Regulation of Cardiac Cav1.2 Channels by Calmodulin.

Cav1.2 Ca2+ channels, a type of voltage-gated L-type Ca2+ channel, are ubiquitously expressed, and the predominant Ca2+ channel type, in working cardiac myocytes. Cav1.2 channels are regulated by the direct interactions with calmodulin (CaM), a Ca2+-binding protein that causes Ca2+-dependent facilitation (CDF) and inactivation (CDI). Ca2+-free CaM (apoCaM) also contributes to the regulation of Cav1.2 channels. Furthermore, CaM indirectly affects channel activity by activating CaM-dependent enzymes, such as CaM-dependent protein kinase II and calcineurin (a CaM-dependent protein phosphatase). In this article, we review the recent progress in identifying the role of apoCaM in the channel 'rundown' phenomena and related repriming of channels, and CDF, as well as the role of Ca2+/CaM in CDI. In addition, the role of CaM in channel clustering is reviewed.

Apelin/ELABELA-APJ system in cardiac hypertrophy: Regulatory mechanisms and therapeutic potential.

Heart failure is one of the most significant public health problems faced by millions of medical researchers worldwide. And pathological cardiac hypertrophy is considered one of the possible factors of increasing the risk of heart failure. Here, we introduce apelin/ELABELA-APJ system as a novel therapeutic target for cardiac hypertrophy, bringing about new directions in clinical treatment. Apelin has been proven to regulate cardiac hypertrophy through various pathways. And an increasing number of studies on ELABELA, the newly discovered endogenous ligand, suggest it can alleviate cardiac hypertrophy through mechanisms similar or different to apelin. In this review, we elaborate on the role that apelin/ELABELA-APJ system plays in cardiac hypertrophy and the intricate mechanisms that apelin/ELABELA-APJ affect cardiac hypertrophy. We also illuminate and make comparisons of the newly designed peptides and small molecules as agonists and antagonists for APJ, updating the breakthroughs in this field.

The changes of serum zinc, copper, and selenium levels in epileptic patients: a systematic review and meta-analysis.

INTRODUCTION: It is widely accepted that trace elements have been implicated in various metabolic processes. Valproic acid (VPA) is a remarkably safe and effective antiepileptic drug. There is no consensus option regarding the fluctuations in serum zinc (Zn), copper (Cu), and selenium (Se) in epileptic patients treated with VPA. We applied a meta-analysis to systematically assess the effects of VPA on serum ions in these patients. AREAS COVERED: In this study, we performed a meta-analysis of the changes in serum Zn, Cu, and Se levels in human samples of healthy controls, epileptic patients, and patients treated with VPA. Twenty-two published analyzable studies were selected by searching the databases of PubMed, China National Knowledge Infrastructure (CNKI), Google Scholar, Web of Science, EMBASE, WAN FANG and Vip. EXPERT OPINION: Serum Se levels in epileptic patients were decreased compared to healthy controls. Serum Zn levels in patients with VPA treatment were significantly lower than those in epileptic patients. The results of this meta-analysis are instructive for the intake of trace elements such as Zn, Cu, and Se in the diet balance of patients with epilepsy treated with VPA. Meanwhile, this study provides a theoretical basis for the combined use of other drugs that affect the intake and absorption of trace elements and VPA.

Calmodulin mutant in central linker reduces the binding affinity with PreIQ and IQ while interacting with CaV1.2 channels.

Calmodulin (CaM) was reported to interact with PreIQ and IQ of CaV1.2 channels, but to date, no explicit binding sites of CaM were illustrated. Therefore, in the present study, we firstly used MOE (Molecular Operating Environment) for protein-protein docking and we found that the most likely residues of CaM that play an important role in the interface are concentrated in central linker region. Next we examined the binding properties of CaM and its mutants to PreIQ and IQ by GST pull-down assays. Here we confirmed that CaM binds to PreIQ and IQ in a concentration-dependent and [Ca2+]-dependent manner. However, silencing the effect of N-lobe and C-lobe by mutating two Ca2+ binding sites of each lobe abolished [Ca2+]-dependence of CaM binding, but could not influence the combination. And the mutant in central linker reduced the binding of CaM/PreIQ and CaM/IQ especially at low [Ca2+]. We confirmed that N-lobe and C-lobe play vital role in sensing the change of Ca2+, and found that the central linker of CaM is involved in the binding of CaM to CaV1.2 channels in particular at low [Ca2+], not only participates in the combination with PreIQ, but also with IQ.

The LQT-associated calmodulin mutant E141G induces disturbed Ca2+-dependent binding and a flickering gating mode of the CaV1.2 channel.

Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca2+ channel isoform CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the CaV1.2 channel has not been determined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of the CaV1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on CaV1.2 channel activity. We found that the maximum binding (Bmax) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH2-terminal peptide) was decreased (by 17.71-59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca2+-dependent increase in Bmax became slower with the combination of CaM-E141G + PreIQ and IQ but faster in the case of NT. Functionally, CaM-WT and CaM-E141G at 500 nM Ca2+ decreased CaV1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca2+, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the CaV1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the CaV1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the CaV1.2 channel and, thus, the development of LQTS. The present study is the first to investigate the detailed binding properties and single-channel gating mode induced by the interaction of CaM-E141G with the CaV1.2 channel.

