Regulation Of L-type Calcium Channels Research Articles

L-type calcium channel regulation of depression, anxiety and anhedonia-related behavioral phenotypes following chronic stress exposure

Exposure to chronic and unpredictable stressors can precipitate mood-related disorders in humans, particularly in individuals with pre-existing mental health challenges. L-type calcium channels (LTCCs) have been implicated in numerous neuropsychiatric disorders, as LTCC encoding genes have been identified as candidate risk factors for neuropsychiatric illnesses. In these sets of experiments, we sought to examine the ability of LTCC blockade to alter depression, anxiety, and anhedonic-related behavioral responses to chronic unpredictable stress (CUS) exposure in female and male rats. Rats first underwent either 21 days of CUS or no exposure to chronic stressors, serving as home cage controls (HCC). Then rats were examined for anhedonia-related behavior, anxiety and depression-like behavioral responses as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and forced swim test (FST). CUS exposed females and males showed anhedonic and anxiogenic-like behavioral responses on the SPT and EPM, respectively, when compared to HCCs. In female and male rats, systemic administration of the LTCC blocker isradipine (0.4 mg/kg and 1.2 mg/kg, I.P.) attenuated the CUS-induced decrease in sucrose preference and reversed the CUS-induced decrease in open arm time. In the FST, systemic isradipine decreased immobility time across all groups, consistent with an antidepressant-like response. However, there were no significant differences in forced swim test immobility time between HCC and CUS exposed animals. Taken together, these data point to a role of LTCCs in the regulation of mood disorder-related behavioral phenotype responses to chronic stress exposure.

Abstract P1086: Lrrc10 Is A Multifaceted Modulator Of Cardiac Calcium And Sodium Channels

L-type calcium channels (LTCC) are vital for heart physiology as they allow entry of calcium to the cardiomyocyte, ultimately leading to contraction. These channels are exquisitely modulated by auxiliary subunits and a vast array of regulatory proteins. Heart-specific leucine-rich repeat-containing protein 10 (Lrrc10) plays a key role as a regulator of LTCC. Previous work has shown that Lrrc10 knockdown in zebrafish disrupts development of the cardiac tube, while ablation of Lrrc10 in mice results in heart dysfunction and maladaptation to increased cardiac afterload. Moreover, human mutations in Lrrc10 gene have been linked to dilated cardiomyopathy, and sudden unexplained nocturnal death syndrome (SUNDS). Still, the molecular mechanisms underlying Lrrc10 regulation of LTCC is not fully established. Beyond this, it is unknown whether Lrrc10 modulates other cardiac ion channels. Here, we hypothesized that Lrrc10 serves as a versatile regulator of four-domain voltage-gated ion channels, while mutations in Lrrc10 differentially disrupt channel regulation. Using whole-cell and single-channel electrophysiology, we found that Lrrc10: 1) upregulates LTCC currents by increasing peak open probability; 2) accelerates calcium-dependent inactivation; and 3) evokes a hyperpolarizing-shift in voltage-dependence of activation. Flow cytometric FRET 2-hybrid assay showed that Lrrc10 interacts with LTCC cytoplasmic subunits, specifically the N- and C-termini. We further used FRET 2-hybrid analysis to evaluate the interaction of Lrrc10 with related cardiac ion channels. We observed robust Lrrc10 interaction with Na V 1.5 channels. Multi-channel electrophysiology recordings revealed increased late sodium currents, suggesting that Lrrc10 also modulates Na V 1.5 activity. Additionally, electrophysiological studies showed that Lrrc10 variants linked to dilated cardiomyopathy and SUNDS disparately alter modulation of calcium and sodium channels. In conclusion, Lrrc10 is a multifaceted regulator of four-domain, voltage-gated cardiac ion channels, a finding that will lend critical insights into heart function and disease.

