Anchored PKA synchronizes adrenergic phosphoregulation of cardiac Cav1.2 channels.

Adrenergic modulation of voltage gated Ca2+ currents is a context specific process. In the heart Cav1.2 channels initiate excitation-contraction coupling. This requires PKA phosphorylation of the small GTPase Rad (Ras associated with diabetes) and involves direct phosphorylation of the Cav1.2 α1 subunit at Ser1700. A contributing factor is the proximity of PKA to the channel through association with A-kinase anchoring proteins (AKAPs). Disruption of PKA anchoring by the disruptor peptide AKAP-IS prevents upregulation of Cav1.2 currents in tsA-201 cells. Biochemical analyses demonstrate that Rad does not function as an AKAP. Electrophysiological recording shows that channel mutants lacking phosphorylation sites (Cav1.2 STAA) lose responsivity to the second messenger cAMP. Measurements in cardiomyocytes isolated from Rad-/- mice show that adrenergic activation of Cav1.2 is attenuated but not completely abolished. Whole animal electrocardiography studies reveal that cardiac selective Rad KO mice exhibited higher baseline left ventricular ejection fraction, greater fractional shortening, and increased heart rate as compared to control animals. Yet, each parameter of cardiac function was slightly elevated when Rad-/- mice were treated with the adrenergic agonist isoproterenol. Thus, phosphorylation of Cav1.2 and dissociation of phospho-Rad from the channel are local cAMP responsive events that act in concert to enhance L-type calcium currents. This convergence of local PKA regulatory events at the cardiac L-type calcium channel may permit maximal ß-adrenergic influence on the fight-or-flight response.

The serine hydrolase ABHD6 controls survival and thermally induced seizures in a mouse model of Dravet syndrome.

Evidence suggests that inhibition of α/ß hydrolase-domain containing 6 (ABHD6) reduces seizures; however, the molecular mechanism of this therapeutic response remains unknown. We discovered that heterozygous expression of Abhd6 (Abhd6+/-) significantly reduced the premature lethality of Scn1a+/- mouse pups, a genetic mouse model of Dravet Syndrome (DS). Both Abhd6+/- mutation and pharmacological inhibition of ABHD6 reduced the duration and incidence of thermally induced seizures in Scn1a+/- pups. Mechanistically, the in vivo anti-seizure response resulting from ABHD6 inhibition is mediated by potentiation of gamma-aminobutyric acid receptors Type-A (GABAAR). Brain slice electrophysiology showed that blocking ABHD6 potentiates extrasynaptic (tonic) GABAAR currents that reduce dentate granule cell excitatory output without affecting synaptic (phasic) GABAAR currents. Our results unravel an unexpected mechanistic link between ABHD6 activity and extrasynaptic GABAAR currents that controls hippocampal hyperexcitability in a genetic mouse model of DS. BRIEF SUMMARY: This study provides the first evidence for a mechanistic link between ABHD6 activity and the control of extrasynaptic GABAAR currents that controls hippocampal hyperexcitability in a genetic mouse model of Dravet Syndrome and can be targeted to dampened seizures.

Combined Antiseizure Efficacy of Cannabidiol and Clonazepam in a Conditional Mouse Model of Dravet Syndrome.

Dravet Syndrome (DS) is a severe childhood epilepsy caused by heterozygous loss-of-function mutations in the SCN1A gene encoding brain type-I voltage-gated sodium channel Nav1.1. DS is a devastating disease that typically begins at six to nine months of age. Symptoms include recurrent intractable seizures and premature death with severe neuropsychiatric comorbidities, including hyperactivity, sleep disorder, anxiety-like behaviors, impaired social interactions, and cognitive deficits. There is an urgent unmet need for therapeutic approaches that control and cure DS, as available therapeutic interventions have poor efficacy, intolerance, or other side effects. Here we investigated the therapeutic potential of combining the benzodiazepine clonazepam (CLZ) with the nonpsychotropic phytocannabinoid cannabidiol (CBD) against thermally induced febrile seizures in a conditional mouse model of DS. Our results show that a low dose of CLZ alone or combined with CBD elevated the threshold temperature for the thermal induction of seizures. Combination of CLZ with CBD significantly reduced seizure duration compared to the vehicle or CLZ alone, but did not affect seizure severity, indicating potential additive actions of CLZ and CBD on the duration of seizures. Our findings provide preclinical evidence supporting combination therapy of CLZ and CBD for treatment of febrile seizures in DS.

