L-type Channels Research Articles

TENS-like Stimulation Downregulates Inflammatory Cytokines in a PC-12 Cell Line

ObjectivesThe purpose of this study was to evaluate the effects of transcutaneous electrical nerve stimulation (TENS)–like stimulation on the expression of the proinflammatory cytokines tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 in PC-12 cells, which are commonly used as neuronal cell models. MethodsNerve growth factor–differentiated PC-12 cells were exposed to electrical stimulation for 15 minutes at 1 mA, 200 μs, and 100 Hz. Cell lysate from stimulated and control cells was assayed for TNF-α, IL-1β, and IL-6. In 6 trials, cells were preincubated with the L-type ion channel blocker nicardipine. Cultured cells were also incubated with Alexa Fluor 488 and visualized by fluorescence microscopy to determine the nuclear vs cytoplasmic distribution of the p65 sub-unit of NF-κB ResultsCompared with control (unstimulated) cells, the stimulated cells had a downregulation of the assayed cytokines. However, preincubation with the L-type ion channel blocker nicardipine blocked this effect of stimulation. Additionally, it was noted that TENS-like stimulation promoted a relative sequestration of the p65 subunit of NF-κB in the cytoplasm vs the nucleus. ConclusionsIt appears that in this cell line and with these stimulation parameters, TENS-like stimulation attenuated the expression of the assayed proinflammatory cytokines, in part by promoting the relative sequestration of the p65 subunit of NF-κB in the cytoplasm, and that voltage-dependent calcium channels have a role in the cascade of events initiated by the TENS-like stimulation.

Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, KV2.1, KV2.2, L-type CaV channels and H/K ATPases) or completely (GlyR, GABAAR, KV1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K+ channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H+ pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair.

Regulation of L-type CaV1.3 channel activity and insulin secretion by the cGMP-PKG signaling pathway

cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated CaV1.3L-type Ca2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant CaV1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG signaling cascade. Our results indicate that the CaVα1 ion-conducting subunit of the CaV1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca2+ macroscopic currents and impair insulin release stimulated with high K+. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphorylation sites to residues resistant to phosphorylation showed that the relevant PKG sites for CaV1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the CaVα1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate CaV1.3 channels and contribute to regulate insulin secretion.

Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes.

Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II–III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

The Marine Guanidine Alkaloid Crambescidin 816 Induces Calcium Influx and Cytotoxicity in Primary Cultures of Cortical Neurons through Glutamate Receptors.

Crambescidin 816 is a guanidine alkaloid produced by the sponge Crambe crambe with known antitumoral activity. While the information describing the effects of this alkaloid in central neurons is scarce, Cramb816 is known to block voltage dependent calcium channels being selective for L-type channels. Moreover, Cramb816 reduced neuronal viability through an unknown mechanism. Here, we aimed to describe the toxic activity of Cramb816 in cortical neurons. Since calcium influx is considered the main mechanism responsible for neuronal cell death, the effects of Cramb816 in the cytosolic calcium concentration of cortical neurons were studied. The alkaloid decreased neuronal viability and induced a dose-dependent increase in cytosolic calcium that was also related to the presence of calcium in the extracellular media. The increase in calcium influx was age dependent, being higher in younger neurons. Moreover, this effect was prevented by glutamate receptor antagonists, which did not fully block the cytotoxic effect of Cramb816 after 24 h of treatment but completely prevented Cramb816 cytotoxicity after 10 min exposure. Therefore, the findings presented herein provide new insights into the cytotoxic effect of Cramb816 in cortical neurons.