Rad-GTPase contributes to heart rate via L-type calcium channel regulation

Sinoatrial node cardiomyocytes (SANcm) possess automatic, rhythmic electrical activity. SAN rate is influenced by autonomic nervous system input, including sympathetic nerve increases of heart rate (HR) via activation of β-adrenergic receptor signaling cascade (β-AR). L-type calcium channel (LTCC) activity contributes to membrane depolarization and is a central target of β-AR signaling. Recent studies revealed that the small G-protein Rad plays a central role in β-adrenergic receptor directed modulation of LTCC. These studies have identified a conserved mechanism in which β-AR stimulation results in PKA-dependent Rad phosphorylation: depletion of Rad from the LTCC complex, which is proposed to relieve the constitutive inhibition of CaV1.2 imposed by Rad association. Here, using a transgenic mouse model permitting conditional cardiomyocyte selective Rad ablation, we examine the contribution of Rad to the control of SANcm LTCC current (ICa,L) and sinus rhythm. Single cell analysis from a recent published database indicates that Rad is expressed in SANcm, and we show that SANcm ICa,L was significantly increased in dispersed SANcm following Rad silencing compared to those from CTRL hearts. Moreover, cRadKO SANcm ICa,L was not further increased with β-AR agonists. We also evaluated heart rhythm in vivo using radiotelemetered ECG recordings in ambulating mice. In vivo, intrinsic HR is significantly elevated in cRadKO. During the sleep phase cRadKO also show elevated HR, and during the active phase there is no significant difference. Rad-deletion had no significant effect on heart rate variability. These results are consistent with Rad governing LTCC function under relatively low sympathetic drive conditions to contribute to slower HR during the diurnal sleep phase HR. In the absence of Rad, the tonic modulated SANcm ICa,L promotes elevated sinus HR. Future novel therapeutics for bradycardia targeting Rad – LTCC can thus elevate HR while retaining βAR responsiveness.

Effect of Verapamil, an L-Type Calcium Channel Inhibitor, on Caveolin-3 Expression in Septic Mouse Hearts.

Sepsis-induced myocardial dysfunction considerably increases mortality risk in patients with sepsis. Previous studies from our group have shown that sepsis alters the expression of structural proteins in cardiac cells, resulting in cardiomyocyte degeneration and impaired communication between cardiac cells. Caveolin-3 (CAV3) is a structural protein present in caveolae, located in the membrane of cardiac muscle cells, which regulates physiological processes such as calcium homeostasis. In sepsis, there is a disruption of calcium homeostasis, which increases the concentration of intracellular calcium, which can lead to the activation of potent cellular enzymes/proteases which cause severe cellular injury and death. The purpose of the present study was to test the hypotheses that sepsis induces CAV3 overexpression in the heart, and the regulation of L-type calcium channels directly relates to the regulation of CAV3 expression. Severe sepsis increases the expression of CAV3 in the heart, as immunostaining in our study showed CAV3 presence in the cardiomyocyte membrane and cytoplasm, in comparison with our control groups (without sepsis) that showed CAV3 presence predominantly in the plasma membrane. The administration of verapamil, an L-type calcium channel inhibitor, resulted in a decrease in mortality rates of septic mice. This effect was accompanied by a reduction in the expression of CAV3 and attenuation of cardiac lesions in septic mice treated with verapamil. Our results indicate that CAV3 has a vital role in cardiac dysfunction development in sepsis and that the regulation of L-type calcium channels may be related to its expression.

Hydroxysafflor Yellow A Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Calcium Overload and Apoptosis.

Myocardial ischemia/reperfusion injury (MI/RI) is an urgent problem with a great impact on health globally. However, its pathological mechanisms have not been fully elucidated. Hydroxysafflor yellow A (HSYA) has a protective effect against MI/RI. This study is aimed at further clarifying the relationship between HSYA cardioprotection and calcium overload as well as the underlying mechanisms. We verified the protective effect of HSYA on neonatal rat primary cardiomyocytes (NPCMs) and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from hypoxia-reoxygenation (HR) injury. To explore the cardioprotective mechanism of HSYA, we employed calcium fluorescence, TUNEL assay, JC-1 staining, and western blotting. Finally, cardio-ECR and patch-clamp experiments were used to explain the regulation of L-type calcium channels (LTCC) in cardioprotection mediated by HSYA. The results showed that HSYA reduced the levels of myocardial enzymes and protected NPCMs from HR injury. HSYA also restored the contractile function of hiPSC-CMs and field potential signal abnormalities caused by HR and exerted a protective effect on cardiac function. Further, we demonstrated that HSYA protects cardiomyocytes from HR injury by decreasing mitochondrial membrane potential and inhibiting apoptosis and calcium overload. Patch-clamp results revealed that MI/RI caused a sharp increase in calcium currents, which was inhibited by pretreatment with HSYA. Furthermore, we found that HSYA restored contraction amplitude, beat rate, and field potential duration of hiPSC-CMs, which were disrupted by the LTCC agonist Bay-K8644. Patch-clamp experiments also showed that HSYA inhibits Bay-K8644-induced calcium current, with an effect similar to that of the LTCC inhibitor nisoldipine. Therefore, our data suggest that HSYA targets LTCC to inhibit calcium overload and apoptosis of cardiomyocytes, thereby exerting a cardioprotective effect and reducing MI/RI injury.

