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.     

Functional embryonic cardiomyocytes after disruption of the L-type alpha1C (Cav1.2) calcium channel gene in the mouse

The L-type alpha(1C) (Ca(v)1.2) calcium channel is the major calcium entry pathway in cardiac and smooth muscle. We inactivated the Ca(v)1.2 gene in two independent mouse lines that had indistinguishable phenotypes. Homozygous knockout embryos (Ca(v)1. 2-/-) died before day 14.5 postcoitum (p.c.). At day 12.5 p.c., the embryonic heart contracted with identical frequency in wild type (+/+), heterozygous (+/-), and homozygous (-/-) Ca(v)1.2 embryos. Beating of isolated embryonic cardiomyocytes depended on extracellular calcium and was blocked by 1 microm nisoldipine. In (+/+), (+/-), and (-/-) cardiomyocytes, an L-type Ba(2+) inward current (I(Ba)) was present that was stimulated by Bay K 8644 in all genotypes. At a holding potential of -80 mV, nisoldipine blocked I(Ba) of day 12.5 p.c. (+/+) and (+/-) cells with two IC(50) values of approximately 0.1 and approximately 1 microm. Inhibition of I(Ba) of (-/-) cardiomyocytes was monophasic with an IC(50) of approximately 1 microm. The low affinity I(Ba) was also present in cardiomyocytes of homozygous alpha(1D) (Ca(v)1.3) knockout embryos at day 12.5 p.c. These results indicate that, up to day 14 p.c., contraction of murine embryonic hearts requires an unidentified, low affinity L-type like calcium channel.     

L-type calcium channels as drug targets in CNS disorders

L-type calcium channels are present in most electrically excitable cells and are needed for proper brain, muscle, endocrine and sensory function. There is accumulating evidence for their involvement in brain diseases such as Parkinson disease, febrile seizures and neuropsychiatric disorders. Pharmacological inhibition of brain L-type channel isoforms, Cav1.2 and Cav1.3, may therefore be of therapeutic value. Organic calcium channels blockers are clinically used since decades for the treatment of hypertension, cardiac ischemia, and arrhythmias with a well-known and excellent safety profile. This pharmacological benefit is mainly mediated by the inhibition of Cav1.2 channels in the cardiovascular system. Despite their different biophysical properties and physiological functions, both brain channel isoforms are similarly inhibited by existing calcium channel blockers. In this review we will discuss evidence for altered L-type channel activity in human brain pathologies, new therapeutic implications of existing blockers and the rationale and current efforts to develop Cav1.3-selective compounds.     

Different roles attributed to Cav1 channel subtypes in spontaneous action potential firing and fine tuning of exocytosis in mouse chromaffin cells

This study examines the Cav1 isoforms expressed in mouse chromaffin cells and compares their biophysical properties and roles played in cell excitability and exocytosis. Using immunocytochemical and electrophysiological techniques in mice lacking the Cav1.3α1 subunit (Cav1.3(-/-) ) or the high sensitivity of Cav1.2α1 subunits to dihydropyridines, Cav1.2 and Cav1.3 channels were identified as the only Cav1 channel subtypes expressed in mouse chromaffin cells. Cav1.3 channels were activated at more negative membrane potentials and inactivated more slowly than Cav1.2 channels. Cav1 channels, mainly Cav1.2, control cell excitability by functional coupling to BK channels, revealed by nifedipine blockade of BK channels in wild type (WT) and Cav1.3(-/-) cells (53% and 35%, respectively), and by the identical change in the shape of the spontaneous action potentials elicited by the dihydropyridine in both strains of mice. Cav1.2 channels also play a major role in spontaneous action potential firing, supported by the following evidence: (i) a similar percentage of WT and Cav1.3(-/-) cells fired spontaneous action potentials; (ii) firing frequency did not vary between WT and Cav1.3(-/-) cells; (iii) mostly Cav1.2 channels contributed to the inward current preceding the action potential threshold; and (iv) in the presence of tetrodotoxin, WT or Cav1.3(-/-) cells exhibited spontaneous oscillatory activity, which was fully abolished by nifedipine perfusion. Finally, Cav1.2 and Cav1.3 channels were essential for controlling the exocytotic process at potentials above and below -10 mV, respectively. Our data reveal the key yet differential roles of Cav1.2 and Cav1.3 channels in mediating action potential firing and exocytotic events in the neuroendocrine chromaffin cell.     