A more efficient conditional mouse model of Dravet syndrome: Implications for epigenetic selection and sex-dependent behaviors.

BACKGROUND: Dravet Syndrome (DS) is an epileptic disorder characterized by spontaneous and thermally-induced seizures, hyperactivity, cognitive deficits, autistic-like behaviors, and Sudden Unexpected Death in Epilepsy (SUDEP). DS is caused by de novo loss-of-function mutations in the SCN1A gene. Selective loss of GABAergic interneuron excitability is the primary cause of the disease. Up to 60% of Scn1a+/- mice die from SUDEP before sexual maturity. NEW METHOD: We used Cre-Lox technology to conditionally delete Scn1a in all epiblast-derived somatic cells by crossing a floxed Scn1a mouse with a mouse expressing Cre under the Meox2 promoter. RESULTS: Parental Scn1a flox (F) mice, parental Meox2 Cre+ mice, and their F/+:Meox2-Cre- offspring were phenotypically normal and did not prematurely die. In contrast, F/+:Meox2-Cre+ offspring recapitulated DS seizure and behavioral phenotypes. Unexpectedly, male F/+:Meox2-Cre+ mice demonstrated impaired social interaction, while females did not. COMPARISON WITH EXISTING METHOD: In the previous models, colony maintenance required breeding SUDEP survivors, which greatly increased colony size required to sustain experimental animal production, and raised the concern that surviving breeders have epigenetic traits that impart new phenotypes to their offspring. Our method greatly facilitates breeding, recapitulates DS phenotypes, eliminates concerns about parents that are survivors, and provides initial evidence for unexpected sex-dependent social interaction impairment. CONCLUSIONS: We introduce a more efficient mouse model of human DS that demonstrates an efficient breeding strategy free from potential inherited epigenetic changes and reveals an unexpected sex-specific impairment of social interaction in DS. This new model should have great value to investigators of DS.

The AKAP Cypher/Zasp contributes to ß-adrenergic/PKA stimulation of cardiac CaV1.2 calcium channels.

Stimulation of the L-type Ca2+ current conducted by CaV1.2 channels in cardiac myocytes by the ß-adrenergic/protein kinase A (PKA) signaling pathway requires anchoring of PKA to the CaV1.2 channel by an A-kinase anchoring protein (AKAP). However, the AKAP(s) responsible for regulation in vivo remain unknown. Here, we test the role of the AKAP Cypher/Zasp in ß-adrenergic regulation of CaV1.2 channels using physiological studies of cardiac ventricular myocytes from young-adult mice lacking the long form of Cypher/Zasp (LCyphKO mice). These myocytes have increased protein levels of CaV1.2, PKA, and calcineurin. In contrast, the cell surface density of CaV1.2 channels and the basal Ca2+ current conducted by CaV1.2 channels are significantly reduced without substantial changes to kinetics or voltage dependence. ß-adrenergic regulation of these L-type Ca2+ currents is also significantly reduced in myocytes from LCyphKO mice, whether calculated as a stimulation ratio or as net-stimulated Ca2+ current. At 100 nM isoproterenol, the net ß-adrenergic-Ca2+ current conducted by CaV1.2 channels was reduced to 39 ± 12% of wild type. However, concentration-response curves for ß-adrenergic stimulation of myocytes from LCyphKO mice have concentrations that give a half-maximal response similar to those for wild-type mice. These results identify Cypher/Zasp as an important AKAP for ß-adrenergic regulation of cardiac CaV1.2 channels. Other AKAPs may work cooperatively with Cypher/Zasp to give the full magnitude of ß-adrenergic regulation of CaV1.2 channels observed in vivo.