Cannabinoid CB1 and CB2 receptors differentially modulate L- and T-type Ca2+ channels in rat retinal ganglion cells

Endocannabinoid signaling system is involved in regulating multiple neuronal functions in the central nervous system by activating G-protein coupled cannabinoid CB1 and CB2 receptors (CB1Rs and CB2Rs). Growing evidence has shown that CB1Rs and CB2Rs are extensively expressed in retinal ganglion cells (RGCs). Here, modulation of L- and T-types Ca2+ channels by activating CB1Rs and CB2Rs in RGCs was investigated. Triple immunofluorescent staining showed that L-type subunit CaV1.2 was co-localized with T-type subunits (CaV3.1, CaV3.2 and CaV3.3) in rat RGCs. In acutely isolated rat RGCs, the CB1R agonist WIN55212-2 suppressed both peak and steady-state Ca2+ currents in a dose-dependent manner, with IC50 being 9.6 μM and 8.4 μM, respectively. It was further shown that activation of CB1Rs by WIN55212-2 or ACEA, another CB1R agonist, significantly suppressed both L- and T-type Ca2+ currents, and shifted inactivation curve of T-type one toward hyperpolarization direction. While the effect on L-type Ca2+ channels was mediated by intracellular cAMP/protein kinase A (PKA), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent protein kinase II (CaMKII) signaling pathways, only CaMKII signaling pathway was involved in the effect on T-type Ca2+ channels. Furthermore, CB65 and HU308, two specific CB2R agonists, significantly suppressed T-type Ca2+ channels, which was mediated by intracellular cAMP/PKA and CaMKII signaling pathways, but had no effect on L-type channels. These results imply that endogenous cannabinoids may modulate the excitability and the output of RGCs by differentially suppressing the activity of L- and T-type Ca2+ channels through activation of CB1Rs and CB2Rs.This article is part of the Special Issue entitled “A New Dawn in Cannabinoid Neurobiology”.

123 - Role of L-type Cav1.3 Ca2+ channels in Ca2+ handling and SAN pacemaker activity altered by external conditions

Introduction Membrane currents and Ca2+ handling generate Sinoatrial node (SAN) automaticity. Several studies showed the negative effect of membrane current impairment, while it is unknown how abnormal Ca2+ affect pacemaking. Purpose To investigate SAN automaticity when Ca2+ handling is altered by external conditions. Methods We video recorded contraction amplitude and spontaneous rate of SAN cells. Results In WT, 0.9 mM external Ca2+ ([Ca2+]0) reduces cell shortening, while 3 mM increased it, compared to control solution (1.8 mM [Ca2+]0). Lowering [Ca2+]0 to 0.9 mM did not affect WT automaticity, while high [Ca2+]0 dysregulate it without changing the average rate. L-type channels (LTCCs) are the main Ca2+ source in SAN cells, with Cav1.3 as the dominant isoform and Cav1.2 less expressed. To discern their role during abnormal [Ca2+]0, we used mice lacking Cav1.3 channels (Cav1.3−/−). As in WT, 0.9 mM [Ca2+]0 decreased cell shortening in Cav1.3−/−, while only a tendency to increase was determined by 3 mM [Ca2+]0. Also, high [Ca2+]0 significantly reduced spontaneous rate in Cav1.3−/− cells. Stimulation of WT cells with the LTCC dihydropyridine agonist BayK 8644 (BayK) increased cell shortening and spontaneous rate. Under BayK, Cav1.3−/− cells showed similar increase of contraction amplitude but reduced rate acceleration than WT. We repeated this protocol with a mouse where Cav1.2 is dihydropyridines insensitive (Cav1.2DHP−/−). Cav1.2DHP−/− SAN cells responded like WT to abnormal [Ca2+]0. Instead, BayK caused rate increase equivalent to WT but reduced contraction shortening in Cav1.2DHP−/−. Conclusion Abnormal increase of Ca2+ influx dysregulates automaticity in WT cells, likely through hyper-stimulation of Ca2+ activated currents. Moreover, selective stimulation of Cav1.2 and/or Cav1.3 with BayK suggests that Cav1.2 maintain the bulk of Ca2+ allowing proper contraction, while Cav1.3 mainly carry the influx of Ca2+ needed to trigger SAN automaticity.

Pharmacological approach to understanding the control of insulin secretion in human islets.