Decreasing expression of alpha1C calcium L-type channel subunit mRNA in rat ventricular myocytes upon manganese exposure

Manganese is an essential trace element found in many enzymes. As it is the case of many essential trace elements, excessive level of manganese is toxic. It has been proven that excessive manganese could cause heart problems. In order to understand the mechanism of manganese toxicity in the heart, the effects of manganese on isolated rat ventricular myocytes were studied. The L-type calcium channel current was measured by whole-cell patch clamp recording mode. In the electrophysiology experiments, both 50 microM Mn2+ and 100 microM Mn2+ could effectively decrease the channel current amplitude density by 35.7% and 68.2%, respectively. Moreover, Mn2+ shifted the steady-state activation curve toward more positive potential and the steady-state inactivation curve toward more negative potential. Investigation by RT-PCR showed that the mRNA expression of alpha1C/Cav1.2 treated with manganese was decreased depending on its concentration, while the mRNA expression of alpha1D/Cav1.3 was almost unchanged. Fluo-3/AM was utilized for real-time free calcium scanning with laser scanning confocal microscopy (LSCM), and the results showed that Mn2+ could elicit a slow and continuous increase of [Ca2+]i in a concentration-dependent manner. These results have suggested that manganese could interfere with the function of the L-type calcium channel, downregulate the mRNA expression of alpha1C/Cav1.2, and thus causing long-lasting molecular changes of L-type calcium channel which have probably been triggered by overloading of calcium in myocytes.     

PKCdelta mediates testosterone-induced increases in coronary smooth muscle Cav1.2

Sex hormones have emerged as important modulators of cardiovascular physiology and pathophysiology. Our previous studies demonstrated that testosterone increases expression and activity of L-type, voltage-gated calcium channels (Cav1.2) in coronary arteries of males. The purpose of the present study was to determine whether testosterone (T) alters coronary protein kinase C delta (PKCdelta) expression and whether PKCdelta plays a role in coronary Cav1.2 expression. For in vitro studies, porcine right coronary arteries (RCA) and post-confluent (passages 3-6) 5-day, serum-restricted coronary smooth muscle cell cultures (CSMC) were incubated in the presence and absence of T or dihydrotestosterone (10 and 100 nm) for 18 h at 37 degrees C in a humidified chamber. For sex and endogenous testosterone-dependent effects, RCA were obtained from intact males, castrated males, castrated males with T replacement, and intact females. In vitro T and dihydrotestosterone caused an approximately 2-3-fold increase in PKCdelta protein levels, approximately 1.5-2-fold increase in PKCdelta kinase activity, and localization of PKCdelta toward the plasma membrane and nuclear envelope. PKCdelta protein levels were higher in coronary arteries of intact males compared with intact females. Elimination of endogenous testosterone by castration reduced RCA PKCdelta protein levels, an effect partially (approximately 45%) reversed by exogenous T (castrated males with T replacement). In CSMC, PKC inhibition with either the general PKC inhibitor, cheylerythrine, or the putative PKCdelta inhibitor, rottlerin, completely inhibited the T-mediated increase in coronary Cav1.2 protein levels. Conversely, Go6976, a conventional PKC isoform inhibitor, failed to inhibit T-induced increases in coronary Cav1.2 protein levels. PKCdelta short interference RNA completely blocked T-induced increases in Cav1.2 protein levels in CSMC. These results demonstrate for the first time that 1) endogenous T is a primary modulator of coronary PKCdelta protein and activity in males and 2) T increases Cav1.2 protein expression in a PKCdelta-dependent manner.     