Cannabidiol attenuates seizures and social deficits in a mouse model of Dravet syndrome.

Worldwide medicinal use of cannabis is rapidly escalating, despite limited evidence of its efficacy from preclinical and clinical studies. Here we show that cannabidiol (CBD) effectively reduced seizures and autistic-like social deficits in a well-validated mouse genetic model of Dravet syndrome (DS), a severe childhood epilepsy disorder caused by loss-of-function mutations in the brain voltage-gated sodium channel NaV1.1. The duration and severity of thermally induced seizures and the frequency of spontaneous seizures were substantially decreased. Treatment with lower doses of CBD also improved autistic-like social interaction deficits in DS mice. Phenotypic rescue was associated with restoration of the excitability of inhibitory interneurons in the hippocampal dentate gyrus, an important area for seizure propagation. Reduced excitability of dentate granule neurons in response to strong depolarizing stimuli was also observed. The beneficial effects of CBD on inhibitory neurotransmission were mimicked and occluded by an antagonist of GPR55, suggesting that therapeutic effects of CBD are mediated through this lipid-activated G protein-coupled receptor. Our results provide critical preclinical evidence supporting treatment of epilepsy and autistic-like behaviors linked to DS with CBD. We also introduce antagonism of GPR55 as a potential therapeutic approach by illustrating its beneficial effects in DS mice. Our study provides essential preclinical evidence needed to build a sound scientific basis for increased medicinal use of CBD.

Loss of ß-adrenergic-stimulated phosphorylation of CaV1.2 channels on Ser1700 leads to heart failure.

L-type Ca2+ currents conducted by voltage-gated calcium channel 1.2 (CaV1.2) initiate excitation-contraction coupling in the heart, and altered expression of CaV1.2 causes heart failure in mice. Here we show unexpectedly that reducing ß-adrenergic regulation of CaV1.2 channels by mutation of a single PKA site, Ser1700, in the proximal C-terminal domain causes reduced contractile function, cardiac hypertrophy, and heart failure without changes in expression, localization, or function of the CaV1.2 protein in the mutant mice (SA mice). These deficits were aggravated with aging. Dual mutation of Ser1700 and a nearby casein-kinase II site (Thr1704) caused accelerated hypertrophy, heart failure, and death in mice with these mutations (STAA mice). Cardiac hypertrophy was increased by voluntary exercise and by persistent ß-adrenergic stimulation. PKA expression was increased, and PKA sites Ser2808 in ryanodine receptor type-2, Ser16 in phospholamban, and Ser23/24 in troponin-I were hyperphosphorylated in SA mice, whereas phosphorylation of substrates for calcium/calmodulin-dependent protein kinase II was unchanged. The Ca2+ pool in the sarcoplasmic reticulum was increased, the activity of calcineurin was elevated, and calcineurin inhibitors improved contractility and ameliorated cardiac hypertrophy. Cardio-specific expression of the SA mutation also caused reduced contractility and hypertrophy. These results suggest engagement of compensatory mechanisms, which initially may enhance the contractility of individual myocytes but eventually contribute to an increased sensitivity to cardiovascular stress and to heart failure in vivo. Our results demonstrate that normal regulation of CaV1.2 channels by phosphorylation of Ser1700 in cardiomyocytes is required for cardiovascular homeostasis and normal physiological regulation in vivo.

K(ATP) channel gain-of-function leads to increased myocardial L-type Ca(2+) current and contractility in Cantu syndrome.