To understand better the control of insulin secretion by human β cells and to identify similarities to and differences from rodent models. Dynamic insulin secretion was measured in perifused human islets treated with pharmacological agents of known modes of action. Glucokinase activation (Ro28-1675) lowered the glucose threshold for stimulation of insulin secretion to 1 mmol/L (G1), augmented the response to G3-G5 but not to G8-G15, whereas tolbutamide remained active in G20, which indicates that not all KATP channels were closed by high glucose concentrations. An almost 2-fold greater response to G15 than to supramaximal tolbutamide in G3 or to KCl+diazoxide in G15 vs G3 quantified the contribution of metabolic amplification to insulin secretion. Both disruption (latrunculin-B) and stabilization (jasplakinolide) of microfilaments augmented insulin secretion without affecting metabolic amplification. Tolbutamide-induced insulin secretion was consistently greater in G10 than G3, with a threshold at 1 and maximum at 10 µmol/L tolbutamide in G10, vs 10 and 25 µmol/L in G3. Sulphonylurea effects were thus clearly glucose-dependent. Insulin secretion was also increased by inhibiting K channels other than KATP channels: Kv or BK channels (tetraethylammonium), TASK-1 channels (ML-365) and SK4 channels (TRAM-34). Opening KATP channels with diazoxide inhibited glucose-induced insulin secretion with half maximum inhibitory concentrations of 9.6 and 24 µmol/L at G7 and G15. Blockade of L-type Ca channels (nimodipine) abolished insulin secretion, whereas a blocker of T-type Ca channels (NNC-55-0396) was ineffective at specific concentrations. Blockade of Na channels (tetrodotoxin) did not affect glucose-induced insulin secretion. In addition to sharing a KATP channel-dependent triggering pathway and a metabolic amplifying pathway, human and rodent β cells were found to display more similarities than differences in the control of insulin secretion.

Novel 1, 4-dihydropyridines for L-type calcium channel as antagonists for cadmium toxicity

The present study, we design and synthesize the novel dihydropyridine derivatives, i.e., 3 (a-e) and 5 (a-e) and evaluated, anticonvulsant activity. Initially due to the lacuna of LCC, we modeled the protein through modeller 9.15v and evaluated through servers. Docking studies were performed with the synthesized compounds and resulted two best compounds, i.e., 5a, 5e showed the best binding energies. The activity of intracellular Ca2+ measurements was performed on two cell lines: A7r5 (rat aortic smooth muscle cells) and SH-SY5Y (human neuroblastoma cells). The 5a and 5e compounds was showing the more specific activity on L-type calcium channels, i.e. A7r5 (IC50 = 0.18 ± 0.02 and 0.25 ± 0.63 μg/ml, respectively) (containing only L-type channels) than SH-SY5Y (i.e. both L-type and T-type channels) (IC50 = 8 ± 0.23 and 10 ± 0.18 μg/ml, respectively) with intracellular calcium mobility similar to amlodipine. Finally, both in silico and in vitro results exploring two derivatives 5a and 5e succeeded to treat cadmium toxicity.

Insights into electrosensory organ development, physiology and evolution from a lateral line-enriched transcriptome.

The anamniote lateral line system, comprising mechanosensory neuromasts and electrosensory ampullary organs, is a useful model for investigating the developmental and evolutionary diversification of different organs and cell types. Zebrafish neuromast development is increasingly well understood, but neither zebrafish nor Xenopus is electroreceptive and our molecular understanding of ampullary organ development is rudimentary. We have used RNA-seq to generate a lateral line-enriched gene-set from late-larval paddlefish (Polyodon spathula). Validation of a subset reveals expression in developing ampullary organs of transcription factor genes critical for hair cell development, and genes essential for glutamate release at hair cell ribbon synapses, suggesting close developmental, physiological and evolutionary links between non-teleost electroreceptors and hair cells. We identify an ampullary organ-specific proneural transcription factor, and candidates for the voltage-sensing L-type Cav channel and rectifying Kv channel predicted from skate (cartilaginous fish) ampullary organ electrophysiology. Overall, our results illuminate ampullary organ development, physiology and evolution.

