Ca Currents Research Articles

Impaired Ca-Calmodulin-Dependent Inactivation of CaV 1.2 Contributes to Loss of SR Ca Release Refractoriness in Mice Lacking Casq2

In cardiac muscle, L-type Ca current (CaV 1.2, ICa) induced Ca release from sarcoplasmic reticulum (SR) is reduced with successively shorter coupling intervals of premature stimuli, a phenomenon known as SR Ca release restitution. We previously reported that myocytes lacking the SR luminal Ca binding protein calsequestrin (Casq2 KO) exhibited less SR Ca release restitution and hence largely lack Ca release refractoriness. Here, we test the hypothesis that altered CaV 1.2 channel gating contributes to altered SR Ca release restitution in Casq2 KO myocytes. Methods and Results: ICa was recorded in voltage-clamped ventricular myocytes isolated from Casq2 KO mice and wild-type (WT) littermates. Cells were pre-treated with ryanodine (50 μM) and thapsigargin (10 μM) to eliminate SR Ca release. Compared to WT, ICa peak currents were unchanged, but the inactivation time course was significantly slower in KO myocytes (tau=157 ms vs. 46 ms in WT, p<0.01). using Ba as a charge carrier abolished the differences in inactivation, suggesting that the underlying defect lies not in the voltage-dependent but rather in the Ca-dependent ICa inactivation. Addition of apo-calmodulin (CaM, 0.35 mg/ml) to highly-buffered (14 mM EGTA) pipette solution accelerated ICa inactivation in KO myocytes (tau=52 ms). To test the effect of CaM on ICa restitution, ICa was measured by applying premature stimuli (S2) at successively shorter S1-S2 coupling intervals after a 1 Hz pacing train (S1). Intracellular dialysis with apo-CaM rendered ICa restitution curves of KO myocytes similar to those obtained in WT myocytes. Conclusion: Impaired Ca-dependent inactivation of ICa in Casq2 KO mice can be reversed by excess apo-CaM, suggesting that impaired Ca-CaM-dependent inactivation of CaV 1.2 contributes to loss of SR Ca release refractoriness in Casq2 KO mice.

A Population Density Domain Model for Calcium-Inactivation of L-Type Calcium Channels

We present a minimal whole cell model of stochastic domain Ca-mediated inactivation of low-density L-type Ca channels. Our approach avoids the computationally demanding task of resolving spatial aspects of global Ca signaling by using probability densities and associated moment equations to represent heterogeneous local Ca signals [Williams et al. Biophys J. 92(7):2311-28, 2007; Biophys J. 95(4):1689-703, 2008]. using a minimal Markov chain model of an L-type Ca channel, simulated whole cell responses to a two-pulse voltage clamp protocol yield an inactivation function for the whole cell Ca current that is similar to - but may deviate from - that obtained by assuming instantaneous formation and collapse of Ca domains [Sherman, Keizer, Rinzel. Biophys J. 58(4):985-95, 1990]. Parameter studies reveal that when domain Ca formation and collapse are slow compared to channel kinetics (e.g., fast voltage-dependent gating), the population density description is required to accurately represent the dynamics of Ca inactivation of whole cell L-type currents. When channel kinetics are slow, the population density approach agrees with - and thus generalizes - “instantaneous domain” models of Ca inactivation.

Isolation and characterization of the 5´-upstream region of the human voltage-gated Ca2+ channel α2δ-1 auxiliary subunit gene: promoter analysis and regulation by transcription factor Sp1

The α2δ proteins are auxiliary subunits of high-voltage-activated Ca(2+) channels associated with alterations of surface expression, kinetics, and voltage-dependent properties of the channel complex. Four mammalian genes and several splice α2δ subunit variants have been cloned and described, though very little information concerning the transcriptional mechanisms that regulate their expression is available. Here, we report the identification and characterization of the human α2δ-1 subunit gene promoter and its regulation by specific transcription factor 1 (Sp1). Transient transfection of human neuroblastoma SH-SY5Y cells with a promoter/luciferase reporter construct revealed a ~1.5 kb 5´-UTR fragment of the CACNA2D1 gene that produced high levels of luciferase activity. Deletional analysis of this sequence showed that the minimal promoter was located within a 413-bp region (nt -326 to +98) with respect to the transcription start site. In this region, no canonical TATA box was present, but a high GC content and five potential Sp1 binding sites were found. The ability of two of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Likewise, Sp1 overexpression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of α2δ protein expressed in the SH-SY5Y cells, as well as reduced the amplitude of whole-cell patch clamp Ca(2+) currents in dorsal root ganglion neurons. This study thus represents the first identification of the transcriptional control region in the gene encoding the Ca(2+) channel α2δ-1 auxiliary subunit.