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) KATP channel subunits. We show that patients with CS, as well as mice with constitutive (cGOF) or tamoxifen-induced (icGOF) cardiac-specific Kir6.1 GOF subunit expression, have enlarged hearts, with increased ejection fraction and increased contractility. Whole-cell voltage-clamp recordings from cGOF or icGOF ventricular myocytes (VM) show increased basal L-type Ca(2+) current (LTCC), comparable to that seen in WT VM treated with isoproterenol. Mice with vascular-specific expression (vGOF) show left ventricular dilation as well as less-markedly increased LTCC. Increased LTCC in KATP GOF models is paralleled by changes in phosphorylation of the pore-forming α1 subunit of the cardiac voltage-gated calcium channel Cav1.2 at Ser1928, suggesting enhanced protein kinase activity as a potential link between increased KATP current and CS cardiac pathophysiology.

Phosphorylation of Cav1.2 on S1928 uncouples the L-type Ca2+ channel from the ß2 adrenergic receptor.

Agonist-triggered downregulation of ß-adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of ß2AR signaling highly specific for Cav1.2. We find that ß2-AR binding to Cav1.2 residues 1923-1942 is required for ß-adrenergic regulation of Cav1.2. Despite the prominence of PKA-mediated phosphorylation of Cav1.2 S1928 within the newly identified ß2AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the ß2AR from Cav1.2 upon ß-adrenergic stimulation rendering Cav1.2 refractory for several minutes from further ß-adrenergic stimulation. This effect is lost in S1928A knock-in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, ß2AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the ß2AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized ß2AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT-LTP) depends on Cav1.2 and its regulation by channel-associated ß2AR.

Dissecting the phenotypes of Dravet syndrome by gene deletion.

Neurological and psychiatric syndromes often have multiple disease traits, yet it is unknown how such multi-faceted deficits arise from single mutations. Haploinsufficiency of the voltage-gated sodium channel Nav1.1 causes Dravet syndrome, an intractable childhood-onset epilepsy with hyperactivity, cognitive deficit, autistic-like behaviours, and premature death. Deletion of Nav1.1 channels selectively impairs excitability of GABAergic interneurons. We studied mice having selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons. In brain slices, these deletions cause increased threshold for action potential generation, impaired action potential firing in trains, and reduced amplification of postsynaptic potentials in those interneurons. Selective deletion of Nav1.1 in parvalbumin- or somatostatin-expressing interneurons increases susceptibility to thermally-induced seizures, which are strikingly prolonged when Nav1.1 is deleted in both interneuron types. Mice with global haploinsufficiency of Nav1.1 display autistic-like behaviours, hyperactivity and cognitive impairment. Haploinsufficiency of Nav1.1 in parvalbumin-expressing interneurons causes autistic-like behaviours, but not hyperactivity, whereas haploinsufficiency in somatostatin-expressing interneurons causes hyperactivity without autistic-like behaviours. Heterozygous deletion in both interneuron types is required to impair long-term spatial memory in context-dependent fear conditioning, without affecting short-term spatial learning or memory. Thus, the multi-faceted phenotypes of Dravet syndrome can be genetically dissected, revealing synergy in causing epilepsy, premature death and deficits in long-term spatial memory, but interneuron-specific effects on hyperactivity and autistic-like behaviours. These results show that multiple disease traits can arise from similar functional deficits in specific interneuron types.

Sleep impairment and reduced interneuron excitability in a mouse model of Dravet Syndrome.

Dravet Syndrome (DS) is caused by heterozygous loss-of-function mutations in voltage-gated sodium channel NaV1.1. Our mouse genetic model of DS recapitulates its severe seizures and premature death. Sleep disturbance is common in DS, but its mechanism is unknown. Electroencephalographic studies revealed abnormal sleep in DS mice, including reduced delta wave power, reduced sleep spindles, increased brief wakes, and numerous interictal spikes in Non-Rapid-Eye-Movement sleep. Theta power was reduced in Rapid-Eye-Movement sleep. Mice with NaV1.1 deleted specifically in forebrain interneurons exhibited similar sleep pathology to DS mice, but without changes in circadian rhythm. Sleep architecture depends on oscillatory activity in the thalamocortical network generated by excitatory neurons in the ventrobasal nucleus (VBN) of the thalamus and inhibitory GABAergic neurons in the reticular nucleus of the thalamus (RNT). Whole-cell NaV current was reduced in GABAergic RNT neurons but not in VBN neurons. Rebound firing of action potentials following hyperpolarization, the signature firing pattern of RNT neurons during sleep, was also reduced. These results demonstrate imbalance of excitatory vs. inhibitory neurons in this circuit. As predicted from this functional impairment, we found substantial deficit in homeostatic rebound of slow wave activity following sleep deprivation. Although sleep disorders in epilepsies have been attributed to anti-epileptic drugs, our results show that sleep disorder in DS mice arises from loss of NaV1.1 channels in forebrain GABAergic interneurons without drug treatment. Impairment of NaV currents and excitability of GABAergic RNT neurons are correlated with impaired sleep quality and homeostasis in these mice.