Selective changes in cytosolic β-adrenergic cAMP signals and L-type Calcium Channel regulation by Phosphodiesterases during cardiac hypertrophy

Background In cardiomyocytes, phosphodiesterases (PDEs) type 3 and 4 are the predominant enzymes that degrade cAMP generated by β-adrenergic receptors (β-ARs), impacting notably the regulation of the L-type Ca2+ current (ICa,L). Cardiac hypertrophy (CH) is accompanied by a reduction in PDE3 and PDE4, however, whether this affects the dynamic regulation of cytosolic cAMP and ICa,L is not known.Methods and Results CH was induced in rats by thoracic aortic banding over a time period of five weeks and was confirmed by anatomical measurements. Left ventricular myocytes (LVMs) were isolated from CH and sham-operated (SHAM) rats and transduced with an adenovirus encoding a Förster resonance energy transfer (FRET)-based cAMP biosensor or subjected to the whole-cell configuration of the patch-clamp technique to measure ICa,L. Aortic stenosis resulted in a 46% increase in heart weight to body weight ratio in CH compared to SHAM. In SHAM and CH LVMs, a short isoprenaline stimulation (Iso, 100 nM, 15 s) elicited a similar transient increase in cAMP with a half decay time (t1/2off) of ~50 s. In both groups, PDE4 inhibition with Ro 20-1724 (10 μM) markedly potentiated the amplitude and slowed the decline of the cAMP transient, this latter effect being more pronounced in SHAM (t1/2off ~ 250 s) than in CH (t1/2off ~ 150 s, P < 0.01). In contrast, PDE3 inhibition with cilostamide (1 μM) had no effect on the amplitude of the cAMP transient and a minimal effect on its recovery in SHAM, whereas it potentiated the amplitude and slowed the decay in CH (t1/2off ~ 80 s). Iso pulse stimulation also elicited a similar transient increase in ICa,L in SHAM and CH, although the duration of the rising phase was delayed in CH. Inhibition of PDE3 or PDE4 potentiated ICa,L amplitude in SHAM but not in CH. Besides, while only PDE4 inhibition slowed down the decline of ICa,L in SHAM, both PDE3 and PDE4 contributed in CH.Conclusion These results identify selective alterations in cytosolic cAMP and ICa,L regulation by PDE3 and PDE4 in CH, and show that the balance between PDE3 and PDE4 for the regulation of β-AR responses is shifted toward PDE3 during CH.

Nanoscale regulation of L-type calcium channels differentiates between ischemic and dilated cardiomyopathies.

BackgroundSubcellular localization and function of L-type calcium channels (LTCCs) play an important role in regulating contraction of cardiomyocytes. Understanding how this is affected by the disruption of transverse tubules during heart failure could lead to new insights into the disease.MethodsCardiomyocytes were isolated from healthy donor hearts, as well as from patients with cardiomyopathies and with left ventricular assist devices. Scanning ion conductance and confocal microscopy was used to study membrane structures in the cells. Super-resolution scanning patch-clamp was used to examine LTCC function in different microdomains. Computational modeling predicted the impact of these changes to arrhythmogenesis at the whole-heart level.FindingsWe showed that loss of structural organization in failing myocytes leads to re-distribution of functional LTCCs from the T-tubules to the sarcolemma. In ischemic cardiomyopathy, the increased LTCC open probability in the T-tubules depends on the phosphorylation by protein kinase A, whereas in dilated cardiomyopathy, the increased LTCC opening probability in the sarcolemma results from enhanced phosphorylation by calcium-calmodulin kinase II. LVAD implantation corrected LTCCs pathophysiological activity, although it did not improve their distribution. Using computational modeling in a 3D anatomically-realistic human ventricular model, we showed how LTCC location and activity can trigger heart rhythm disorders of different severity.InterpretationOur findings demonstrate that LTCC redistribution and function differentiate between disease aetiologies. The subcellular changes observed in specific microdomains could be the consequence of the action of distinct protein kinases.FundingThis work was supported by 10.13039/100000002NIH grant (ROI-HL 126802 to NT-JG) and British Heart Foundation (grant RG/17/13/33173 to JG, project grant PG/16/17/32069 to RAC). Funders had no role in study design, data collection, data analysis, interpretation, writing of the report