Astragaloside IV Inhibits Membrane Ca[Formula: see text] Current but Enhances Sarcoplasmic Reticulum Ca[Formula: see text] Release.

Astragaloside IV (AS-IV) is one of the active ingredients in Astragalus membrananceus (Huangqi), a traditional Chinese medicine. The present study investigated the effects of AS-IV on Ca[Formula: see text] handling in cardiac myocytes to elucidate its possible mechanism in the treatment of cardiac disease. The results showed that AS-IV at 1 and 10[Formula: see text][Formula: see text]M reduced KCl-induced [Ca[Formula: see text]]i increase ([Formula: see text] from 1.33[Formula: see text][Formula: see text][Formula: see text]0.04 (control, [Formula: see text] 28) to 1.22[Formula: see text][Formula: see text][Formula: see text]0.02 ([Formula: see text], [Formula: see text] 29) and 1.22[Formula: see text][Formula: see text][Formula: see text]0.02 ([Formula: see text] 0.01, [Formula: see text]), but it enhanced Ca[Formula: see text] release from SR ([Formula: see text] from 1.04[Formula: see text][Formula: see text][Formula: see text]0.01 (control, [Formula: see text]) to 1.44[Formula: see text][Formula: see text][Formula: see text]0.03 ([Formula: see text], [Formula: see text]) and 1.60[Formula: see text][Formula: see text][Formula: see text]0.04 ([Formula: see text] 0.01, [Formula: see text]0), in H9c2 cells. Similar results were obtained in native cardiomyocytes. AS-IV at 1 and 10[Formula: see text][Formula: see text]M inhibited L-type Ca[Formula: see text] current ([Formula: see text] from [Formula: see text]4.42[Formula: see text][Formula: see text][Formula: see text]0.58 pA/pF of control to [Formula: see text]2.25[Formula: see text][Formula: see text][Formula: see text]0.12 pA/pF ([Formula: see text] 0.01, [Formula: see text] 5) and [Formula: see text]1.78[Formula: see text][Formula: see text][Formula: see text]0.28 pA/pF ([Formula: see text] 0.01, [Formula: see text] 5) respectively, when the interference of [Ca[Formula: see text]]i was eliminated due to the depletion of SR Ca[Formula: see text] store by thapsigargin, an inhibitor of Ca[Formula: see text] ATPase. Moreover, when BAPTA, a rapid Ca[Formula: see text] chelator, was used, CDI (Ca[Formula: see text]-dependent inactivation) of [Formula: see text] was eliminated, and the inhibitory effects of AS-IV on ICaL were significantly reduced at the same time. These results suggest that AS-IV affects Ca[Formula: see text] homeostasis through two opposite pathways: inhibition of Ca[Formula: see text] influx through L-type Ca[Formula: see text] channel, and promotion of Ca[Formula: see text] release from SR.

Mg(2+)-dependent facilitation and inactivation of L-type Ca(2+) channels in guinea pig ventricular myocytes.

This study aimed to investigate the intracellular Mg(2+) regulation of the L-type Ca(2+) channels in guinea pig ventricular myocytes. By adopting the inside-out configuration of the patch clamp technique, single channel currents of the L-type Ca(2+) channels were recorded at different intracellular Mg(2+) concentrations ([Mg(2+)]i). At free [Mg(2+)]i of 0, 10(-9), 10(-7), 10(-5), 10(-3), and 10(-1) M, 1.4 µM CaM + 3 mM ATP induced channel activities of 44%, 117%, 202%, 181%, 147%, and 20% of the control activity in cell-attached mode, respectively, showing a bell-shaped concentration-response relationship. Moreover, the intracellular Mg(2+) modulated the Ca(2+) channel gating properties, accounting for alterations in channel activities. These results imply that Mg(2+) has a dual effect on the L-type Ca(2+) channels: facilitation and inhibition. Lower [Mg(2+)]i maintains and enhances the basal activity of Ca(2+) channels, whereas higher [Mg(2+)]i inhibits channel activity. Taken together, our data from the application of an [Mg(2+)]i series suggest that the dual effect of Mg(2+) upon the L-type Ca(2+) channels exhibits long open-time dependence.

Electrophysiological effect and the gating mechanism of astragaloside IV on L-type Ca(2+) channels of guinea-pig ventricular myocytes.