Low pHo boosts burst firing and catecholamine release by blocking TASK-1 and BK channels while preserving Cav1 channels in mouse chromaffin cells.

Mouse chromaffin cells (MCCs) generate spontaneous burst-firing that causes large increases of Ca2+ -dependent catecholamine release, and is thus a key mechanism for regulating the functions of MCCs. With the aim to uncover a physiological role for burst-firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo ) from 7.4 to 6.6 induces cell depolarizations of 10-15 mV that generate bursts of ∼330ms at 1-2Hz and a 7.4-fold increase of cumulative catecholamine-release. Burst-firing originates from the inhibition of the pH-sensitive TASK-1-channels and a 60% reduction of BK-channel conductance at pHo 6.6. Blockers of the two channels (A1899 and paxilline) mimic the effects of pHo 6.6, and this is reverted by the Cav1 channel blocker nifedipine. MCCs act as pH-sensors. At low pHo , they depolarize, undergo burst-firing and increase catecholamine-secretion, generating an effective physiological response that may compensate for the acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue. Mouse chromaffin cells (MCCs) generate action potential (AP) firing that regulates the Ca2+ -dependent release of catecholamines (CAs). Recent findings indicate that MCCs possess a variety of spontaneous firing modes that span from the common 'tonic-irregular' to the less frequent 'burst' firing. This latter is evident in a small fraction of MCCs but occurs regularly when Nav1.3/1.7 channels are made less available or when the Slo1β2-subunit responsible for BK channel inactivation is deleted. Burst firing causes large increases of Ca2+ -entry and potentiates CA release by ∼3.5-fold and thus may be a key mechanism for regulating MCC function. With the aim to uncover a physiological role for burst-firing we investigated the effects of acidosis on MCC activity. Lowering the extracellular pH (pHo ) from 7.4 to 7.0 and 6.6 induces cell depolarizations of 10-15mV that generate repeated bursts. Bursts at pHo 6.6 lasted ∼330ms, occurred at 1-2Hz and caused an ∼7-fold increase of CA cumulative release. Burst firing originates from the inhibition of the pH-sensitive TASK-1/TASK-3 channels and from a 40% BK channel conductance reduction at pHo 7.0. The same pHo had little or no effect on Nav, Cav, Kv and SK channels that support AP firing in MCCs. Burst firing of pHo 6.6 could be mimicked by mixtures of the TASK-1 blocker A1899 (300nm) and BK blocker paxilline (300nm) and could be prevented by blocking L-type channels by adding 3μm nifedipine. Mixtures of the two blockers raised cumulative CA-secretion even more than low pHo (∼12-fold), showing that the action of protons on vesicle release is mainly a result of the ionic conductance changes that increase Ca2+ -entry during bursts. Our data provide direct evidence suggesting that MCCs respond to low pHo with sustained depolarization, burst firing and enhanced CA-secretion, thus mimicking the physiological response of CCs to acute acidosis and hyperkalaemia generated during heavy exercise and muscle fatigue.