Late‐onset bursts evoked by mossy fibre bundle stimulation in unipolar brush cells: evidence for the involvement of H‐ and TRP‐currents

Synaptic transmission at central synapses has usually short latency and graded amplitude, thereby regulating threshold crossing and the probability of action potential generation. In the granular layer of the vestibulo-cerebellum, unipolar brush cells (UBCs) receive a giant synapse generating a stereotyped excitatory postsynaptic potential (EPSP)-burst complex with early-onset (∼2 ms) and high reliability. By using patch-clamp recordings in cerebellar slices of the rat vestibulo-cerebellum, we found that mossy fibre bundle stimulation also evoked (in ∼80% of cases) a late-onset burst (after tens to hundreds of milliseconds) independent of EPSP generation. Different from the early-onset, the late-onset burst delay decreased and its duration increased by raising stimulation intensity or the number of impulses. Although depending on synaptic activity, the late-onset response was insensitive to perfusion of APV ((2R)-5-amino-phosphonopentanoate), NBQX (2,3-dioxo-6-nitro-tetrahydrobenzo(f)quinoxaline-7-sulfonamide) and MCPG ((RS)-α-methyl-4-carboxyphenylglycine) and did not therefore depend on conventional glutamatergic transmission mechanisms. The late-onset response was initiated by a slow depolarizing ramp driven by activation of an H-current (sensitive to ZD7288 and Cs(+)) and of a TRP- (transient receptor potential) current (sensitive to SKF96365), while the high voltage-activated and high voltage-activated Ca(2+) currents (sensitive to nimodipine and mibefradil, respectively) played a negligible role. The late-onset burst was occluded by intracellular cAMP. These results indicate that afferent activity can regulate H- and TRP-current gating in UBCs generating synaptically driven EPSP-independent responses, in which the delay rather than amplitude is graded with the intensity of the input pattern. This modality of synaptic transmission may play an important role in regulating UBC activation and granular layer functions in the vestibulo-cerebellum.

Functional expression of SK channels in murine detrusor PDGFRα+ cells

We sought to characterize molecular expression and ionic conductances in a novel population of interstitial cells (PDGFRα(+) cells) in murine bladder to determine how these cells might participate in regulation of detrusor excitability. PDGFRα(+) cells and smooth muscle cells (SMCs) were isolated from detrusor muscles of PDGFRα(+)/eGFP and smMHC/Cre/eGFP mice and sorted by FACS. PDGFRα(+) cells were highly enriched in Pdgfra (12 fold vs. unsorted cell) and minimally positive for Mhc (SMC marker), Kit (ICC marker) and Pgp9.5 (neuronal marker). SK3 was dominantly expressed in PDGFRα(+) cells in comparison to SMCs. αSlo (BK marker) was more highly expressed in SMCs. SK3 protein was observed in PDGFRα(+) cells by immunohistochemistry but could not be resolved in SMCs. Depolarization evoked voltage-dependent Ca(2+) currents in SMCs, but inward current conductances were not activated in PDGFRα(+) cells under the same conditions. PDGFRα(+) cells displayed spontaneous transient outward currents (STOCs) at potentials positive to -60 mV that were inhibited by apamin. SK channel modulators, CyPPA and SKA-31, induced significant hyperpolarization of PDGFRα(+) cells and activated SK currents under voltage clamp. Similar responses were not resolved in SMCs at physiological potentials. Single channel measurements confirmed the presence of functional SK3 channels (i.e. single channel conductance of 10 pS and sensitivity to intracellular Ca(2+)) in PDGFRα(+) cells. The apamin-sensitive stabilizing factor regulating detrusor excitability is likely to be due to the expression of SK3 channels in PDGFRα(+) cells because SK agonists failed to elicit resolvable currents and hyperpolarization in SMCs at physiological potentials.

