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.     

Calcium release-activated calcium (CRAC) channels mediate the β2-adrenergic regulation of Na,K-ATPase

β₂-Adrenergic agonists have been shown to regulate Na,K-ATPase in the alveolar epithelium by recruiting Na,K-ATPase-containing vesicles to the plasma membrane of alveolar epithelial cells (AEC). Here, we provide evidence that β₂-agonists induce store-operated calcium entry (SOCE) in AECs. This calcium entry is necessary for β₂-agonist-induced recruitment of Na,K-ATPase to the plasma membrane of AECs. Specifically, we show that β₂-agonists induce SOCE via stromal interaction molecule 1 (STIM1)-associated calcium release-activated calcium (CRAC) channels. We also demonstrate that the magnitude of SOCE affects the abundance of Na,K-ATPase at the plasma membrane of AECs.     

alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation

Smooth muscle exhibitsmechanosensitivity independent of neural input, suggesting thatmechanosensitive pathways reside within smooth muscle cells. The nativeL-type calcium current recorded from human intestinal smooth muscle ismodulated by stretch. To define mechanosensitive mechanisms involved inthe regulation of smooth muscle calcium entry, we cloned the_1C L-type calcium channel subunit (Ca_V1.2)from human intestinal smooth muscle and expressed the channel in aheterologous system. This channel subunit retained mechanosensitivitywhen expressed alone or coexpressed with a ₂ calciumchannel subunit in HEK-293 or Chinese hamster ovary cells. Theheterologously expressed human cardiac _1C splice formalso demonstrated mechanosensitivity. Inhibition of kinase signalingdid not affect mechanosensitivity of the native channel. Truncation of the _1C COOH terminus, which containsan inhibitory domain and a proline-rich domain thought to mediatemechanosensitive signaling from integrins, did not disruptmechanosensitivity of the expressed channel. These data demonstratemechanical regulation of calcium entry through molecularly identifiedL-type calcium channels in mammalian cells and suggest that themechanosensitivity resides within the pore forming_1C-subunit.

     

hKv4.3 channel characterization and regulation by calcium channel antagonists

Relative expression pattern of short and long isoforms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were performed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110 +/- 0.004 mM and 492.9 +/- 15.1 microM, respectively). Verapamil also accelerated current inactivation. Another calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potassium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward potassium channel properties in native tissues.     

CRAC channel is inhibited by neomycin in a Ptdlns(4,5)P2‐independent manner

Depletion of intracellular Ca²⁺ stores evokes store‐operated Ca²⁺ entry through the Ca²⁺ release‐activated Ca²⁺ (CRAC) channels. In this study, we found that the store‐operated Ca²⁺ entry was inhibited by neomycin, an aminoglycoside that strongly binds phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Patch clamp recordings revealed that neomycin blocked the CRAC currents reconstituted by co‐expression of Orai1 and Stim1 in HEK293 cells. Using a rapamycin‐inducible PtdIns(4,5)P2‐specific phosphatase (Inp54p) system to manipulate the PtdIns(4,5)P2 in the plasma membrane, we found that the CRAC current was not altered by PtdIns(4,5)P2 depletion. This result suggests that PtdIns(4,5)P2 is not required for CRAC channel activity, and thereby, neomycin inhibits CRAC channels in a manner that is independent of neomycin–PtdIns(4,5)P2 binding. Copyright © 2015 John Wiley & Sons, Ltd.     

Structural and stoichiometric determinants of Ca release-activated Ca (CRAC) channel Ca-dependent inactivation

Depletion of intracellular Ca^2 + stores in mammalian cells results in Ca^2 + entry across the plasma membrane mediated primarily by Ca^2 + release-activated Ca^2 + (CRAC) channels. Ca^2 + influx through these channels is required for the maintenance of homeostasis and Ca^2 + signaling in most cell types. One of the main features of native CRAC channels is fast Ca^2 +-dependent inactivation (FCDI), where Ca^2 + entering through the channel binds to a site near its intracellular mouth and causes a conformational change, closing the channel and limiting further Ca^2 + entry. Early studies suggested that FCDI of CRAC channels was mediated by calmodulin. However, since the discovery of STIM1 and Orai1 proteins as the basic molecular components of the CRAC channel, it has become apparent that FCDI is a more complex phenomenon. Data obtained using heterologous overexpression of STIM1 and Orai1 suggest that, in addition to calmodulin, several cytoplasmic domains of STIM1 and Orai1 and the selectivity filter within the channel pore are required for FCDI. The stoichiometry of STIM1 binding to Orai1 also has emerged as an important determinant of FCDI. Consequently, STIM1 protein expression levels have the potential to be an endogenous regulator of CRAC channel Ca^2 + influx. This review discusses the current understanding of the molecular mechanisms governing the FCDI of CRAC channels, including an evaluation of further experiments that may delineate whether STIM1 and/or Orai1 protein expression is endogenously regulated to modulate CRAC channel function, or may be dysregulated in some pathophysiological states.     