Dominant role of smooth muscle L-type calcium channel Cav1.2 for blood pressure regulation

Blood pressure is regulated by a number of key molecules involving G-protein-coupled receptors, ion channels and monomeric small G-proteins. The relative contribution of these different signaling pathways to blood pressure regulation remains to be determined. Tamoxifen-induced, smooth muscle-specific inactivation of the L-type Cav1.2 Ca2+ channel gene in mice (SMAKO) reduced mean arterial blood pressure (MAP) in awake, freely moving animals from 120 +/- 4.5 to 87 +/- 8 mmHg. Phenylephrine (PE)- and angiotensin 2 (AT2)-induced MAP increases were blunted in SMAKO mice, whereas the Rho-kinase inhibitor Y-27632 reduced MAP to the same extent in control and SMAKO mice. Depolarization-induced contraction was abolished in tibialis arteries of SMAKO mice, and development of myogenic tone in response to intravascular pressure (Bayliss effect) was absent. Hind limb perfusion experiments suggested that 50% of the PE-induced resistance is due to calcium influx through the Cav1.2 channel. These results show that Cav1.2 calcium channels are key players in the hormonal regulation of blood pressure and development of myogenic tone.     

Moderate calcium channel dysfunction in adult mice with inducible cardiomyocyte-specific excision of the cacnb2 gene

The major L-type voltage-gated calcium channels in heart consist of an α1C (Ca(V)1.2) subunit usually associated with an auxiliary β subunit (Ca(V)β2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of Ca(V)β2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the Ca(V)β2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, Ca(V)β2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include Ca(V)β2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other Ca(V)β proteins or changes of Ca(V)1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, Ca(V)β2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, Ca(V)β2 down-regulation in cardiomyocytes is well tolerated by the adult mice.     

Differential modulation of L-type calcium channel subunits by oleate

Nonesterified fatty acids such as oleate and palmitate acutely potentiate insulin secretion from pancreatic islets in a glucose-dependent manner. In addition, recent studies show that fatty acids elevate intracellular free Ca(2+) and increase voltage-gated Ca(2+) current in mouse beta-cells, although the mechanisms involved are poorly understood. Here we utilized a heterologous system to express subunit-defined voltage-dependent L-type Ca(2+) channels (LTCC) and demonstrate that beta-cell calcium may increase in part from an interaction between fatty acid and specific calcium channel subunits. Distinct functional LTCC were assembled in both COS-7 and HEK-293 cells by expressing either one of the EYFP-tagged L-type alpha(1)-subunits (beta-cell Cav1.3 or lung Cav1.2) and ERFP-tagged islet beta-subunits (ibeta(2a) or ibeta(3)). In COS-7 cells, elevations in intracellular Ca(2+) mediated by LTCC were enhanced by an oleate-BSA complex. To extend these findings, Ca(2+) current was measured in LTCC-expressing HEK-293 cells that revealed an increase in peak Ca(2+) current within 2 min after addition of the oleate complex, with maximal potentiation occurring at voltages <0 mV. Both Cav1.3 and Cav1.2 were modulated by oleate, and the presence of different auxiliary beta-subunits resulted in differential augmentation. The potentiating effect of oleate on Cav1.2 was abolished by the pretreatment of cells with triacsin C, suggesting that long-chain CoA synthesis is necessary for Ca(2+) channel modulation. These results show for the first time that two L-type Ca(2+) channels expressed in beta-cells (Cav1.3 and Cav1.2) appear to be targeted by nonesterified fatty acids. This effect may account in part for the acute potentiation of glucose-dependent insulin secretion by fatty acids.     

A macromolecular trafficking complex composed of β2-adrenergic receptors,A-Kinase Anchoring Proteins and L-type calcium channels

Abstract

Sympathetic modulation of cardiac L-type calcium channels is an important mechanism for regulating heart rate and cardiac contractility. At the molecular level, activation of β-adrenergic receptors (βAR) increases calcium influx into cardiac myocytes by activating protein kinase A (PKA), leading to subsequent phosphorylation of L-type calcium channels. In the case of the β₂AR, this process is facilitated by the presence of A-Kinase Anchoring Proteins (AKAPs) that serve as scaffolding proteins for the L-type calcium channel and the β₂AR complex. Our work has shown that, in addition to facilitating PKA phosphorylation of the channel, AKAPs also promote an increase in the Cav1.2 channel surface expression. Here we review the molecular mechanisms of β₂AR/AKAP/L-type channel interactions and trafficking.     