Diallyl trisulfide regulates rat colonic smooth muscle contractions by inhibiting L-type calcium channel currents

Diallyl trisulfide (DATS) is an active organosulfide component of allicin and has several beneficial effects, including antimicrobial, antioxidant, cardioprotective and anticancer effects. Few studies have shown the modulatory effect of DATS on L-type calcium channels in rat colonic smooth muscle cells and colonic motility. To investigate the modulatory effect of DATS on L-type calcium channels in rat colonic smooth muscle and colonic contraction, L-type calcium channel currents were recorded, and colonic contractility in longitudinal and circular smooth muscle strips was measured. DATS attenuated L-type calcium channel currents without affecting steady-state activation or inactivation kinetics and inhibited the spontaneous contractions of both longitudinal and circular smooth muscle strips dose-dependently. In conclusion, DATS has an inhibitory effect on the contractions of colonic muscle strips that is related to its regulation of L-type calcium channels.

Mechanical stretch increases L-type calcium channel stability in cardiomyocytes through a polycystin-1/AKT-dependent mechanism

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavβ2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gβγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Giβγ-AKT-Cavβ2 pathway, suggesting a new mechanism for regulation of cardiac function.

Mechanisms of the cyclic nucleotide cross-talk signaling network in cardiac L-type calcium channel regulation

Regulation of L-type Calcium (Ca2+) Channel (LCC) gating is critical to shaping the cardiac action potential (AP) and triggering the initiation of excitation-contraction (EC) coupling in cardiac myocytes. The cyclic nucleotide (cN) cross-talk signaling network, which encompasses the β-adrenergic and the Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) pathways and their interaction (cross-talk) through distinctively-regulated phosphodiesterase isoenzymes (PDEs), regulates LCC current via Protein Kinase A- (PKA) and PKG-mediated phosphorylation. Due to the tightly-coupled and intertwined biochemical reactions involved, it remains to be clarified how LCC gating is regulated by the signaling network from receptor to end target. In addition, the large number of EC coupling-related phosphorylation targets of PKA and PKG makes it difficult to quantify and isolate changes in L-type Ca2+ current (ICaL) responses regulated by the signaling network. We have developed a multi-scale, biophysically-detailed computational model of LCC regulation by the cN signaling network that is supported by experimental data. LCCs are modeled with functionally distinct PKA- and PKG-phosphorylation dependent gating modes. The model exhibits experimentally observed single channel characteristics, as well as whole-cell LCC currents upon activation of the cross-talk signaling network. Simulations show 1) redistribution of LCC gating modes explains changes in whole-cell current under various stimulation scenarios of the cN cross-talk network; 2) NO regulation occurs via potentiation of a gating mode characterized by prolonged closed times; and 3) due to compensatory actions of cross-talk and antagonizing functions of PKA- and PKG-mediated phosphorylation of LCCs, the effects of individual inhibitions of PDEs 2, 3, and 4 on ICaL are most pronounced at low levels of β-adrenergic stimulation. Simulations also delineate the contribution of the following two mechanisms to overall LCC regulation, which have otherwise been challenging to distinguish: 1) regulation of PKA and PKG activation via cN cross-talk (Mechanism 1); and 2) LCC interaction with activated PKA and PKG (Mechanism 2). These results provide insights into how cN signals transduced via the cN cross-talk signaling network are integrated via LCC regulation in the heart.

Science Signaling Podcast for 24 January 2017: Tissue-specific regulation of L-type calcium channels.

