Calreticulin negatively regulates the surface expression of Cav1.3 L-type calcium channel

Background

The neuroendocrine Cav1.3 L-type Ca channels have been recently found in the Human fetal heart and shown to play a vital role in Ca entry from the sarcolemma into the cell and in Ca homeostasis. Calreticulin, a Ca binding endoplasmic reticulum (ER) resident protein, has been recently shown to translocate to the cell surface where its role and function are just emerging. Here, we demonstrated a novel mechanism of Cav1.3 and calreticulin interaction resulting in downregulation of Cav1.3 channel densities in native Human fetal cardiac cells and Human Embryonic Kidney cell lines (tsA201).

Methods and results

Cell surface and cytoplasmic staining of calreticulin was demonstrated first in cultured human fetal cardiomyocytes (HFC), gestational age 18–24 weeks, using confocal microscopy thereby establishing that calreticulin is present at the cell surface in HFC. Co-immunoprecipitation from HFC using anti-Cav1.3 Ca channel antibody, and probing with anti-calreticulin antibody revealed a 46 kDa band corresponding to calreticulin suggesting that Cav1.3 Ca channel and calreticulin co-assemble in a macromolecular complex. Co-expression of Cav1.3 and calreticulin in tsA201 cells resulted in a decrease in surface expression of Cav1.3 Ca channels. These findings were consistent with the electrophysiological studies showing that co-transfection of Cav1.3 Ca channel and calreticulin resulted in 55% reduction of Cav1.3 Ca current densities recorded from tsA201 cells.

Conclusions

The results show the first evidence that calreticulin: (1) is localized outside the ER on the cell surface of HFC; (2) coimmunoprecipitates with Cav1.3 L-type Ca channel; (3) negatively regulates Cav1.3 surface expression thus resulting in decreased Cav1.3 Ca current densities. The data demonstrate a novel mechanism of modulation of Cav1.3 Ca channel by calreticulin, which may be involved in pathological settings such as autoimmune associated congenital heart block where Cav1.3 Ca channels are downregulated.     

Activity-dependent modulation of neuronal KV channels by retinoic acid enhances CaV channel activity

The metabolite of vitamin A, retinoic acid (RA), is known to affect synaptic plasticity in the nervous system and to play an important role in learning and memory. A ubiquitous mechanism by which neuronal plasticity develops in the nervous system is through modulation of voltage-gated Ca²⁺ (Ca_V) and voltage-gated K⁺ channels. However, how retinoids might regulate the activity of these channels has not been determined. Here, we show that RA modulates neuronal firing by inducing spike broadening and complex spiking in a dose-dependent manner in peptidergic and dopaminergic cell types. Using patch-clamp electrophysiology, we show that RA-induced complex spiking is activity dependent and involves enhanced inactivation of delayed rectifier voltage-gated K⁺ channels. The prolonged depolarizations observed during RA-modulated spiking lead to an increase in Ca²⁺ influx through Ca_V channels, though we also show an opposing effect of RA on the same neurons to inhibit Ca²⁺ influx. At physiological levels of Ca²⁺, this inhibition is specific to Ca_V2 (not Ca_V1) channels. Examining the interaction between the spike-modulating effects of RA and its inhibition of Ca_V channels, we found that inhibition of Ca_V2 channels limits the Ca²⁺ influx resulting from spike modulation. Our data thus provide novel evidence to suggest that retinoid signaling affects both delayed rectifier K⁺ channels and Ca_V channels to fine-tune Ca²⁺ influx through Ca_V2 channels. As these channels play important roles in synaptic function, we propose that these modulatory effects of retinoids likely contribute to synaptic plasticity in the nervous system.     

AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit

L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.     

AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit

L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca_V1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.¹ Altier C, Dubel SJ, Barrère C, Jarvis SE, Stotz SC, Spaetgens RL, et al. Trafficking of L-type calcium channels mediated by the postsynaptic scaffolding protein AKAP79. J Biol Chem 2002; 277:33598 - 603; http://dx.doi.org/10.1074/jbc.M202476200; PMID: 12114507 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Cav1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca_V1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca_V1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Cav1.2 intracellular regions.     

