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.     

Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle

Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current (ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7-8 mo; 35-45 kg) were obtained from the breeder. Males were left intact (IM, n=8), castrated (CM, n=8), or castrated with testosterone replacement (CMT, n=8; 10 mg/day Androgel). Females remained gonad intact (n=8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.     

Myocardin调控心肌H9C2细胞Ca2+通道机制研究

目的：研究同样在维持心脏正常结构和功能过程中发挥着重要作用的转录因子心肌素Myocardin对L型Ca²⁺通道Cav1.2的转录调控作用及分子机制。方法：全细胞膜片钳技术记录心肌细胞膜Ca²⁺电流,慢病毒包装技术制备Myocardin-GFP慢病毒用于感染心肌细胞以过表达Myocardin,Real-time PCR定量检测Cav1.2基因mRNA水平,Western blotting检测Cav1.2蛋白表达水平。PCR介导的定点突变技术得到Ca²⁺通道启动子区特定CarGbox位点突变的突变体。荧光素酶报告系统检测野生型WT和突变体MU启动子活性,以确定Myocardin在Cav1.2基因启动子区的作用位点。结果：全细胞膜片钳技术表明Myocardin激活Cav1.2而增加心肌细胞膜Ca²⁺电流,real-time PCR和Western blotting结果表明,Myocardin激活Cav1.2基因的转录和表达,荧光素酶报告系统检测突变体启动子活性,发现Myocardin激活Cav1.2基因的转录依赖其启动子区的CarGbox。结论：Myocardin通过与Cav1.2基因启动子区CarGbox结合进而激活其转录和表达,促进Ca²⁺通道蛋白装配到心肌细胞膜上,加强Ca²⁺内流,增强膜电流。     

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.     

Osteoblast Ca(2+) permeability and voltage-sensitive Ca(2+) channel expression is temporally regulated by 1,25-dihydroxyvitamin D(3)

The cardiac subtype of the L-type voltage-sensitive Ca²⁺ channel (VSCC) Cav1.2 (_1C) is the primary voltage-sensitive channel responsible for Ca²⁺ influx into actively proliferating osteoblasts. This channel also serves as the major transducer of Ca²⁺ signals in growth-phase osteoblasts in response to hormone treatment. In this study, we have demonstrated that 24-h treatment of MC3T3-E1 preosteoblasts with 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], a coupling factor for bone resorption, coordinately downregulates Cav1.2 (_1C) and uniquely upregulates T-type channel Cav3.2 (_1H). No other voltage-sensitive channel -subunit of the 10 that were surveyed was upregulated by 1,25(OH)₂D₃. The shift from predominantly L-type to T-type channel expression has been demonstrated to occur at both mRNA and protein levels detected using quantitative PCR and immunohistochemistry with antibodies specific for each channel type. Functional and pharmacological studies using specific inhibitors have revealed that treatment with 1,25(OH)₂D₃ also alters the Ca²⁺ permeability properties of the osteoblast membrane from a state of primarily L-current sensitivity to T-current sensitivity. We conclude that the L-type channel is likely to support proliferation of osteoblast cells, whereas T-type channels are more likely to be involved in supporting differentiated functions after 1,25(OH)₂D₃-mediated reversal of remodeling has occurred. This latter observation is consistent with the unique expression of the T-type VSCC Cav3.2 (_1H) in terminally differentiated osteocytes as we recently reported. calcium influx; bone     

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.     

Ligand-activated signal transduction in the 2-cell embryo

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.     

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.     

RhoA GTPase regulates L-type Ca2+ currents in cardiac myocytes

Regulation of ionic channels plays a pivotal role in controlling cardiac function. Here we show that the Rho family of small G proteins regulates L-type Ca2+ currents in ventricular cardiomyocytes. Ventricular myocytes isolated from transgenic (TG) mice that overexpress the specific GDP dissociation inhibitor Rho GDI-alpha exhibited significantly decreased basal L-type Ca2+ current density (approximately 40%) compared with myocytes from nontransgenic (NTG) mice. The Ca2+ channel agonist BAY K 8644 and the beta-adrenergic agonist isoproterenol increased Ca2+ currents in both NTG and TG myocytes to a similar maximal level, and no changes in mRNA or protein levels were observed in the Ca2+ channel alpha1-subunits. These results suggest that the channel activity but not the expression level was altered in TG myocytes. In addition, the densities of inward rectifier and transient outward K+ currents were unchanged in TG myocytes. The amplitudes and rates of basal twitches and Ca2+ transients were also similar between the two groups. When the protein was delivered directly into adult ventricular myocytes via TAT-mediated protein transduction, Rho GDI-alpha significantly decreased Ca2+ current density, which supports the idea that the defective Ca2+ channel activity in TG myocytes was a primary effect of the transgene. In addition, expression of a dominant-negative RhoA but not a dominant-negative Rac-1 or Cdc42 also significantly decreased Ca2+ current density, which indicates that inhibition of Ca2+ channel activity by overexpression of Rho GDI-alpha is mediated by inhibition of RhoA. This study points to the L-type Ca2+ channel activity as a novel downstream target of the RhoA signaling pathway.     