Differential regulation of calcium channel coding genes by prolonged depolarization

Calcium channels must be subjected to a very precise regulation in order to preserve cell function and viability. Voltage gated calcium channels (VGCC) represent the main pathway for calcium entry in excitable cells. This explains why depolarization induces a rapid-onset and short-term inactivation of calcium currents. Contrarily to this well-documented mechanism to maintain calcium below toxic levels, the regulatory pathways inducing longer-lasting changes and cell surface expression of functional calcium channels are largely unknown. Since calcium is a main player in the activity-dependent regulation of many genes, we hypothesize that calcium channel coding genes could be also subjected to activity-dependent regulation. We have used prolonged depolarization to analyze the effects of sustained intracellular calcium elevation on the mRNAs coding for the different alpha(1) pore-forming subunits of the calcium channels expressed in chromaffin cells. Our findings reveal that persistent depolarization is accompanied by a prolonged intracellular calcium elevation and reduction of calcium current. This calcium current inhibition could be mediated, at least partially, by the downregulation of the mRNAs coding for several alpha(1) subunits. Thus, we show here that depolarization inhibits the expression of Ca(V)1.1, Ca(V)1.2, Ca(V)1.3, Ca(V)2.2 and Ca(V)2.3 mRNAs, while the Ca(V)2.1 mRNA remains unmodified. Moreover, such downregulation of channels depends on calcium entry through the L-type calcium channel, as both mRNA and calcium current changes induced by depolarization are abrogated by L-type channel specific blockers.     

Novel potent and selective calcium-release-activated calcium (CRAC) channel inhibitors. Part 2: Synthesis and inhibitory activity of aryl-3-trifluoromethylpyrazoles

To identify potent and selective calcium-release-activated calcium (CRAC) channel inhibitors, we examined the structure-activity relationships of the pyrazole and thiophene moieties in compound 4. Compound 25b was found to exhibit highly potent and selective inhibitory activity for CRAC channels and further modifications of the pyrazole and benzoyl moieties of compound 25b produced compound 29. These compounds were potent inhibitors of IL-2 production in vitro and also acted as inhibitors in pharmacological models of diseases resulting from T-lymphocyte activation, after oral administration.     

The epithelial calcium channel, ECaC, is activated by hyperpolarization and regulated by cytosolic calcium.

The recently cloned epithelial Ca(2+) channel, ECaC, which is expressed in the apical membrane of 1,25-dihydroxyvitamin D(3)-responsible epithelia, was characterized in Xenopus laevis oocytes by measuring the Ca(2+)-activated Cl(-) current which is a sensitive read-out of the Ca(2+) influx. ECaC-expressing oocytes responded to a voltage ramp with a maximal inward current of -2.1 +/- 0.3 microA at a holding potential of -99 +/- 1 mV. The inward current decreased progressively at less negative potentials and at +50 mV a small Ca(2+)-induced outward current was observed. The Ca(2+) influx-evoked current at a hyperpolarizing pulse to -100 mV displayed a fast activation followed by a rapid but partial inactivation. Loading of the oocytes with the Ca(2+) chelator BAPTA delayed the activation and blocked the inactivation of ECaC. When a series of brief hyperpolarizing pulses were given a significant decline in the peak response and subsequent plateau phase was observed. In conclusion, the distinct electrophysiological features of ECaC are hyperpolarization-dependent activation, Ca(2+)-dependent regulation of channel conductance and desensitization during repetitive stimulation.     

Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by G? 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.     