Astragaloside IV (AS-IV) is one of the main active ingredients of Astragalus membranaceus. This study is aimed to investigate AS-IV׳s effects on Ca(2+) channel activity of single cardiomyocytes and single Ca(2+) channels. Whole-cell Ca(2+) currents in freshly dissociated cardiomyocytes were measured using the whole-cell patch-clamp technique. Single Ca(2+) channel currents were examined in cell-attached patches and inside-out patches. In the whole-cell recording, AS-IV reduced the amplitude of L-type Ca(2+) currents (ICaL) in a concentration-dependent manner. Although AS-IV did not alter the steady-state activation curves, the voltage dependence of the current inactivation curves was negatively shifted by AS-IV in a concentration dependent manner. Consistent with the results of the whole-cell recording, in the inside-out configuration the ensemble average of single Ba(2+) current via L-type Ca(2+) channel was dose-dependently reduced by AS-IV. The reduction of unitary Ba(2+) current at 0.1 or 1 µM AS-IV was accounted for a decrease in the channel activity (NPo). In addition to the decrease in NPo, there was a reduction of Po without a change in channel number or an apparent change in single channel current. Furthermore, we found that the open-closed kinetics of the channel were affected by AS-IV. AS-IV induced the shift of L-type Ca(2+) channels from either brief openings (mode 1) or long-lasting openings (mode 2) to no active opening (mode 0). Our results suggest that AS-IV blocks the currents through Ca(2+) channels in guinea-pig ventricular myocytes by affecting the open-closed kinetics of L-type Ca(2+) channels to inhibit the channel activities. This study could provide theoretical basis for the drug exploiting of the monomer of Astragalus membranaceus.

The individual N- and C-lobes of calmodulin tether to the Cav1.2 channel and rescue the channel activity from run-down in ventricular myocytes of guinea-pig heart.

The present study examined the binding of the individual N- and C-lobes of calmodulin (CaM) to Cav1.2 at different Ca(2+) concentration ([Ca(2+)]) from ≈ free to 2mM, and found that they may bind to Cav1.2 Ca(2+)-dependently. In particular, using the patch-clamp technique, we confirmed that the N- or C-lobes can rescue the basal activity of Cav1.2 from run-down, demonstrating the functional relevance of the individual lobes. The data imply that at resting [Ca(2+)], CaM may tether to the channel with its single lobe, leading to multiple CaM molecule binding to increase the grade of Ca(2+)-dependent regulation of Cav1.2.

The Ca(2+)-dependent interaction of calpastatin domain L with the C-terminal tail of the Cav1.2 channel.

To demonstrate the interaction of calpastatin (CS) domain L (CSL) with Cav1.2 channel, we investigated the binding of CSL with various C-terminus-derived peptides at≈free, 100 nM, 10 µM, and 1mM Ca(2+) by using the GST pull-down assay method. Besides binding with the IQ motif, CSL was also found to bind with the PreIQ motif. With increasing [Ca(2+)], the affinity of the CSL-IQ interaction gradually decreased, and the affinity of the CSL-PreIQ binding gradually increased. The results suggest that CSL may bind with both the IQ and PreIQ motifs of the Cav1.2 channel in different Ca(2+)-dependent manners.

Lobe-related concentration- and Ca(2+)-dependent interactions of calmodulin with C- and N-terminal tails of the CaV1.2 channel.

This study examined the bindings of calmodulin (CaM) and its mutants with the C- and N-terminal tails of the voltage-gated Ca(2+) channel CaV1.2 at different CaM and Ca(2+) concentrations ([Ca(2+)]) by using the pull-down assay method to obtain basic information on the binding mode, including its concentration- and Ca(2+)-dependencies. Our data show that more than one CaM molecule could bind to the CaV1.2 C-terminal tail at high [Ca(2+)]. Additionally, the C-lobe of CaM is highly critical in sensing the change of [Ca(2+)] in its binding to the C-terminal tail of CaV1.2, and the binding between CaM and the N-terminal tail of CaV1.2 requires high [Ca(2+)]. Our data provide new details on the interactions between CaM and the CaV1.2 channel.

The up-regulation of voltage-gated sodium channels subtypes coincides with an increased sodium current in hippocampal neuronal culture model.

Voltage-gated sodium channels (VGSC) have been linked to inherited forms of epilepsy. The expression and biophysical properties of VGSC in the hippocampal neuronal culture model have not been clarified. In order to evaluate mechanisms of epileptogenesis that are related to VGSC, we examined the expression and function of VGSC in the hippocampal neuronal culture model in vitro and spontaneously epileptic rats (SER) in vivo. Our data showed that the peak amplitude of transient, rapidly-inactivating Na(+) current (I(Na,T)) in model neurons was significantly increased compared with control neurons, and the activation curve was shifted to the negative potentials in model neurons in whole cell recording by patch-clamp. In addition, channel activity of persistent, non-inactivating Na(+) current (I(Na,P)) was obviously increased in the hippocampal neuronal culture model as judged by single-channel patch-clamp recording. Furthermore, VGSC subtypes Na(V)1.1, Na(V)1.2 and Na(V)1.3 were up-regulated at the protein expression level in model neurons and SER as assessed by Western blotting. Four subtypes of VGSC proteins in SER were clearly present throughout the hippocampus, including CA1, CA3 and dentate gyrus regions, and neurons expressing VGSC immunoreactivity were also detected in hippocampal neuronal culture model by immunofluorescence. These findings suggested that the up-regulation of voltage-gated sodium channels subtypes in neurons coincided with an increased sodium current in the hippocampal neuronal culture model, providing a possible explanation for the observed seizure discharge and enhanced excitability in epilepsy.