releases from the sarcoplasmic reticulum. In the present study, we explored the remaining mechanisms for the inhibition of I_Ca by FP. Since FP significantly increases H⁺ concentration and H⁺ is known to inhibit I_Ca, we examined whether pH regulation plays a role in the inhibitory effect by FP on I_Ca. A flow of pressurized (∼16.3 dyne/cm²) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes for which the I_Ca was monitored using whole-cell patch-clamp under HEPES-buffered conditions. Extracellular application of the alkalizing agent, NH₄Cl (20 mM), enhanced I_Ca by ∼34% in the control conditions while increasing I_Ca significantly less (by ∼21%) in FP-pretreated myocytes, suggesting an inhibition of the effect of NH₄Cl on I_Ca possibly by FP-induced acidosis. Application of DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid, 500 μM), which blocks exchange but not Cl⁻–OH⁻ exchange, did not alter the inhibitory effect of FP on I_Ca. Replacement of external Cl⁻ with aspartate attenuated the inhibitory effect of FP on I_Ca. In highly Ca²⁺-buffered cells, where Ca²⁺-dependent inhibition of I_Ca was minimized, the external Cl⁻ removal eliminated the inhibitory effect of FP on I_Ca. These results suggest that the decrease of I_Ca in the presence of FP is at least partly caused by intracellular acidosis via activation of Cl⁻–OH⁻ exchange in rat ventricular myocytes.     

Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca²⁺ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca²⁺ currents (I_Ca(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type I_Ca was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type I_Ca (I_Ca(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between I_Ca(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of I_Ca(T) as well as the large window current and proportion of myocytes showing I_Ca(T) may explain in part Ca²⁺ overload observed in cardiomyopathic heart cells of the hamster.     

Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner [1]. To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.     

Methamphetamine directly accelerates beating rate in cardiomyocytes by increasing Ca entry via L-type Ca channel

Methamphetamine induces several cardiac dysfunctions, which leads to arrhythmia, cardiac failure and sudden cardiac death. Although these cardiac alterations elicited by methamphetamine were thought to be due to an indirect action of methamphetamine, namely, an excessive catecholamine release from synaptic terminals, while it seems likely that methamphetamine directly modulates the functioning of cardiomyocytes independent of neurotransmitters. However, the direct effects of methamphetamine on cardiomyocytes are still not clear. We show that methamphetamine directly accelerates the beating rate and alters Ca²⁺ oscillation pattern in cultured neonatal rat cardiomyocytes. Adrenergic receptor antagonists did not block the methamphetamine-induced alterations in cardiomyocytes. Treatment with a ryanodine receptor type 2 inhibitor and a sarcoplasmic reticulum Ca²⁺-ATPase inhibitor did not affect these responses, either. In contrast, the L-type Ca²⁺ channel inhibitor nifedipine eradicated these responses. Furthermore, methamphetamine elevated the internal free Ca²⁺ concentration in HEK-293T cells stably transfected with the L-type Ca²⁺ channel α1C subunit. In neonatal rat cardiomyocytes, methamphetamine accelerates beating rate and alters Ca²⁺ oscillation pattern by increasing Ca²⁺ entry via the L-type Ca²⁺ channels independent of any neurotransmitters.     

H-mediated coupling of transmembrane Ca fluxes in vegetative Trichoderma viride mycelia suggested by the study of ageing and adaptation to extreme Ca concentrations

The adaptation to extreme concentrations of Ca²⁺ and its consequence on the properties of the ⁴⁵Ca²⁺ transport were studied in submerged mycelia of Trichoderma viride. The adaptation to low [Ca²⁺]_o did not cause changes in kinetic parameters of the ⁴⁵Ca²⁺ influx but the adaptation to high [Ca²⁺]_o increased the K_M(Ca2+). The V_max of the ⁴⁵Ca²⁺ influx decreased with the age of (non-adapted) mycelia with concomitant decrease of the K_M(Ca2+) these changes were prevented in mycelia adapted to high Ca²⁺. High [Ca²⁺]_o decreased the stimulation by the uncoupler, 3, 3′, 4′, 5-tetrachloro salicylanilide (TCS) (30 μM), as compared to the control, whereas the Ca²⁺ chelator, EGTA, stimulated it. In the aged mycelia, the stimulation by TCS of the ⁴⁵Ca²⁺ influx faded away, in parallel with the activity of the H⁺-ATPase. The ⁴⁵Ca²⁺ efflux from mycelia was affected by TCS in a similar way as the ⁴⁵Ca²⁺ influx. The results demonstrate the adaptive responses of transport processes participating in the mycelial Ca²⁺ homeostasis and ageing are in agreement with a notion that both Ca²⁺-influx and-efflux are coupled by the H⁺-homeostasis at the plasma membrane.     