Calmodulin and Stac3 Enhance Functional Expression of CaV1.1

CaV1.1 is a prominent L-type voltage-gated calcium channel (VGCC) that plays an integral role in mediating skeletal muscle excitation-contraction coupling. Unlike other homologous L-type channels (e.g. CaV1.3), in depth biophysical analysis of CaV1.1 is challenging as functional expression of these channels has been generally restricted to cell types with a muscular lineage. Interestingly, in contrast to CaV1.3, the carboxy terminus of CaV1.1 has a low affinity for the calcium binding protein, calmodulin (CaM) suggesting that the loss of CaM pre-association may be responsible for the poor functional expression of CaV1.1 in recombinant systems. Here, we explicitly demonstrate that restoration of CaM to the channel complex enables functional expression of CaV1.1 in HEK293 cells. Further mechanistic analysis shows that CaM substantially enhances surface membrane trafficking of CaV1.1. In conjunction with recent studies that showed Stac3 (SH3 and cysteine rich domain 3) adaptor proteins also enable CaV1.1 currents in recombinant systems (Polster et al (2015) PNAS 112:602), our results argue that multiple cell signaling molecules can evoke similar functional outcomes and points to redundancy in molecular pathways that orchestrate CaV1.1 surface expression. These results also suggest there may be an overlap between channel regulation and trafficking of L-type VGCC with Stac3 and CaM. In all, our findings furnish a convenient platform to probe CaV1.1 function and related pharmacology.

Phosphorylation of Ser1928 mediates the enhanced activity of the L-type Ca2+ channel Cav1.2 by the β2-adrenergic receptor in neurons.

The L-type Ca2+ channel Cav1.2 controls multiple functions throughout the body including heart rate and neuronal excitability. It is a key mediator of fight-or-flight stress responses triggered by a signaling pathway involving β-adrenergic receptors (βARs), cyclic adenosine monophosphate (cAMP), and protein kinase A (PKA). PKA readily phosphorylates Ser1928 in Cav1.2 in vitro and in vivo, including in rodents and humans. However, S1928A knock-in (KI) mice have normal PKA-mediated L-type channel regulation in the heart, indicating that Ser1928 is not required for regulation of cardiac Cav1.2 by PKA in this tissue. We report that augmentation of L-type currents by PKA in neurons was absent in S1928A KI mice. Furthermore, S1928A KI mice failed to induce long-term potentiation in response to prolonged theta-tetanus (PTT-LTP), a form of synaptic plasticity that requires Cav1.2 and enhancement of its activity by the β2-adrenergic receptor (β2AR)-cAMP-PKA cascade. Thus, there is an unexpected dichotomy in the control of Cav1.2 by PKA in cardiomyocytes and hippocampal neurons.

Effects of L- and N-Type Ca Channel Blocker Cilnidipine on Changes in Heart Rate and QT Interval During Dialysis

Background/Aims: Hemodialysis patients have poor prognosis due to increased prevalence of cardiovascular diseases. Treatment to suppress increases in sympathetic nerve activity and QT prolongation may have the potential to reduce the occurrence of these events. The L/N-type Calcium (Ca) channel blocker cilnidipine has unique inhibitory action to inhibit sympathetic nerve activity and in a canine model ameliorates QT prolongation. In this study, we investigated whether cilnidipine has inhibitory effects on heart rate, an index of sympathetic nerve activity, and QT prolongation in patients undergoing dialysis. Methods: An L-type Ca channel blocker amlodipine was administered for 4 weeks followed by cilnidipine treatment for 4 weeks. On the last day of each period, heart rate and corrected QT interval were estimated and compared between the two periods. Results: Cilnidipine showed greater suppression of heart rate during dialysis than did amlodipine. The corrected QT interval in one dialysis session was significantly increased, and 3 of 17 patients showed prominent QT prolongation during administration of amlodipine but not cilnidipine. Conclusion: These data suggested that cilnidipine may inhibit increases in heart rate and QT interval. Cilnidipine may have beneficial effects in reducing cardiovascular events, resulting from increased sympathetic nerve activity and lethal arrhythmias in hemodialysis patients.