Use 'em and lose 'em-activity-induced removal of calcium channels from the plasma membrane

Calcium influx via L-type (Cav1.2 and Cav1.3) calcium channels is tightly regulated to ensure optimal intracellular calcium levels. Although much is known about acute modulation of these channels by second messengers, the mechanisms that control their trafficking to and from the plasma membrane remain poorly understood. In this issue of Neuron, Green and colleagues demonstrate that the opening of L-type calcium channels results in negative feedback regulation due to their calcium-dependent internalization.     

Silencing of Cav1.2 gene in neonatal cardiomyocytes by lentiviral delivered shRNA

Cav1.2 (α_1C) and Cav1.3 (α_1D) L-type Ca channels are co-expressed in the heart. To date, there are no pharmacological or biophysical tools to separate α_1D from α_1C Ca currents (I_Ca-L) in cardiomyocytes. Here, we established a physiological model to study α_1D I_Ca-L in native myocytes using RNA interference. Transfection of rat neonatal cardiomyocytes (RNC) with α_1C specific siRNA resulted in low silencing efficiency (50-60%) at the mRNA and protein levels. The use of lentivirus shRNA resulted in 100% transfection efficiency and 92% silencing of the α_1C gene by real-time PCR and Western blot. Electrophysiological experiments showed that the total I_Ca-L was similarly reduced by 80% in lentivirus transfected cells. Both biochemical and functional data demonstrated high transfection and silencing efficiency in the cardiomyocytes using lentiviral shRNA. This novel approach allows for the assessments of the roles of α_1C and α_1D Ca channels in native myocytes and could be used to examine their roles in physiological and pathological settings.     

Localization of the calcium channel subunits Cav1.2 (alpha1C) and Cav2.3 (alpha1E) in the mouse organ of Corti

Voltage-activated Ca2+ channels play an important role in synaptic transmission, signal processing and development. The immunohistochemical localization of Cav1.2 (alpha1C) and Cav2.3 (alpha1E) Ca2+ channels was studied in the developing and adult mouse organ of Corti using subunit-specific antibodies and fluorescent secondary antibodies with cochlear cryosections. Cav1.2 immunoreactivity has been detected from postnatal day 14 (P14) onwards at the synapses between cholinergic medial efferents and outer hair cells as revealed by co-staining with anti-synaptophysin and anti-choline acetyltransferase. Most likely the Cav1.2 immunoreactivity was located presynaptically at the site of contact of the efferent bouton with the outer hair cell which suggests a role for class C L-type Ca2+ channels in synaptic transmission of the medial efferent system. The localization of the second Ca2+ channel tested, Cav2.3, showed a pronounced change during cochlear development. From P2 until P10, Cav2.3 immunoreactivity was found in the outer spiral bundle followed by the inner spiral bundle, efferent endings and by medial efferent fibers. Around P14, Cav2.3 immunoreactivity disappeared from these structures and from P19 onwards it was observed in the basal poles of the outer hair cell membranes.     

Apo calmodulin binding to the L-type voltage-gated calcium channel Cav1.2 IQ peptide

The influx of calcium through the L-type voltage-gated calcium channels (LTCCs) is the trigger for the process of calcium-induced calcium release (CICR) from the sarcoplasmic reticulum, an essential step for cardiac contraction. There are two feedback mechanisms that regulate LTCC activity: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF), both of which are mediated by calmodulin (CaM) binding. The IQ domain (aa 1645-1668) housed within the cytoplasmic domain of the LTCC Cav1.2 subunit has been shown to bind both calcium-loaded (Ca2+CaM ) and calcium-free CaM (apoCaM). Here, we provide new data for the structural basis for the interaction of apoCaM with the IQ peptide using NMR, revealing that the apoCaM C-lobe residues are most significantly perturbed upon complex formation. In addition, we have employed transmission electron microscopy of purified LTCC complexes which shows that both apoCaM and Ca2+CaM can bind to the intact channel.     

Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain

Chronic pain states are characterized by long-term sensitization of spinal cord neurons that relay nociceptive information to the brain. Among the mechanisms involved, up-regulation of Cav1.2-comprising L-type calcium channel (Cav1.2-LTC) in spinal dorsal horn have a crucial role in chronic neuropathic pain. Here, we address a mechanism of translational regulation of this calcium channel. Translational regulation by microRNAs is a key factor in the expression and function of eukaryotic genomes. Because perfect matching to target sequence is not required for inhibition, theoretically, microRNAs could regulate simultaneously multiple mRNAs. We show here that a single microRNA, miR-103, simultaneously regulates the expression of the three subunits forming Cav1.2-LTC in a novel integrative regulation. This regulation is bidirectional since knocking-down or over-expressing miR-103, respectively, up- or down-regulate the level of Cav1.2-LTC translation. Functionally, we show that miR-103 knockdown in naive rats results in hypersensitivity to pain. Moreover, we demonstrate that miR-103 is down-regulated in neuropathic animals and that miR-103 intrathecal applications successfully relieve pain, identifying miR-103 as a novel possible therapeutic target in neuropathic chronic pain.     

The role of L-type voltage-gated calcium channels Cav1.2 and Cav1.3 in normal and pathological brain function

The use of specific activators and inhibitors that penetrate the central nervous system has suggested an essential functional role of L-type calcium channels (LTCC) in several important physiological processes of the brain, including the modulation of the mesoaccumbal dopamine signalling pathway, synaptic transmission of auditory stimuli and synaptic plasticity of neutral and aversive learning and memory processes. However, the lack of selectivity of available pharmacological agents towards the most prominent LTCC isoforms in the brain, namely Ca_v1.2 and Ca_v1.3, has hampered the elucidation of the precise contribution made by each specific channel isoform within these specific physiological processes. Modern genetic approaches, both in rodents and in human, have recently enhanced our understanding of the selective functional roles of Ca_v1.2 and Ca_v1.3 channels. In rodents, the characterisation of global and conditional isoform-specific knockouts suggests a contribution of Ca_v1.2 channels in spatial memory formation, whereas Ca_v1.3 channels seem to be involved in the consolidation of fear memories and in neurodegenerative mechanisms associated with the development of Parkinson’s disease. With regard to the molecular mechanisms underlying drug addiction, Ca_v1.3 channels are necessary for the development and Ca_v1.2 channels for the expression of cocaine and amphetamine behavioural sensitisation. In humans, both the identification of naturally occurring LTCC variants (“channelopathies”) and unbiased genome-wide association studies have linked LTCCs to working memory performance in healthy individuals and schizophrenic patients. Individually, CACNA1C polymorphisms and CACNA1D variants have been linked to a variety of psychiatric diseases and to congenital deafness, respectively. However, the contribution of individual LTCCs and their polymorphisms to human brain function and diseases remains unclear, necessitating the use of isoform-specific pharmacological agents.     

Caldendrin, a neuron-specific modulator of Cav/1.2 (L-type) Ca2+ channels

EF-hand Ca2+-binding proteins such as calmodulin and CaBP1 have emerged as important regulatory subunits of voltage-gated Ca2+ channels. Here, we show that caldendrin, a variant of CaBP1 enriched in the brain, interacts with and distinctly modulates Cav1.2 (L-type) voltage-gated Ca2+ channels relative to other Ca2+-binding proteins. Caldendrin binds to the C-terminal IQ-domain of the pore-forming alpha1-subunit of Cav1.2 (alpha(1)1.2) and competitively displaces calmodulin and CaBP1 from this site. Compared with CaBP1, caldendrin causes a more modest suppression of Ca2+-dependent inactivation of Cav1.2 through a different subset of molecular determinants. Caldendrin does not bind to the N-terminal domain of alpha11.2, a site that is critical for functional interactions of the channel with CaBP1. Deletion of the N-terminal domain inhibits CaBP1, but spares caldendrin modulation of Cav1.2 inactivation. In contrast, mutations of the IQ-domain abolish physical and functional interactions of caldendrin and Cav1.2, but do not prevent channel modulation by CaBP1. Using antibodies specific for caldendrin and Cav1.2, we show that caldendrin coimmunoprecipitates with Cav1.2 from the brain and colocalizes with Cav1.2 in somatodendritic puncta of cortical neurons in culture. Our findings reveal functional diversity within related Ca2+-binding proteins, which may enhance the specificity of Ca2+ signaling by Cav1.2 channels in different cellular contexts.