Bisphenol A Inhibits Voltage-Activated Ca2+ Channels in Vitro: Mechanisms and Structural Requirements

Bisphenol A (BPA), a high volume production chemical compound attracts growing attention as a health-relevant xenobiotic in humans. It can directly bind to hormone receptors, enzymes, and ion channels to become biologically active. In this study we show that BPA acts as a potent blocker of voltage-activated Ca(2+) channels. We determined the mechanisms of block and the structural elements of BPA essential for its action. Macroscopic Ba(2+) / Ca(2+) currents through native L-, N-, P/Q-, T-type Ca(2+) channels in rat endocrine GH(3) cells, mouse dorsal root ganglion neurons or cardiac myocytes, and recombinant human R-type Ca(2+) channels expressed in human embryonic kidney (HEK) 293 cells were rapidly and reversibly inhibited by BPA with similar potency (EC(50) values: 26-35 μM). Pharmacological and biophysical analysis of R-type Ca(2+) channels revealed that BPA interacts with the extracellular part of the channel protein. Its action does not require intracellular signaling pathways, is neither voltage- nor use-dependent, and does not affect channel gating. This indicates that BPA interacts with the channel in its resting state by directly binding to an external site outside the pore-forming region. Structure-effect analyses of various phenolic and bisphenolic compounds revealed that 1) a double-alkylated (R-C(CH(3))(2)-R, R-C(CH(3))(CH(2)CH(3))-R), or double-trifluoromethylated sp(3)-hybridized carbon atom between the two aromatic rings and 2) the two aromatic moieties in angulated orientation are optimal for BPA's effectiveness. Since BPA highly pollutes the environment and is incorporated into the human organism, our data may provide a basis for future studies relevant for human health and development.

Genetic inhibition of Na+-Ca2+ exchanger current disables fight or flight sinoatrial node activity without affecting resting heart rate.

The sodium-calcium exchanger 1 (NCX1) is predominantly expressed in the heart and is implicated in controlling automaticity in isolated sinoatrial node (SAN) pacemaker cells, but the potential role of NCX1 in determining heart rate in vivo is unknown. To determine the role of Ncx1 in heart rate. We used global myocardial and SAN-targeted conditional Ncx1 knockout (Ncx1(-/-)) mice to measure the effect of the NCX current on pacemaking activity in vivo, ex vivo, and in isolated SAN cells. We induced conditional Ncx1(-/-) using a Cre/loxP system. Unexpectedly, in vivo and ex vivo hearts and isolated SAN cells showed that basal rates in Ncx1(-/-) (retaining ≈20% of control level NCX current) and control mice were similar, suggesting that physiological NCX1 expression is not required for determining resting heart rate. However, increases in heart rate and SAN cell automaticity in response to isoproterenol or the dihydropyridine Ca(2+) channel agonist BayK8644 were significantly blunted or eliminated in Ncx1(-/-) mice, indicating that NCX1 is important for fight or flight heart rate responses. In contrast, the pacemaker current and L-type Ca(2+) currents were equivalent in control and Ncx1(-/-) SAN cells under resting and isoproterenol-stimulated conditions. Ivabradine, a pacemaker current antagonist with clinical efficacy, reduced basal SAN cell automaticity similarly in control and Ncx1(-/-) mice. However, ivabradine decreased automaticity in SAN cells isolated from Ncx1(-/-) mice more effectively than in control SAN cells after isoproterenol, suggesting that the importance of NCX current in fight or flight rate increases is enhanced after pacemaker current inhibition. Physiological Ncx1 expression is required for increasing sinus rates in vivo, ex vivo, and in isolated SAN cells, but not for maintaining resting heart rate.

Phosphodiesterase type 3A regulates basal myocardial contractility through interacting with sarcoplasmic reticulum calcium ATPase type 2a signaling complexes in mouse heart.