This Podcast features an interview with Johannes Hell and Manuel Navedo, senior authors of two Research Articles that appear in the 24 January 2017 issue of Science Signaling, about tissue-specific regulation of the L-type calcium channel CaV1.2. This channel is present in many tissues, including the heart, vasculature, and brain, and allows calcium to flow into cells when it is activated. Signaling through the β-adrenergic receptor (βAR) stimulates CaV1.2 activity in heart cells and neurons to accelerate heart rate and increase neuronal excitability, respectively. Using mouse models, Qian et al found that βAR-mediated enhancement of CaV1.2 activity in the brain required phosphorylation of Ser1928, whereas βAR-mediated enhancement of CaV1.2 activity in the heart did not require phosphorylation of this residue. In a related study, Nystoriak et al demonstrated that phosphorylation of Ser1928 in arterial myocytes was required for vasoconstriction during acute hyperglycemia and in diabetic mice. These findings demonstrate tissue-specific differences in CaV1.2 regulation and suggest that it may be possible to design therapies to target this channel in specific tissues.Listen to Podcast.

Microdomain-Specific Remodelling of Autonomic Regulation of L-Type Calcium Channels Revealed by Super-Resolution Scanning Patch Clamp in Rat Atrial Myocytes in Heart Failure

It has recently become evident that discrete clusters of L-type Ca2+ channels (LTCCs) exist along the cardiomyocyte sarcolemma in association with distinct membrane structures and receptors forming macromolecular signalling complexes. Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiomyocyte structure, may contribute to the pathophysiology of heart failure (HF) altering cardiomyocyte function. Here, we used Ca2+ imaging and super-resolution scanning patch-clamp to examine microdomain-specific regulation of LTCCs by β1 adrenergic (β1ARs) and adenosine A1 receptors (AdoA1Rs) in atrial myocytes isolated from control and 16-weeks post-MI HF rats. In control, β1ARs stimulation (ISO 100 nM and β2AR antagonist ICI 50 nM) enhanced spontaneous Ca2+ activity which was abolished by AdoA1Rs stimulation (2′-MeCCPA 200 nM). Accordingly, activation of single LTCCs in both t-tubules and on the crest of sarcolemma from non-localized β1ARs stimulation (ISO 2 µM and ICI 50 nM in external solution) was also abolished by local AdoA1R stimulation (10 µM). In HF, both β1ARs and AdoA1R effects on LTCCs were preserved in t-tubules and lost on the crest. Similar, the effect of β1AR stimulation on spontaneous Ca2+ activities was significantly reduced (28% vs 134% in HF vs control, P<0.01) and the anti-adrenergic effect of AdoA1R was lost (8% vs 84% in HF vs control, P<0.01). In addition, the effects of both β1ARs and AdoA1Rs on normalized cell shortening were significantly reduced in HF. Our findings provide the first direct evidence for microdomain-specific remodeling of LTCC regulation by adrenergic and adenosine receptors in HF which could be linked to local disruption in the interactions between LTCCs and their cellular microenvironment.

Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium Channels.

Mitochondria are key integrators of convergent intracellular signaling pathways. Two important second messengers modulated by mitochondria are calcium and reactive oxygen species. To date, coherent mechanisms describing mitochondrial integration of calcium and oxidative signaling in arterial smooth muscle are incomplete. To address and add clarity to this issue, we tested the hypothesis that mitochondria regulate subplasmalemmal calcium and hydrogen peroxide microdomain signaling in cerebral arterial smooth muscle. Using an image-based approach, we investigated the impact of mitochondrial regulation of L-type calcium channels on subcellular calcium and reactive oxygen species signaling microdomains in isolated arterial smooth muscle cells. Our single-cell observations were then related experimentally to intact arterial segments and to living animals. We found that subplasmalemmal mitochondrial amplification of hydrogen peroxide microdomain signaling stimulates L-type calcium channels, and that this mechanism strongly impacts the functional capacity of the vasoconstrictor angiotensin II. Importantly, we also found that disrupting this mitochondrial amplification mechanism in vivo normalized arterial function and attenuated the hypertensive response to systemic endothelial dysfunction. From these observations, we conclude that mitochondrial amplification of subplasmalemmal calcium and hydrogen peroxide microdomain signaling is a fundamental mechanism regulating arterial smooth muscle function. As the principle components involved are fairly ubiquitous and positioning of mitochondria near the plasma membrane is not restricted to arterial smooth muscle, this mechanism could occur in many cell types and contribute to pathological elevations of intracellular calcium and increased oxidative stress associated with many diseases.