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca channels in the membrane

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca²⁺ homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca²⁺ in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca²⁺polyP) complexes. PHB(Ca²⁺polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA⁻ strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.     

Characterization of cardiomyocyte excitation-contraction coupling in the FVB/N-C57BL/6 intercrossed "chocolate" brown mice

Mice are extensively used for gene modification research and isolated cardiomyocytes are essential for evaluation of cardiac function without interference from non-myocyte contribution. This study was designed to characterize cardiomyocyte excitation-contraction coupling in FVB/N-C57BL/6 intercrossed brown mice. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix softedge system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening and relengthening (+/- dL/dt), intracellular Ca(2+) rise and decay rate. Resting cell length was longer in age- and gender-matched C57BL/6 and brown mice compared to FVB strain. PS and +/- dL/dt were significantly lower in brown mice compared to FVB/N and C57BL/6 groups. TPS was shortened in C57BL/6 mice and TR(90) was prolonged in brown mice compared to other groups. Resting intracellular Ca(2+) level and single exponential intracellular Ca(2+) decay constant were comparable among all three mouse lines. Rise in intracellular Ca(2+) in response to electrical stimulus was higher in C57BL/6 mouse myocytes whereas bi-exponential intracellular Ca(2+) decay was faster in brown mice. Myocytes from all three groups exhibited similar fashion of reduction in PS in response to increased stimulus frequency. These data suggest that inherent differences in cardiomyocyte excitation-contraction coupling exist between strains, which may warrant caution when comparing data from these mouse lines.     

Effect of Isoproterenol on the L-type Ca2+ Current in Cardiac Cells from Rats and Hybernating Ground Squirrels

The perforated patch clamp method was used to study the effect of the agonist of beta-adrenoreceptors isoproterenol on L-type Ca²⁺ current in cardiocytes of rats and ground squirrels in two states: active and hibernating. It is shown that isoproterenol exerts a dual effect on Ca²⁺ currents of rats and ground squirrels in the active state: at V _h = –50 mV, the current increases, whereas at V _h = –30 mV, it decreases. In hibernating ground squirrels, the dual effect of isoproterenol is not observed: isoproterenol increases Ca²⁺ current at any V _h values. The hypothesis is put forward that, during the entrance of ground squirrels into hibernation, the phosphorylation of one of the sites (not cAMP-dependent) of L-type Ca²⁺ channels is blocked.     

Calpain2 protease: A new member of the Wnt/Ca pathway modulating convergent extension movements in Xenopus

Calpains are a family of calcium-dependent intracellular cysteine proteases that regulate several physiological processes by limited cleavage of different substrates. The role of Calpain2 in embryogenesis is not clear with conflicting evidence from a number of mouse knockouts. Here we report the temporal and spatial expression of Calpain2 in Xenopus laevis embryos and address its role in Xenopus development. We show that Calpain2 is expressed maternally with elevated expression in neural tissues and that Calpain2 activity is spatially and temporally regulated. Using a Calpain inhibitor, a dominant negative and a morpholino oligonoucleotide we demonstrate that impaired Calpain2 activity results in defective convergent extension both in mesodermal and neural tissues. Specifically, Calpain2 downregulation results in loss of tissue polarity and blockage of mediolateral intercalation in Keller explants without affecting adherens junction turnover. We further show that Calpain2 is activated in response to Wnt5a and that the inhibitory effect of Wnt5a expression on animal cap elongation can be rescued by blocking Calpain2 function. This suggests that Calpain2 activity needs to be tightly regulated during convergent extension. Finally we show that expression of Xdd1 blocks the membrane translocation of Calpain2 suggesting that Calpain2 activation is downstream of Dishevelled. Overall our data show that Calpain2 activation through the Wnt/Ca²⁺ pathway and Dishevelled can modulate convergent extension movements.     

Insecticidal effects of Flourensia oolepis Blake (Asteraceae) essential oil

Flourensia oolepis Blake (Asteraceae) essential oil had a complex chemical composition with τ-muurolene (6.14%), santolinetriene (6.22%), 2-methylene-4,8,8-trimethyl-4-vinyl-bicyclo[5.2.0]nonane (10.15%), δ-cadinene (10.27%) and γ-gurjunene (20.69%) comprising more than 50% of the oil. This oil had repellent and toxic effects on Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) adults, acting as a contact toxin. Myzus persicae (Sulzer) (Homoptera: Aphididae) and Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) adults showed behavioral sensibility to this oil.