T48 - Molecule-Based Genetic Association Studies On Psychiatric Disorders

Background GWAS have successfully detected genetic variants associated with schizophrenia [Ripke et al., 2014]. However, only a small fraction of heritability can be explained. Gene-set/pathway based methods can overcome limitations arising from single SNP-based analysis, but most of them place constraints on size which may exclude highly specific and functional sets [Ramanan et al., 2012], like macromolecules. Ion channels, belonging to macromolecules, are created by polymerization of several subunits whose encoding genes are located far away or even on different chromosomes. We combined such molecules information with GWAS genotype data to investigate how functional channels associated with psychiatric disorders. Methods We defined a biologically meaningful gene-set based on channel structure and performed association study applying the SNP-set (Sequence) Kernel Association Test [Wu et al., 2010] to the Psychiatric Genomics Consortium (PGC) genotype data from bipolar disorder and schizophrenia. Results In the first stage of study (Voltage-gated calcium (Cav) channels vs schizophrenia), we identified 8 out 9 subtypes of Cav channels significantly associated with schizophrenia, including the L-type channels (Cav1.1, Cav1.2, Cav1.3), P-/Q-type Cav2.1, N-type Cav2.2, R-type Cav2.3, T-type Cav3.1 and Cav3.3. Only genes from Cav1.2 and Cav3.3 have been implicated by the largest GWAS (N = 82,315). In the second stage of study, more ion channels (K+ channels, Na+ channels, …) have been analyzed and data from bipolar disorder was investigated. The results will be presented. Discussion The results suggest that abnormalities of Cav channels may play an important role in the pathophysiology of schizophrenia. Analyzing subunit-encoding genes of a macromolecule in aggregate is a more powerful approach to identify the genetic architecture of polygenic diseases. Molecule-based genetic association study offers the potential of power for discovery and natural connections to biological mechanisms of psychiatric disorders. Significant channels may represent appropriate drug targets for therapeutics.

AB317. SPR-44 Pharmacological activation of individual KCNQ channel subtypes in detrusor smooth muscle represents a promising novel approach for overactive bladder treatment

ObjectiveOur recent studies have demonstrated voltage-gated KCNQ channels (KCNQ1-KCNQ5) as key regulators of detrusor smooth muscle (DSM) function. Despite emerging developments, the physiological role of individual KCNQ channel subtypes remains less clear. Here, we utilized the novel compound ML-213, a potent activator of KCNQ2, KCNQ4, and KCNQ5 channels, to elucidate their physiological roles in guinea pig DSM function.MethodsUsing isometric DSM tension recordings, Ca2+ imaging, and amphotericin-B perforated patch-clamp electrophysiology, we elucidated the role of ML-213-sensitive KCNQ channels in regulating DSM excitability and contractility.ResultsML-213 concentration-dependently (100 nM–30 µM) inhibited spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in DSM isolated strips. ML-213 (10 µM) decreased the global intracellular Ca2+ concentrations in DSM isolated strips, effects blocked by the L-type voltage-gated Ca2+ (CaV) channel inhibitor nifedipine (1 µM) and the KCNQ1-KCNQ5 channel inhibitor XE991 (10 µM). These data suggest that ML-213 decreases the global intracellular Ca2+ concentration by inhibiting L-type CaV channels through an indirect mechanism downstream from KCNQ channel activation. In addition, ML-213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in DSM cells, effects reversible by washout. We next aimed to examine the effects of ML-213 on whole cell KCNQ currents. To isolate KCNQ currents, the bath solution contained the large conductance voltage- and Ca2+-activated K+ channel inhibitor paxilline (1 µM) and gadolinium chloride (GdCl3, 50 µM), which blocks L-type CaV channels and non-selective cation channels. Under these experimental conditions, ML-213 (10 µM) enhanced whole cell KCNQ currents. These findings suggest that the modulation of K+ transport through ML-213-sensitive KCNQ channels underlies ML-213-induced cell membrane hyperpolarization to decrease the global intracellular Ca2+ concentration and DSM contractility.ConclusionsThese data using the novel compound ML-213, suggest that KCNQ2-, KCNQ4-, and KCNQ5-containing channels are essential regulators of the excitability, intracellular Ca2+ concentration, and contractility of DSM by virtue of their control of the membrane potential. Moreover, these new findings provide a foundational basis for future investigations on KCNQ channel functional roles in human DSM excitability and contractility to confirm their potential as novel therapeutic targets for overactive bladder.Source of FundingNIH grant R01-DK106964 to GV Petkov and F31-DK104528 to A Provence.