Genetic background modulates impaired excitability of inhibitory neurons in a mouse model of Dravet syndrome.

Dominant loss-of-function mutations in voltage-gated sodium channel NaV1.1 cause Dravet Syndrome, an intractable childhood-onset epilepsy. NaV1.1(+/-) Dravet Syndrome mice in C57BL/6 genetic background exhibit severe seizures, cognitive and social impairments, and premature death. Here we show that Dravet Syndrome mice in pure 129/SvJ genetic background have many fewer seizures and much less premature death than in pure C57BL/6 background. These mice also have a higher threshold for thermally induced seizures, fewer myoclonic seizures, and no cognitive impairment, similar to patients with Genetic Epilepsy with Febrile Seizures Plus. Consistent with this mild phenotype, mutation of NaV1.1 channels has much less physiological effect on neuronal excitability in 129/SvJ mice. In hippocampal slices, the excitability of CA1 Stratum Oriens interneurons is selectively impaired, while the excitability of CA1 pyramidal cells is unaffected. NaV1.1 haploinsufficiency results in increased rheobase and threshold for action potential firing and impaired ability to sustain high-frequency firing. Moreover, deletion of NaV1.1 markedly reduces the amplification and integration of synaptic events, further contributing to reduced excitability of interneurons. Excitability is less impaired in inhibitory neurons of Dravet Syndrome mice in 129/SvJ genetic background. Because specific deletion of NaV1.1 in forebrain GABAergic interneuons is sufficient to cause the symptoms of Dravet Syndrome in mice, our results support the conclusion that the milder phenotype in 129/SvJ mice is caused by lesser impairment of sodium channel function and electrical excitability in their forebrain interneurons. This mild impairment of excitability of interneurons leads to a milder disease phenotype in 129/SvJ mice, similar to Genetic Epilepsy with Febrile Seizures Plus in humans.

Basal and ß-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700.

L-type calcium (Ca(2+)) currents conducted by voltage-gated Ca(2+) channel CaV1.2 initiate excitation-contraction coupling in cardiomyocytes. Upon activation of ß-adrenergic receptors, phosphorylation of CaV1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca(2+) entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700-Ala in the CaV1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type CaV1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3-4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca(2+) current and regulation by ß-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.

Soma size and Cav1.3 channel expression in vulnerable and resistant motoneuron populations of the SOD1G93A mouse model of ALS.

Although the loss of motoneurons is an undisputed feature of amyotrophic lateral sclerosis (ALS) in man and in its animal models (SOD1 mutant mice), how the disease affects the size and excitability of motoneurons prior to their degeneration is not well understood. This study was designed to test the hypothesis that motoneurons in mutant SOD1(G93A) mice exhibit an enlargement of soma size (i.e., cross-sectional area) and an increase in Cav1.3 channel expression at postnatal day 30, well before the manifestation of physiological symptoms that typically occur at p90 (Chiu et al. 1995). We made measurements of spinal and hypoglossal motoneurons vulnerable to degeneration, as well as motoneurons in the oculomotor nucleus that are resistant to degeneration. Overall, we found that the somata of motoneurons in male SOD1(G93A) mutants were larger than those in wild-type transgenic males. When females were included in the two groups, significance was lost. Expression levels of the Cav1.3 channels were not differentiated by genotype, sex, or any interaction of the two. These results raise the intriguing possibility of an interaction between male sex steroid hormones and the SOD1 mutation in the etiopathogenesis of ALS.