Bidirectional regulation of macrophage function by TGF-β

The dual role of transforming growth factor-beta (TGF-β) in modulating macrophage function is an important concept gaining increasing recognition. In addition to its role as a ‘macrophage-deactivating' agent, TGF-β functions as a monocyte activator, inducing cytoke production and mediating host defence. These functions are context-dependent, modulated by the differentiation state of the cell, the local cytokine environment, and the local levels of TGF-β in itself. In general, during the initial stages of inflammation, TGF-β locally acts as a proinflammatory agent by recruiting and activating resting monocytes. As these cells differentiate specific immunosuppressive actions of TGF-β predominate, leading to resolution of the inflammatory response. Increasing our understanding of the bidirectional regulation of macrophage function will facilitate prediction of the ultimate outcome of modulating TGF-β levels in vivo.     

Mitochondrial regulation of CRAC channel-driven cellular responses

One widespread and conserved Ca(2+) entry pathway in eukaryotic cells is the store-operated CRAC channel and Ca(2+) entry through these channels activates a disparate array of cellular responses. Under physiological conditions, mitochondria are important regulators of CRAC channel gating. Through these actions, mitochondria help control a range of spatially and temporally distinct CRAC channel-driven responses including enzyme activation, secretion, intercellular communication through paracrine signals and gene expression.     

Bidirectional regulation of macrophage function by TGF-beta

The dual role of transforming growth factor-beta (TGF-beta) in modulating macrophage function is an important concept gaining increasing recognition. In addition to its role as a 'macrophage-deactivating' agent, TGF-beta functions as a monocyte activator, inducing cytoke production and mediating host defence. These functions are context-dependent, modulated by the differentiation state of the cell, the local cytokine environment, and the local levels of TGF-beta in itself. In general, during the initial stages of inflammation, TGF-beta locally acts as a proinflammatory agent by recruiting and activating resting monocytes. As these cells differentiate specific immunosuppressive actions of TGF-beta predominate, leading to resolution of the inflammatory response. Increasing our understanding of the bidirectional regulation of macrophage function will facilitate prediction of the ultimate outcome of modulating TGF-beta levels in vivo.     

Hormonal and environmental regulation of epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss)

We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC mRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+]=20-30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (approximately 7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an approximately 100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. Immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells.     

Cell cycle-dependent regulation of store-operated I(CRAC) and Mg2+-nucleotide-regulated MagNuM (TRPM7) currents

Calcium signaling is a central mechanism for numerous cellular functions and particularly relevant for immune cell proliferation. However, the role of calcium influx in mitotic cell cycle progression is largely unknown. We here report that proliferating rat mast cells RBL-2H3 tightly control their major store-operated calcium influx pathway, I(CRAC), during cell cycle progression. While I(CRAC) is maintained at control levels during the first gap phase (G1), the current is significantly up-regulated in preparation for and during chromatin duplication. However, mitosis strongly suppresses I(CRAC). Non-proliferating cells deprived of growth hormones strongly down-regulate I(CRAC) while increasing cell volume. We further show that the other known calcium (and magnesium) influx pathway in mast cells, the TRPM7-like magnesium-nucleotide-regulated metal (MagNuM) current, is largely uncoupled from cell cycle regulation except in G1. Taken together, our results demonstrate that both store-operated calcium influx via I(CRAC) and MagNuM are regulated at crucial checkpoints during cell cycle progression.     

Contact-dependent regulation of N-type calcium channel subunits during synaptogenesis

The developmental regulation of the N-type calcium channel during synaptogenesis was studied using cultured rat hippocampal neurons to elucidate the roles of extrinsic versus intrinsic cues in the expression and distribution of this channel. Prior to synapse formation, α_1B and β₃ subunits of the N-type calcium channel were distributed diffusely throughout neurites, growth cones, and somata. As synaptogenesis proceeded, the subunit distributions became punctate and colocalized with the synaptic vesicle protein synaptotagmin. Isolated neurons were also examined to test for the requirement of extrinsic cues that control N-type calcium channel expression and distribution. These neurons expressed N-type calcium channel subunits, but their distributions remained diffuse. Functional ω-conotoxin GVIA-sensitive channels were expressed in isolated neurons, although the distribution of α_1B subunits was diffuse. The distribution of the α_1B subunit and synaptotagmin only became punctate when neuron-neuron contact was allowed. Thus, the expression of functional N-type calcium channels is the result of an intrinsic program while extrinsic regulatory cues mediated by neuron-neuron contact are required to control their distribution during synaptogenesis. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 198–208, 1998