Antagonism of L-type Ca2+ channels CaV1.3 and CaV1.2 by 1,4-dihydropyrimidines and 4H-pyrans as dihydropyridine mimics

The L-type calcium channel (LTCC) Ca_V1.3 is regarded as a new potential therapeutic target for Parkinson’s disease. Calcium influx through Ca_V1.3 LTCC during autonomous pacemaking in adult dopaminergic neurons of the substantia nigra pars compacta is related to the generation of mitochondrial oxidative stress in animal models. Development of a Ca_V1.3 antagonist selective over Ca_V1.2 is essential because Ca_V1.2 pore-forming subunits are the predominant form of LTCCs and are abundant in the central nervous and cardiovascular systems. We have explored 1,4-dihydropyrimidines and 4H-pyrans to identify potent and selective antagonists of Ca_V1.3 relative to Ca_V1.2 LTCCs. A library of 36 dihydropyridine (DHP)-mimic 1,4-dihydropyrimidines and 4H-pyrans was synthesized, and promising chiral compounds were resolved. The antagonism studies of Ca_V1.3 and Ca_V1.2 LTCCs using DHP mimic compounds showed that dihydropyrimidines and 4H-pyrans are effective antagonists of DHPs for Ca_V1.3 LTCCs. Some 1,4-dihydropyrimidines are more selective than isradipine for Ca_V1.3 over Ca_V1.2, shown here by both calcium flux and patch-clamp electrophysiology experiments, where the ratio of antagonism is around 2–3. These results support the hypothesis that the modified hydrogen bonding donor/acceptors in DHP-mimic dihydropyrimidines and 4H-pyrans can interact differently with DHP binding sites, but, in addition, the data suggest that the binding sites of DHP in Ca_V1.3 and Ca_V1.2 LTCCs are very similar.     

Nitrite exerts potent negative inotropy in the isolated heart via eNOS-independent nitric oxide generation and cGMP-PKG pathway activation

The ubiquitous anion nitrite (NO₂⁻) has recently emerged as an endocrine storage form of nitric oxide (NO) and a signalling molecule that mediates a number of biological responses. Although the role of NO in regulating cardiac function has been investigated in depth, the physiological signalling effects of nitrite on cardiac function have only recently been explored. We now show that remarkably low concentrations of nitrite (1 nM) significantly modulate cardiac contractility in isolated and perfused Langendorff rat heart. In particular, nitrite exhibits potent negative inotropic and lusitropic activities as evidenced by a decrease in left ventricular pressure and relaxation, respectively. Furthermore, we demonstrate that the nitrite-dependent effects are mediated by NO formation but independent of NO synthase (NOS) activity. Specifically, nitrite infusion in the Langendorff system produces NO and cGMP/PKG-dependent negative inotropism, as evidenced by the formation of cellular iron-nitrosyl complexes and inhibition of biological effect by NO scavengers and by PKG inhibitors. These data are consistent with the hypothesis that nitrite represents an eNOS-independent source of NO in the heart which modulates cardiac contractility through the NO-cGMP/PKG pathway. The observed high potency of nitrite supports a physiological function of nitrite as a source of cardiomyocyte NO and a fundamental signalling molecule in the heart.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca²⁺ into cells by Ca_V channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca²⁺ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (Ca_V2.2) channels for permeating Ca²⁺ and Ba²⁺ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when Ca_V currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (Ca_V1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba²⁺ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through Ca_V1 and Ca_V2 channels (i.e. high-voltage activated Ca_V channels). The voltage dependence of Ca_V1 channel permeation is likely a mechanism mediating sustained Ca²⁺ influx during the plateau phase of the cardiac action potential.     

Regulation of insulin secretion and GLUT4 trafficking by the calcium sensor synaptotagmin VII

Insulin regulates blood glucose by promoting uptake by fat and muscle, and inhibiting production by liver. Insulin-stimulated glucose uptake is mediated by GLUT4, which translocates from an intracellular compartment to the plasma membrane. GLUT4 traffic and insulin secretion both rely on calcium-dependent, regulated exocytosis. Deletion of the voltage-gated potassium channel Kv1.3 results in constitutive expression of GLUT4 at the plasma membrane. Inhibition of channel activity stimulated GLUT4 translocation through a calcium dependent mechanism. The synaptotagmins (Syt) are calcium sensors for vesicular traffic, and Syt VII mediates lysosomal and secretory granule exocytosis. We asked if Syt VII regulates insulin secretion by pancreatic beta cells, and GLUT4 translocation in insulin-sensitive tissues mouse model. Syt VII deletion (Syt VII -/-) results in glucose intolerance and a marked decrease in glucose-stimulated insulin secretion in vivo. Pancreatic islet cells isolated from Syt VII -/- cells secreted significantly less insulin than islets of littermate controls. Syt VII deletion disrupted GLUT4 traffic as evidenced by constitutive expression of GLUT4 present at the plasma membrane of fat and skeletal muscle cells and unresponsiveness to insulin. These data document a key role for Syt VII in peripheral glucose homeostasis through its action on both insulin secretion and GLUT4 traffic.     