cAMP is an important regulator of myocardial function, and regulation of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs) is a critical determinant of the amplitude, duration, and compartmentation of cAMP-mediated signaling. The role of different PDE isozymes, particularly PDE3A vs PDE3B, in the regulation of heart function remains unclear. To determine the relative contribution of PDE3A vs PDE3B isozymes in the regulation of heart function and to dissect the molecular basis for this regulation. Compared with wild-type littermates, cardiac contractility and relaxation were enhanced in isolated hearts from PDE3A(-/-), but not PDE3B(-/-), mice. Furthermore, PDE3 inhibition had no effect on PDE3A(-/-) hearts but increased contractility in wild-type (as expected) and PDE3B(-/-) hearts to levels indistinguishable from PDE3A(-/-). The enhanced contractility in PDE3A(-/-) hearts was associated with cAMP-dependent elevations in Ca(2+) transient amplitudes and increased sarcoplasmic reticulum (SR) Ca(2+) content, without changes in L-type Ca(2+) currents of cardiomyocytes, as well as with increased SR Ca(2+)-ATPase type 2a activity, SR Ca(2+) uptake rates, and phospholamban phosphorylation in SR fractions. Consistent with these observations, PDE3 activity was reduced ≈8-fold in SR fractions from PDE3A(-/-) hearts. Coimmunoprecipitation experiments further revealed that PDE3A associates with both SR calcium ATPase type 2a and phospholamban in a complex that also contains A-kinase anchoring protein-18, protein kinase type A-RII, and protein phosphatase type 2A. Our data support the conclusion that PDE3A is the primary PDE3 isozyme modulating basal contractility and SR Ca(2+) content by regulating cAMP in microdomains containing macromolecular complexes of SR calcium ATPase type 2a-phospholamban-PDE3A.

Distinct localization and modulation of Cav1.2 and Cav1.3 L‐type Ca2+ channels in mouse sinoatrial node

Dysregulation of L-type Ca(2+) currents in sinoatrial nodal (SAN) cells causes cardiac arrhythmia. Both Ca(v)1.2 and Ca(v)1.3 channels mediate sinoatrial L-type currents. Whether these channels exhibit differences in modulation and localization, which could affect their contribution to pacemaking, is unknown. In this study, we characterized voltage-dependent facilitation (VDF) and subcellular localization of Ca(v)1.2 and Ca(v)1.3 channels in mouse SAN cells and determined how these properties of Ca(v)1.3 affect sinoatrial pacemaking in a mathematical model. Whole cell Ba(2+) currents were recorded from SAN cells from mice carrying a point mutation that renders Ca(v)1.2 channels relatively insensitive to dihydropyridine antagonists. The Ca(v)1.2-mediated current was isolated in the presence of nimodipine (1 μm), which was subtracted from the total current to yield the Ca(v)1.3 component. With strong depolarizations (+80 mV), Ca(v)1.2 underwent significantly stronger inactivation than Ca(v)1.3. VDF of Ca(v)1.3 was evident during recovery from inactivation at a time when Ca(v)1.2 remained inactivated. By immunofluorescence, Ca(v)1.3 colocalized with ryanodine receptors in sarcomeric structures while Ca(v)1.2 was largely restricted to the delimiting plasma membrane. Ca(v)1.3 VDF enhanced recovery of pacemaker activity after pauses and positively regulated pacemaking during slow heart rate in a numerical model of mouse SAN automaticity, including preferential coupling of Ca(v)1.3 to ryanodine receptor-mediated Ca(2+) release. We conclude that strong VDF and colocalization with ryanodine receptors in mouse SAN cells are unique properties that may underlie a specific role for Ca(v)1.3 in opposing abnormal slowing of heart rate.

Zonisamide: its pharmacology, efficacy and safety in clinical trials

Zonisamide is a benzisoxazole derivative, chemically unrelated to other antiepileptic drugs, that appears to have multiple mechanisms of action, including inhibition of Na(+) channels and reduction of T-type Ca(2+) currents. It is currently licensed in Europe and the USA for adjunctive treatment of partial seizures in adults, and in Europe as monotherapy for treatment of partial seizures in adults with newly diagnosed epilepsy. Zonisamide displays predictable, dose-dependent pharmacokinetics and has a half-life of ~60h, allowing once- or twice-daily administration. It has a low potential for interactions with other medications, including oral contraceptives. The clinical efficacy of adjunctive zonisamide therapy has been established in four pivotal, phase III, randomized, double-blind, placebo-controlled trials, which together included approximately 850 patients, aged 12-77years, with refractory partial epilepsy. In all four trials, zonisamide 300-600mg/day resulted in significant reductions in median total seizure rates vs placebo, and zonisamide was generally well tolerated; the most frequently reported adverse events being somnolence, dizziness and anorexia/weight loss. Subanalysis of the primary European trial indicated that zonisamide was effective when administered as first-line adjunctive treatment, and a long-term extension to the same trial demonstrated that the efficacy and safety/tolerability of adjunctive zonisamide was sustained for up to 36months. Once-daily monotherapy with zonisamide (200-500mg/day) has been shown to be non-inferior to, and as well tolerated as, twice-daily monotherapy with controlled-release carbamazepine (400-1200mg/day) in adults with newly diagnosed partial epilepsy. Zonisamide has also been shown to have favourable long-term retention rates, an important indication of its overall effectiveness.