Cardiac N-methyl D-aspartate Receptors as a Pharmacological Target.

This study focuses on characterization of the cardiac N-methyl D-aspartate receptors (NMDARs) as a target for endogenous and synthetic agonists and antagonists. Using isolated perfused rat hearts, we have shown that intracoronary administration of the NMDAR agonists and antagonists has a pronounced effect on autonomous heart function. Perfusion of rat hearts with autologous blood supplemented with NMDAR agonists was associated with induction of tachycardia, sinus arrhythmia, and ischemia occurring within physiological plasma concentration range for glutamate and glycine. Intracoronary administration of the NMDAR antagonists exerted an antiarrhythmic effect and resulted in bradycardia and improvement of capillary perfusion. Action of antagonists eliprodil, Ro25-6981, memantine, ketamine, and MK-801 on autonomous heart function diverged strikingly from that of L-type Ca channel blockers. Cardiac NMDAR subunit composition differed from that of neuronal receptors and was age specific and chamber specific. Transcripts of the GluN3A and GluN2D were found in all heart chambers, whereas expression of GluN1 and GluN2A and 2C was restricted to the atria. Expression of the GluN2B protein in ventricles increased markedly with age of the animals. The obtained data reveal that NMDARs are expressed in rat heart contributing to the autonomic heart rate regulation and the function of the cardiac conduction system.

Junctophilin-2 gene therapy rescues heart failure by normalizing RyR2-mediated Ca2 + release

BackgroundJunctophilin-2 (JPH2) is the primary structural protein for the coupling of transverse (T)-tubule associated cardiac L-type Ca channels and type-2 ryanodine receptors on the sarcoplasmic reticulum within junctional membrane complexes (JMCs) in cardiomyocytes. Effective signaling between these channels ensures adequate Ca-induced Ca release required for normal cardiac contractility. Disruption of JMC subcellular domains, a common feature of failing hearts, has been attributed to JPH2 downregulation. Here, we tested the hypothesis that adeno-associated virus type 9 (AAV9) mediated overexpression of JPH2 could halt the development of heart failure in a mouse model of transverse aortic constriction (TAC). Methods and resultsFollowing TAC, a progressive decrease in ejection fraction was paralleled by a progressive decrease of cardiac JPH2 levels. AAV9-mediated expression of JPH2 rescued cardiac contractility in mice subjected to TAC. AAV9-JPH2 also preserved T-tubule structure. Moreover, the Ca2+ spark frequency was reduced and the Ca2+ transient amplitude was increased in AAV9-JPH2 mice following TAC, consistent with JPH2-mediated normalization of SR Ca2+ handling. ConclusionsThis study demonstrates that AAV9-mediated JPH2 gene therapy maintained cardiac function in mice with early stage heart failure. Moreover, restoration of JPH2 levels prevented loss of T-tubules and suppressed abnormal SR Ca2+ leak associated with contractile failure following TAC. These findings suggest that targeting JPH2 might be an attractive therapeutic approach for treating pathological cardiac remodeling during heart failure.

Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 μM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon).

Effect of ICa,L Blockade on Adrenergic Stimulation in Developing Heart.

The effect of verapamil-induced blockade of L-type calcium ionic currents (ICa,L) on the action of non-selective adrenergic cardiac stimulation by norepinephrine was examined during different periods of early postnatal ontogeny. In 1-week-old rats, intravenous norepinephrine induced a short-term tachycardia both with and without preliminary injected verapamil. In 3-week-old rats, norepinephrine alone produced no chronotropic effect; in contrast, it induced a biphasic tachycardia in verapamil-treated rats. In 6- and 20-month-old rats, norepinephrine induced a short-term tachycardia, which could be prevented by verapamil. The age-related peculiarities of chronotropic action of non-selective adrenergic stimulation are indicative of the role of L-type calcium ionic channels in the development of sympathetic control over the heart.