Regulation of L-type calcium channel by phospholemman in cardiac myocytes

We evaluated whether phospholemman (PLM) regulates L-type Ca2+ current (ICa) in mouse ventricular myocytes. Expression of α1-subunit of L-type Ca2+ channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak ICa (at −10mV) from KO myocytes was ~41% larger, the inactivation time constant (τinact) of ICa was ~39% longer, but deactivation time constant (τdeact) was similar. In the presence of isoproterenol (1μM), peak ICa was ~48% larger and τinact was ~144% higher in KO myocytes. With Ba2+ as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τdeact. To dissect the molecular determinants by which PLM regulated ICa, we expressed PLM mutants by adenovirus-mediated gene transfer in cultured KO myocytes. After 24h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak ICa and longer τinact than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylatable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak ICa. Expressing PLM mutants did not alter expression of α1-subunit of L-type Ca2+ channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak ICa amplitude. We conclude that PLM limits Ca2+ influx in cardiac myocytes by reducing maximal ICa and accelerating voltage-dependent inactivation.

ASSA13-03-46 The Effects of Verapamil On SK2 Channel In Myocardium Cells From Human Chronic Atrial Fibrillation

<h3>Objective</h3> Small conductance calcium-activated potassium channel (SK channel) plays an important role in heart. Four subtype of SK channel are classified according to their sensitive to apamin. Electrophysiological properties of SK2 channel are voltage-independent as well as solely activated by changes in intracellular calcium concentration. This study investigated the alteration of SK2 channel currents in cAF patients. Moreover, L-type calcium channel inhibitor verapamil were adopted to observe the change of SK2 channel. We plan to explore the functional relationship between L-type calcium channel and SK2 channels. <h3>Methods</h3> Single human cardiac myocyte was isolated by two-step enzyme digestion. The whole-cell patch clamp technique was used to record the SK2 current in isolated cardiomyocytes from patients with siuns rhythm (SR) or chronic atrial fibrillation (cAF). Verapamil (10 μmol/L) and apamin (100 nmol/L) were added into bath solution respectively to observe the change of current. <h3>Results</h3> The current density (pA/pF) of SK2 channel as well as the ration in the integrated inward currents were up-regulated significantly in cAF groups. At wide range of test potentials, L-type calcium channel inhibitor verapamil reduced SK2 channel currents in cardiomyocytes from patients with SR or cAF. Compared with control group, the apamin-sensitive currents density of the verapamil group showed a significant decrease in human atrial myocytes. It indicates that part of SK2 current can be inhibited by verapamil. <h3>Conclusions</h3> It suggested that the up-regulation of SK2 currents underlies the occurrence and maintenance of cAF. There may be functionally coupling between SK2 channels and L-type calcium channels in human myocardium cells. The regulation of L-type calcium channel involves the remodelling of SK2 channels in the atrial myocytes from SR and cAF patients. This work was supported by the National Natural Science Foundation of China (No. 30870903) and the Natural Science Foundation of Luzhou.

Calcium Concentration Fluctuations and Subspace Volume Influence Calcium-Regulated Calcium Channel Gating and Subspace Dynamics

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough that local signaling may involve 1-100 calcium ions. Therefore, the question arises: is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of subspace calcium concentration and channel open probability (a result that accounts for the small subspace volume and concentration fluctuations) with the corresponding deterministic model (an approximation that assumes large system size and ignores concentration fluctuations). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium-binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast, demonstrating that the calcium concentration fluctuations and subspace volume influence channel gating and subspace dynamics. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers [Tadross, Dick, Yue. Cell 133: 1228-40, 2008]. Additional work will consider the influence of slow and rapid buffers present in the subspace and whether and under what conditions these buffers mitigate the effects of concentration fluctuations on channel gating and subspace dynamics.