ORMDL3 modulates store-operated calcium entry and lymphocyte activation

T lymphocytes rely on a Ca(2+) signal known as store-operated calcium entry (SOCE) for their activation. This Ca(2+) signal is generated by activation of a T-cell receptor, depletion of endoplasmic reticulum (ER) Ca(2+) stores and activation of Ca(2+) release-activated Ca(2+) currents (I(CRAC)). Here, we report that the ER protein orosomucoid like 3 (ORMDL3), the product of the ORMDL3 gene associated with several autoimmune and/or inflammatory diseases, negatively modulates I(CRAC), SOCE, nuclear factor of activated T cells nuclear translocation and interleukin-2 production. ORMDL3 inhibits the Ca(2+) influx mechanism at the outer mitochondrial membrane, resulting in a Ca(2+)-dependent inhibition of I(CRAC) and reduced SOCE. The effect of ORMDL3 could be mimicked by interventions that decreased mitochondrial Ca(2+) influx and reverted by buffering of cytosolic Ca(2+) or activation of mitochondrial Ca(2+) influx. In conclusion, ORMDL3 modifies key steps in the process of T-lymphocyte activation, providing a functional link between the genetic associations of the ORMDL3 gene with autoimmune and/or inflammatory diseases.

The ionic bases of the action potential in isolated mouse cardiac Purkinje cell

Collecting electrophysiological and molecular data from the murine conduction system presents technical challenges. Thus, only little advantage has been taken of numerous genetically engineered murine models to study excitation through the cardiac conduction system of the mouse. To develop an approach for isolating murine cardiac Purkinje cells (PCs), to characterize major ionic currents and to use the data to simulate action potentials (APs) recorded from PCs. Light microscopy was used to isolate and identify PCs from apical and septal cells. Current and voltage clamp techniques were used to record APs and whole cell currents. We then simulated a PC AP on the basis of our experimental data. APs recorded from PCs were significantly longer than those recorded from ventricular cells. The prominent plateau phase of the PC AP was very negative (≈-40 mV). Spontaneous activity was observed only in PCs. The inward rectifier current demonstrated no significant differences compared to ventricular myocytes (VMs). However, sodium current density was larger, and the voltage-gated potassium current density was significantly less in PCs compared with myocytes. T-type Ca(2+) currents (I(Ca,T)) were present in PCs but not VMs. Computer simulations suggest that I(Ca,T) and cytosolic calcium diffusion significantly modulate AP profile recorded in PCs, as compared to VMs. Our study provides the first comprehensive ionic profile of murine PCs. The data show unique features of PC ionic mechanisms that govern its excitation process. Experimental data and numerical modeling results suggest that a smaller voltage-gated potassium current and the presence of I(Ca,T) are important determinants of the longer and relatively negative plateau phase of the APs.

Voltage-activated Ca2+ channels in rat renal afferent and efferent myocytes: no evidence for the T-type Ca2+ current

Based on indirect methods, it has been suggested that both L- and T-type Ca(2+) channels mediate signalling in the renal afferent arteriole and that T-type Ca(2+) channels are involved in signalling in the efferent arteriole. However, Ca(2+) currents have never been studied in these two vessels. Our study was initiated to directly determine the type of Ca(2+) channels in these vessels for the first time, using patch clamp. Native myocytes were obtained from individually isolated rat renal afferent and efferent arterioles and from rat tail arteries (TA). TA myocytes, which possess both L- and T-type Ca(2+) currents, served as a positive control. Inward Ca(2+) and Ba(2+) currents (I(Ca) and I(Ba)) were measured in 1.5 mmol/L Ca(2+) and 10 mmol/L Ba(2+), respectively, using the whole-cell configuration. By exploiting known differences in activation and inactivation characteristics and differing sensitivities to nifedipine and kurtoxin, the presence of both L- and T-type Ca(2+) channels in TA myocytes was readily demonstrated. Afferent arteriolar myocytes exhibited relatively large I(Ca) densities (-2.0 ± 0.2 pA/pF) in physiological Ca(2+) and the I(Ba) was 3.6-fold greater. These currents were blocked by nifedipine, but not by kurtoxin, and did not exhibit the activation and inactivation characteristics of T-type Ca(2+) channels. Efferent arteriolar myocytes did not exhibit a discernible voltage-activated I(Ca) in physiological Ca(2+). Our findings support the physiological role of L-type Ca(2+) channels in the afferent, but not efferent, arteriole, but do not support the premise that functional T-type Ca(2+) channels are present in either vessel.