Dual regulation of the cardiac L-type calcium channel in L6 cells by protein kinase C

The role of protein kinase C (PKC) in the regulation of cardiac L-type Ca²⁺ channel activity (LCC) was investigated in L6 rat neonatal myoblasts. Depolarization of fura-2 loaded cells with 140 mM KCl activated a Ba²⁺ influx pathway that was blocked by nifedipine and stimulated by (−) Bay K 8644. At least two splice variants of the α_1C subunit of the cardiac LCC were identified by PCR; the α_1S subunit of the skeletal muscle LCC was not detected. Peptides that specifically inhibit translocation of the novel, Ca²⁺-independent δ and PKC isozymes reduced Ba²⁺ influx by 27% and 19%, respectively, whereas a corresponding peptide directed against translocation of classical PKC α had no effect. Ingenol 3,20-dibenzoate, an agent reported to selectively activate novel PKCs, increased Ba²⁺ uptake by 31% while ethanol, a PKC agonist, enhanced uptake by 38%. In contrast, selective activation of classical PKCs with thymeleatoxin or an agonist peptide reduced Ba²⁺ influx by 23–33%. Ba²⁺ influx was reduced by 30–40% when cells were treated with either a PKC inhibitor (Gö 6983, bisindolylmaleimide) or the PKC activator phorbol-12-myristate-13-acetate. We propose that novel, Ca²⁺-insensitive PKC(s) enhance cardiac Ca²⁺ channel activity in L6 cells under basal conditions while activation of the classical, Ca²⁺-sensitive PKC(s) inhibits channel activity. These findings provide the first evidence that different PKC isozymes exert class-specific opposing effects on cardiac L-type Ca²⁺ channel activity in L6 myoblasts.     

Regulation of ERK5 by insulin and angiotensin-II in vascular smooth muscle cells

ERK5 is involved in proliferation of vascular smooth muscle cells (VSMC). The proliferative actions of insulin and angiotensin-II (A-II) in VSMC are mediated in part by ERK1/2. We hypothesized that insulin and A-II also regulate ERK5 activity in VSMC. Acute treatment (<60min) with insulin or A-II increased phosphorylation of ERK1/2 at 15min and ERK5 at 5min. Chronic treatment (< or = 8h) with insulin increased ERK1/2 phosphorylation by 4h and ERK5 by 8h. A-II-stimulated phosphorylation of ERK1/2 by 8h and ERK5 by 4h. The EC(50) for insulin treatment effecting ERK1/2 and ERK5 phosphorylation was 1.5 and 0.1nM, whereas the EC(50) for A-II was 2nM, each. Insulin plus A-II induced an additive effect only on ERK5 phosphorylation. Inhibition of insulin- and A-II-stimulated phosphorylation of ERK5 and ERK1/2 by PD98059 and Wortmannin exhibited differential and time-dependent effects. Taken together, these data indicate that insulin and A-II regulate the activity of ERK5, but different from that seen for ERK1/2.     

5-amino-imidazole carboxamide riboside acutely potentiates glucose-stimulated insulin secretion from mouse pancreatic islets by KATP channel-dependent and -independent pathways

AMP-activated protein kinase (AMPK) is an important signaling effector that couples cellular metabolism and function. The effects of AMPK activation on pancreatic beta-cell function remain unresolved. We used 5-amino-imidazole carboxamide riboside (AICAR), an activator of AMPK, to define the signaling mechanisms linking the activation of AMPK with insulin secretion. Application of 300 microM AICAR to mouse islets incubated in 5-14 mM glucose significantly increased AMPK activity and potentiated insulin secretion. AICAR inhibited ATP-sensitive K(+) (K(ATP)) channels and increased the frequency of glucose-induced calcium oscillations in islets incubated in 8-14 mM glucose. At lower glucose concentration (5mM) AICAR did not affect K(ATP) activity or intracellular ([Ca(2+)](i)). AICAR also did not inhibit (86)Rb(+) efflux from islets isolated from Sur1(-/-) mice that lack K(ATP) channels yet significantly potentiated glucose stimulated insulin secretion. Our data suggest that AICAR stimulates insulin secretion by both K(ATP) channel-dependent and -independent pathways.     