Impaired excitability of somatostatin- and parvalbumin-expressing cortical interneurons in a mouse model of Dravet syndrome.

Haploinsufficiency of the voltage-gated sodium channel NaV1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of NaV1.1 in forebrain GABAergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the NaV1.1 sodium channel, Scn1a, revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.

PDE4B mediates local feedback regulation of ß1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes.

Multiple cAMP phosphodiesterase (PDE) isoforms play divergent roles in cardiac homeostasis but the molecular basis for their non-redundant function remains poorly understood. Here, we report a novel role for the PDE4B isoform in ß-adrenergic (ßAR) signaling in the heart. Genetic ablation of PDE4B disrupted ßAR-induced cAMP transients, as measured by FRET sensors, at the sarcolemma but not in the bulk cytosol of cardiomyocytes. This effect was further restricted to a subsarcolemmal compartment because PDE4B regulates ß1AR-, but not ß2AR- or PGE2-induced responses. The spatially restricted function of PDE4B was confirmed by its selective effects on PKA-mediated phosphorylation patterns. PDE4B limited the PKA-mediated phosphorylation of key players in excitation-contraction coupling that reside in the sarcolemmal compartment, including L-type Ca(2+) channels and ryanodine receptors, but not phosphorylation of distal cytosolic proteins. ß1AR- but not ß2AR-ligation induced PKA-dependent activation of PDE4B and interruption of this negative feedback with PKA inhibitors increased sarcolemmal cAMP. Thus, PDE4B mediates a crucial PKA-dependent feedback that controls ß1AR-dependent cAMP signals in a restricted subsarcolemmal domain. Disruption of this feedback augments local cAMP/PKA signals, leading to an increased intracellular Ca(2+) level and contraction rate.

Apolipoprotein CIII hyperactivates ß cell CaV1 channels through SR-BI/ß1 integrin-dependent coactivation of PKA and Src.

Apolipoprotein CIII (ApoCIII) not only serves as an inhibitor of triglyceride hydrolysis but also participates in diabetes-related pathological events such as hyperactivation of voltage-gated Ca(2+) (CaV) channels in the pancreatic ß cell. However, nothing is known about the molecular mechanisms whereby ApoCIII hyperactivates ß cell CaV channels. We now demonstrate that ApoCIII increased CaV1 channel open probability and density. ApoCIII enhanced whole-cell Ca(2+) currents and the CaV1 channel blocker nimodipine completely abrogated this enhancement. The effect of ApoCIII was not influenced by individual inhibition of PKA, PKC, or Src. However, combined inhibition of PKA, PKC, and Src counteracted the effect of ApoCIII, similar results obtained by coinhibition of PKA and Src. Moreover, knockdown of ß1 integrin or scavenger receptor class B type I (SR-BI) prevented ApoCIII from hyperactivating ß cell CaV channels. These data reveal that ApoCIII hyperactivates ß cell CaV1 channels through SR-BI/ß1 integrin-dependent coactivation of PKA and Src.

Phosphorylation sites required for regulation of cardiac calcium channels in the fight-or-flight response.