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.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

Defective calcium inactivation causes long QT in obese insulin-resistant rat

The majority of diabetic patients who are overweight or obese die of heart disease. We suspect that the obesity-induced insulin resistance may lead to abnormal cardiac electrophysiology. We tested this hypothesis by studying an obese insulin-resistant rat model, the obese Zucker rat (OZR). Compared with the age-matched control, lean Zucker rat (LZR), OZR of 16-17 wk old exhibited an increase in QTc interval, action potential duration, and cell capacitance. Furthermore, the L-type calcium current (I(CaL)) in OZR exhibited defective inactivation and lost the complete inactivation back to the closed state, leading to increased Ca(2+) influx. The current density of I(CaL) was reduced in OZR, whereas the threshold activation and the current-voltage relationship of I(CaL) were not significantly altered. L-type Ba(2+) current (I(BaL)) in OZR also exhibited defective inactivation, and steady-state inactivation was not significantly altered. However, the current-voltage relationship and activation threshold of I(BaL) in OZR exhibited a depolarized shift compared with LZR. The total and membrane protein expression levels of Cav1.2 [pore-forming subunit of L-type calcium channels (LTCC)], but not the insulin receptors, were decreased in OZR. The insulin receptor was found to be associated with the Cav1.2, which was weakened in OZR. The total protein expression of calmodulin was reduced, but that of Cavβ2 subunit was not altered in OZR. Together, these results suggested that the 16- to 17-wk-old OZR has 1) developed cardiac hypertrophy, 2) exhibited altered electrophysiology manifested by the prolonged QTc interval, 3) increased duration of action potential in isolated ventricular myocytes, 4) defective inactivation of I(CaL) and I(BaL), 5) weakened the association of LTCC with the insulin receptor, and 6) decreased protein expression of Cav1.2 and calmodulin. These results also provided mechanistic insights into a remodeled cardiac electrophysiology under the condition of insulin resistance, enhancing our understanding of long QT associated with obese type 2 diabetic patients.     

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.     

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.     

Functional expression and characterization of a voltage-gated CaV1.3 (alpha1D) calcium channel subunit from an insulin-secreting cell line.

L-type calcium channels mediate depolarization-induced calcium influx in insulin-secreting cells and are thought to be modulated by G protein-coupled receptors (GPCRs). The major fraction of L-type alpha1-subunits in pancreatic beta-cells is of the neuroendocrine subtype (CaV1.3 or alpha1D). Here we studied the biophysical properties and receptor regulation of a CaV1.3 subunit previously cloned from HIT-T15 cells. In doing so, we compared this neuroendocrine CaV1.3 channel with the cardiac L-type channel CaV1.2a (or alpha1C-a) after expression together with alpha2delta- and beta3-subunits in Xenopus oocytes. Both the current voltage relation and voltage dependence of inactivation for the neuroendocrine CaV1.3 channel were shifted to more negative potentials compared with the cardiac CaV1.2 channel. In addition, the CaV1.3 channel activated and inactivated more rapidly than the CaV1.2a channel. Both subtypes showed a similar sensitivity to the dihydropyridine (+)isradipine. More interestingly, the CaV1.3 channels were found to be stimulated by ligand-bound G(i)/G(o)-coupled GPCRs whereas a neuronal CaV2.2 (or alpha1B) channel was inhibited. The observed receptor-induced stimulation of CaV1.3 channels could be mimicked by phorbol-12-myristate-13-acetate and was sensitive to inhibitors of protein kinases, but not to the phosphoinositol-3-kinase-inhibitor wortmannin, pointing to serine/threonine kinase-dependent regulation. Taken together, we describe a neuroendocrine L-type CaV1.3 calcium channel that is stimulated by G(i)/G(o)-coupled GPCRs and differs significantly in distinct biophysical characteristics from the cardiac subtype (CaV1.2a), suggesting that the channels have different roles in native cells.     

Deletion of the distal C terminus of CaV1.2 channels leads to loss of beta-adrenergic regulation and heart failure in vivo

L-type calcium currents conducted by CaV1.2 channels initiate excitation-contraction coupling in cardiac and vascular smooth muscle. In the heart, the distal portion of the C terminus (DCT) is proteolytically processed in vivo and serves as a noncovalently associated autoinhibitor of CaV1.2 channel activity. This autoinhibitory complex, with A-kinase anchoring protein-15 (AKAP15) bound to the DCT, is hypothesized to serve as the substrate for β-adrenergic regulation in the fight-or-flight response. Mice expressing CaV1.2 channels with the distal C terminus deleted (DCT-/-) develop cardiac hypertrophy and die prematurely after E15. Cardiac hypertrophy and survival rate were improved by drug treatments that reduce peripheral vascular resistance and hypertension, consistent with the hypothesis that CaV1.2 hyperactivity in vascular smooth muscle causes hypertension, hypertrophy, and premature death. However, in contrast to expectation, L-type Ca2+ currents in cardiac myocytes from DCT-/- mice were dramatically reduced due to decreased cell-surface expression of CaV1.2 protein, and the voltage dependence of activation and the kinetics of inactivation were altered. CaV1.2 channels in DCT-/- myocytes fail to respond to activation of adenylyl cyclase by forskolin, and the localized expression of AKAP15 is reduced. Therefore, we conclude that the DCT of CaV1.2 channels is required in vivo for normal vascular regulation, cell-surface expression of CaV1.2 channels in cardiac myocytes, and β-adrenergic stimulation of L-type Ca2+ currents in the heart.