Up-regulation of L-type high voltage-gated calcium channel subunits by sustained exposure to 1,4- and 1,5-benzodiazepines in cerebrocortical neurons

The aim of this study is to examine how sustained exposure to two 1,4-benzodiazepines (BZDs) with different action period, diazepam and brotizolam, and a 1,5-BZD, clobazam, affects L-type high voltage-gated calcium channel (HVCC) functions and its mechanisms using primary cultures of mouse cerebral cortical neurons. The sustained exposure to these three BZDs increased [⁴⁵Ca²⁺] influx, which was due to the enhanced [⁴⁵Ca²⁺] entry through L-type HVCCs but not through of Cav2.1 and Cav2.2. Increase in [³H]diltiazem binding after the exposure to these three BZDs was due to the increase in the binding sites of [³H]diltiazem. Western blot analysis showed increase of Cav1.2 and Cav1.3 in association with the increased expression of α2/δ1 subunit. Similar changes in [³H]diltiazem binding and L-type HVCC subunit expression were found in the cerebral cortex from mouse with BZD physical dependence. These results indicate that BZDs examined here have the potential to increase L-type HVCC functions mediated via the enhanced expression of not only Cav1.2 and Cav1.3 but also α2/δ1 subunit after their sustained exposure, which may participate in the development of physical dependence by these BZDs.     

Regulation of TRPC5 currents by intracellular ATP: Single channel studies

TRPC5 channels are nonselective cation channels activated by G-protein-coupled receptors. It was previously found that recombinant TRPC5 currents are inhibited by intracellular ATP, when studied by whole-cell patch-clamp recording. In the present study, we investigated the mechanism of ATP inhibition at the single-channel level using patches from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. In inside-out patches, application of ATP to the intracellular face of the membrane reduced TRPC5 channel activity at both positive and negative potentials without affecting the unitary current amplitude or open dwell time of the channel. The effect of ATP was rapidly reversible. These results suggest that ATP may bind to the channel protein and affect the ability of the channel to open or to remain in an open, nondesensitized state. The activity of TRPC5 channels may be influenced by cellular metabolism via changes in ATP levels.     

Age-related deficits in neuronal physiology and cognitive function are recapitulated in young mice overexpressing the L-type calcium channel,CaV1.3

The calcium dysregulation hypothesis of brain aging posits that an age-related increase in neuronal calcium concentration is responsible for alterations in a variety of cellular processes that ultimately result in learning and memory deficits in aged individuals. We previously generated a novel transgenic mouse line, in which expression of the L-type voltage-gated calcium, Ca_V1.3, is increased by ~50% over wild-type littermates. Here, we show that, in young mice, this increase is sufficient to drive changes in neuronal physiology and cognitive function similar to those observed in aged animals. Specifically, there is an increase in the magnitude of the postburst afterhyperpolarization, a deficit in spatial learning and memory (assessed by the Morris water maze), a deficit in recognition memory (assessed in novel object recognition), and an overgeneralization of fear to novel contexts (assessed by contextual fear conditioning). While overexpression of Ca_V1.3 recapitulated these key aspects of brain aging, it did not produce alterations in action potential firing rates, basal synaptic communication, or spine number/density. Taken together, these results suggest that increased expression of Ca_V1.3 in the aged brain is a crucial factor that acts in concert with age-related changes in other processes to produce the full complement of structural, functional, and behavioral outcomes that are characteristic of aged animals.     

Neurochemical characterization of the striatum and the nucleus accumbens in L-type Ca(v)1.3 channels knockout mice

L-type Ca(v)1.3 channels control the autonomous pacemaking of the substantia nigra (SN) dopamine (DA) neurons, which maintains the sustained release of DA in the striatum, its target structure. The persistent engagement of L-type channels during pacemaking might lead to increased vulnerability to environmental stressors or degenerative processes, providing a mechanism for the development of Parkinson's disease (PD). Interestingly, L-type channels are not necessary for pacemaking, opening the possible use of calcium channel antagonists as neuroprotective agents for PD without disturbing normal DA function. In this study we aimed to evaluate the consequences of Ca(v)1.3 channels deletion at the neurochemical level. For this purpose, tissue concentrations of DA and their respective metabolites were measured using high performance liquid chromatography (HPLC) in the striatum and the nucleus accumbens (NAcc) of mice lacking the gene for the Ca(v)1.3 channel subunit (CACNA1D) and compared to those in wild-type mice. Striatal DA level did not differ between the two groups. In contrast, the level of serotonin, glutamate, GABA, and taurine were increased by more than 50% in the striatum of Ca(v)1.3 null mice. Neurotransmitters levels in the NAcc did not differ between the different groups. In conclusion, our results neurochemically corroborate the robustness of the nigrostriatal DA neurons in the absence of Ca(v)1.3 channels, but suggest that complete deletion of this channel affected a variety of other transmitter systems.