Investigation of L‐type Ca2+ current in the aganglionic bowel segment in Hirschsprung’s disease

Studies on animal models of Hirschsprung's disease (HD) suggest that L-type Ca(2+) channels are down-regulated in the aganglionic bowel segment, however, this has yet to be confirmed in HD patients. The objective of this study was to test the hypothesis that L-type Ca(2+) current density is decreased in smooth muscle cells (SMC) obtained from the aganglionic bowel segment of patients with HD in comparison with those from the ganglionic segment. Smooth muscle cells were freshly isolated from colon samples obtained from HD patients undergoing pull-through surgery. L-type Ca(2+) currents were recorded using the perforated patch configuration of the whole cell voltage clamp technique and the expression levels of CACNA1C transcripts (which encode L-type Ca(2+) channels) in the ganglionic and aganglionic bowel segments were compared using real-time quantitative PCR. All SMC displayed robust currents that had activation/inactivation kinetics typical of L-type Ca(2+) current, were inhibited by nifedipine and enhanced by the L-type Ca(2+) channel agonists FPL 64176 and Bay K 8644. Moreover, FPL 64176 activated currents were also inhibited by nifedipine. However, there was no significant difference in L-type Ca(2+) current density, CACNA1C subunit expression or sensitivity to the pharmacological agents noted above, between SMC isolated from the ganglionic and aganglionic regions of the HD colon. In contrast to studies on genetic animal models of HD, L-type Ca(2+) currents are not down-regulated in the aganglionic bowel segment of HD patients and are therefore unlikely to account for the impaired colonic peristalsis observed in these patients.

P6 Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilation

Hydrogen sulfide (H 2 S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H 2 S induce vasodilation are unclear. We tested the hypothesis that H 2 S dilates piglet cerebral arterioles by modulating local and global intracellular Ca 2+ signals in smooth muscle cells. High-speed confocal imaging revealed that H 2 S increased Ca 2+ spark frequency ∼1.43-fold and decreased global intracellular Ca 2+ concentration ([Ca 2+ ] i ) by ∼37 nM in smooth muscle cells of intact cerebral arteries. In contrast, H 2 S did not alter Ca 2+ wave frequency. In voltage-clamped (−40 mV) cells, H 2 S increased the frequency of Ca 2+ spark-induced transient Ca 2+ -activated K + (K Ca ) currents ∼ 1.8-fold, but did not alter the amplitude of these events. H 2 S did not alter the activity of single K Ca channels recorded in the absence of Ca 2+ sparks in arteriole smooth muscle cells. H 2 S increased SR Ca 2+ load ([Ca 2+ ] SR ), measured as caffeine (10 mM)-induced [Ca 2+ ] i transients, ∼1.5-fold. H 2 S dilated pressurized cerebral arterioles. Iberiotoxin, a K Ca channel blocker, and ryanodine, a ryanodine receptor (RyR) channel inhibitor, reduced H 2 S-induced vasodilation by ∼38 and ∼37%, respectively. In summary, our data indicate that H 2 S elevates [Ca 2+ ] SR , leading to Ca 2+ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient K Ca current frequency leads to a reduction in global [Ca 2+ ] i and vasodilation.