Interleukin-6 inhibits L-type calcium channel activity of cultured cerebellar granule neurons

Our previous work has shown that interleukin-6 (IL-6) implements its neuroprotective effect by inhibiting the intracellular Ca(2+) overload in neurons. Here, we examined whether regulation of L-type calcium channels (LCCs) activities is involved in the neuroprotective action of IL-6. In cultured cerebellar granule neurons (CGNs), patch-clamp recording showed that the whole-cell Ca(2+) current and LCC current were significantly reduced by IL-6 pretreatment (120ng/ml, for 24h). Calcium imaging data indicated that IL-6 significantly suppressed high K(+)-induced intracellular Ca(2+) overload and LCC Ca(2+) influx. Moreover, expression of the LCC subunit, Ca(v)1.2, was remarkably downregulated by IL-6 in cultured CGNs. These findings suggest that IL-6 exerts a neurotrophic effect by preventing Ca(2+) overload, at least partly through inhibition of LCC activity in cultured CGNs.

MicroRNAs Are Essential for Stretch-induced Vascular Smooth Muscle Contractile Differentiation via MicroRNA (miR)-145-dependent Expression of L-type Calcium Channels

Stretch of the vascular wall is an important stimulus to maintain smooth muscle contractile differentiation that is known to depend on L-type calcium influx, Rho-activation, and actin polymerization. The role of microRNAs in this response was investigated using tamoxifen-inducible and smooth muscle-specific Dicer KO mice. In the absence of Dicer, which is required for microRNA maturation, smooth muscle microRNAs were completely ablated. Stretch-induced contractile differentiation and Rho-dependent cofilin-2 phosphorylation were dramatically reduced in Dicer KO vessels. On the other hand, acute stretch-sensitive growth signaling, which is independent of influx through L-type calcium channels, was not affected by Dicer KO. Contractile differentiation induced by the actin polymerizing agent jasplakinolide was not altered by deletion of Dicer, suggesting an effect upstream of actin polymerization. Basal and stretch-induced L-type calcium channel expressions were both decreased in Dicer KO portal veins, and inhibition of L-type channels in control vessels mimicked the effects of Dicer deletion. Furthermore, inhibition of miR-145, a highly expressed microRNA in smooth muscle, resulted in a similar reduction of L-type calcium channel expression. This was abolished by the Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN93, suggesting that Ca(2+)/calmodulin-dependent protein kinase IIδ, a target of miR-145 and up-regulated in Dicer KO, plays a role in the regulation of L-type channel expression. These results show that microRNAs play a crucial role in stretch-induced contractile differentiation in the vascular wall in part via miR-145-dependent regulation of L-type calcium channels.

Discrete-State Stochastic Models of Calcium-Regulated Calcium Influx and Subspace Dynamics Are Not Well-Approximated by ODEs That Neglect Concentration Fluctuations

Cardiac myocyte calcium signaling is often modeled using deterministic ordinary differential equations (ODEs) and mass-action kinetics. However, spatially restricted “domains” associated with calcium influx are small enough (e.g., 10−17 liters) that local signaling may involve 1–100 calcium ions. Is it appropriate to model the dynamics of subspace calcium using deterministic ODEs or, alternatively, do we require stochastic descriptions that account for the fundamentally discrete nature of these local calcium signals? To address this question, we constructed a minimal Markov model of a calcium-regulated calcium channel and associated subspace. We compared the expected value of fluctuating subspace calcium concentration (a result that accounts for the small subspace volume) with the corresponding deterministic model (an approximation that assumes large system size). When subspace calcium did not regulate calcium influx, the deterministic and stochastic descriptions agreed. However, when calcium binding altered channel activity in the model, the continuous deterministic description often deviated significantly from the discrete stochastic model, unless the subspace volume is unrealistically large and/or the kinetics of the calcium binding are sufficiently fast. This principle was also demonstrated using a physiologically realistic model of calmodulin regulation of L-type calcium channels introduced by Yue and coworkers.

Functional regulation of L-type calcium channel via a novel cAMP-independent protein kinase (CIK) in goat epididymal spermatozoa

Functional regulation of L-type calcium channel via a novel cAMP-independent protein kinase (CIK) in goat epididymal spermatozoa