L-type Ca(2+) currents conducted by CaV1.2 channels initiate excitation-contraction coupling in the heart. Their activity is increased by ß-adrenergic/cAMP signaling via phosphorylation by PKA in the fight-or-flight response, but the sites of regulation are unknown. We describe the functional role of phosphorylation of Ser1700 and Thr1704-sites of phosphorylation by PKA and casein kinase II at the interface between the proximal and distal C-terminal regulatory domains. Mutation of both residues to Ala in STAA mice reduced basal L-type Ca(2+) currents, due to a small decrease in expression and a substantial decrease in functional activity. The increase in L-type Ca(2+) current caused by isoproterenol was markedly reduced at physiological levels of stimulation (3-10 nM). Maximal increases in calcium current at nearly saturating concentrations of isoproterenol (100 nM) were also significantly reduced, but the mutation effects were smaller, suggesting that alternative regulatory mechanisms are engaged at maximal levels of stimulation. The ß-adrenergic increase in cell contraction was also diminished. STAA ventricular myocytes exhibited arrhythmic contractions in response to isoproterenol, and up to 20% of STAA cells failed to sustain contractions when stimulated at 1 Hz. STAA mice have reduced exercise capacity, and cardiac hypertrophy is evident at 3 mo. We conclude that phosphorylation of Ser1700 and Thr1704 is essential for regulation of basal activity of CaV1.2 channels and for up-regulation by ß-adrenergic signaling at physiological levels of stimulation. Disruption of phosphorylation at those sites leads to impaired cardiac function in vivo, as indicated by reduced exercise capacity and cardiac hypertrophy.

Differential regulation of cardiac excitation-contraction coupling by cAMP phosphodiesterase subtypes.

AIMS: Multiple phosphodiesterases (PDEs) hydrolyze cAMP in cardiomyocytes, but the functional significance of this diversity is not well understood. Our goal here was to characterize the involvement of three different PDEs (PDE2-4) in cardiac excitation-contraction coupling (ECC). METHODS AND RESULTS: Sarcomere shortening and Ca(2+) transients were recorded simultaneously in adult rat ventricular myocytes and ECC protein phosphorylation by PKA was determined by western blot analysis. Under basal conditions, selective inhibition of PDE2 or PDE3 induced a small but significant increase in Ca(2+) transients, sarcomere shortening, and troponin I phosphorylation, whereas PDE4 inhibition had no effect. PDE3 inhibition, but not PDE2 or PDE4, increased phospholamban phosphorylation. Inhibition of either PDE2, 3, or 4 increased phosphorylation of the myosin-binding protein C, but neither had an effect on L-type Ca(2+) channel or ryanodine receptor phosphorylation. Dual inhibition of PDE2 and PDE3 or PDE2 and PDE4 further increased ECC compared with individual PDE inhibition, but the most potent combination was obtained when inhibiting simultaneously PDE3 and PDE4. This combination also induced a synergistic induction of ECC protein phosphorylation. Submaximal ß-adrenergic receptor stimulation increased ECC, and this effect was potentiated by individual PDE inhibition with the rank order of potency PDE4 = PDE3 > PDE2. Identical results were obtained on ECC protein phosphorylation. CONCLUSION: Our results demonstrate that PDE2, PDE3, and PDE4 differentially regulate ECC in adult cardiomyocytes. PDE2 and PDE3 play a more prominent role than PDE4 in regulating basal cardiac contraction and Ca(2+) transients. However, PDE4 becomes determinant when cAMP levels are elevated, for instance, upon ß-adrenergic stimulation or PDE3 inhibition.

Correlations in timing of sodium channel expression, epilepsy, and sudden death in Dravet syndrome.

Dravet Syndrome (DS) is an intractable genetic epilepsy caused by loss-of-function mutations in SCN1A, the gene encoding brain sodium channel Nav 1.1. DS is associated with increased frequency of sudden unexpected death in humans and in a mouse genetic model of this disease. Here we correlate the time course of declining expression of the murine embryonic sodium channel Nav 1.3 and the rise in expression of the adult sodium channel Nav 1.1 with susceptibility to epileptic seizures and increased incidence of sudden death in DS mice. Parallel studies with unaffected human brain tissue demonstrate similar decline in Nav 1.3 and increase in Nav 1.1 with age. In light of these results, we introduce the hypothesis that the natural loss Nav 1.3 channel expression in brain development, coupled with the failure of increase in functional Nav 1.1 channels in DS, defines a tipping point that leads to disinhibition of neural circuits, intractable seizures, co-morbidities, and premature death in this disease.