TRPC3 overexpression promotes angiotensin II-induced cardiac dysfunction

Results AngII (100 nM) reduced left ventricular pressure (LVP) within 2 min to 64%, +dP/dt to 50% and −dP/dt to 55% of control in TRPC3(+/−) hearts, while even producing a positive inotropic effect in wild-type (WT) hearts. Simultaneously, ECG recordings demonstrated AngII-induced episodes of acute arrhythmogenicity in all TRPC3(+/−) hearts (n = 6), whereas rhythm of WT hearts (n = 6) remained unaffected. The AngII-induced impairment of cardiac functions in TRPC3(+/−) hearts was partially reversed by Pyr3 (30 μM). The amplitude of Ca transient was significantly higher (p < 0.05; n = 60) in myocytes from TRPC3(+/−) mice ([Ca] F/F0 0.354 ± 0.024) as compared to WT ([Ca] F/F0 0.262 ± 0.021). Also, the time constant (τ) of Ca decline was different between WT (0.196 ± 0.009 ms; n = 61) and TRPC3(+/−) (0.170 ± 0.008; n = 67; p < 0.05). Sarcomere shortening showed no significant difference between the two groups (3.80 ± 0.69% vs. 3.52 ± 0.65%; n = 10) whereas the SR-loading estimated from rapid application of caffeine (20 mM) revealed an increased SR loading of up to 40% in TRPC3 (+/−) myocytes as compared to WT (p < 0.05; n = 43). The time constant of Ca decline during caffeine challenge was also significantly changed (p < 0.05) in TRPC3 (+/−) myocytes (3.04 ± 0.44 ms; n = 11) as compared to WT cells (1.65 ± 0.158 ms; n = 16). Importantly, AngII (100 nM) induced a rise in diastolic Ca levels, which was accompanied by irregular contractions in TRPC3(+/−) but not in WT myocytes. The rise in the diastolic Ca levels was significantly suppressed by Pyr3 (10 μM; n = 16), SEA 0400 (1 μM; n = 14) and KN-93 (1 μM; n = 12). Electrophysiological characterization of L-type voltage-gated Ca currents and action potentials revealed that baseline electrophysiological parameters were not affected by TRPC3 overexpression, while AngII induced a transient prolongation of action potential duration only in TRPC3(+/−) myocytes. This TRPC3-dependent response was associated with a higher incidence of delayed afterdepolarizations.

Anchored phosphatases modulate glucose homeostasis

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic β-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-β-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in β-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

Nitric oxide as intracellular modulator: internal production of NO increases neuronal excitability via modulation of several ionic conductances

Nitric oxide (NO) has been shown to regulate neuronal excitability in the nervous system, but little is known as to whether NO, which is synthesized in certain neurons, also serves functional roles within NO-producing neurons themselves. We investigated this possibility by using a nitric oxide synthase (NOS)-expressing neuron, and studied the role of intrinsic NO production on neuronal firing properties in single-cell culture. B5 neurons of the pond snail Helisoma trivolvis fire spontaneous action potentials (APs), but once the intrinsic activity of NOS was inhibited, neurons became hyperpolarized and were unable to fire evoked APs. These striking long-term effects could be attributed to intrinsic NO acting on three types of conductances, a persistent sodium current (I(NaP) ), voltage-gated Ca currents (I(Ca) ) and small-conductance calcium-activated potassium (SK) channels. We show that NOS inhibitors 7-nitroindazole and S-methyl-l-thiocitrulline resulted in a decrease in I(NaP) , and that their hyperpolarizing and inhibiting effects on spontaneous spiking were mimicked by the inhibitor of I(NaP) , riluzole. Moreover, inhibition of NOS, soluble guanylate cyclase (sGC) or protein kinase G (PKG) attenuated I(Ca) , and blocked spontaneous and depolarization-induced spiking, suggesting that intrinsic NO controlled I(Ca) via the sGC/PKG pathway. The SK channel inhibitor apamin partially prevented the hyperpolarization observed after inhibition of NOS, suggesting a downregulation of SK channels by intrinsic NO. Taken together, we describe a novel mechanism by which neurons utilize their self-produced NO as an intrinsic modulator of neuronal excitability. In B5 neurons, intrinsic NO production is necessary to maintain spontaneous tonic and evoked spiking activity.

Short-term memory in the heart: A road map for channel trafficking required

Short-term memory in the heart: A road map for channel trafficking required

Ca2+ release-activated Ca2+ (CRAC) current, structure, and function

Store-operated Ca2+ entry describes the phenomenon that connects a depletion of internal Ca2+ stores to an activation of plasma membrane-located Ca2+ selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca2+ sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca2+-selective pore-forming subunit of Ca2+ release-activated Ca2+ channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca2+ currents.