Density Of L-type Calcium Channels Research Articles

Interaction of Junctophilins and the C-Terminus of Cav1.1 Subunits Regulates Localization and Function of L-Type Calcium Channels in Skeletal Muscle

Junctophilins (JPs) are known to contribute to the stabilization of the junctional membrane (JM) complex by bridging the plasma membrane and sarcoplasmic reticulum; however, the role of JPs on the JM-targeting and function of L-type calcium channels is still unclear. Thus, we explored structural and functional consequences of JP knockdown (KD) in C2C12 and GLT myotubes. JP1 or JP2 KD significantly inhibited the JM-targeting of L-type calcium channels whereas JP2 but not JP1 KD significantly decreased the current density of L-type calcium channels.

Disruption of K 2P 6.1 Produces Vascular Dysfunction and Hypertension in Mice

K(2P)6.1, a member of the 2-pore domain K channel family, is highly expressed in the vascular system; however, its function is unknown. We tested the following hypotheses. K(2P)6.1 regulates the following: (1) systemic blood pressure; (2) the contractile state of arteries; (3) vascular smooth muscle cell migration; (4) proliferation; and/or (5) volume regulation. Mice lacking K(2P)6.1 (KO) were generated by deleting exon 1 of Kcnk6. Mean arterial blood pressure in both anesthetized and awake KO mice was increased by 17±2 and 26±3 mm Hg, respectively (P<0.05). The resting membrane potential in freshly dispersed vascular smooth muscle cells was depolarized by 17±2 mV in the KO compared with wild-type littermates (P<0.05). The contractile responses to KCl (P<0.05) and BAY K 8644 (P<0.01), an activator of L-type calcium channels, were enhanced in isolated segments of aorta from KO mice. However, there was no difference in the current density of L-type calcium channels. Responses to U46619, an agent that activates rho kinase, showed an enhanced contraction in aorta from KO mice (P<0.001). The BAY K 8644-mediated increase in contraction was decreased to wild-type levels when treated with Y27632, a rho kinase inhibitor, (P<0.05). K(2P)6.1 does not appear to be involved with migration, proliferation, or volume regulation in cultured vascular smooth muscle cells. We conclude that K(2P)6.1 deficiency induces vascular dysfunction and hypertension through a mechanism that may involve smooth muscle cell depolarization and enhanced rho kinase activity.

Chillin’ Channels Through Internalization

Voltage-gated calcium channels, and L-type calcium channels (LTCs) in particular, provide a key mechanism by which electrical depolarization of a neuron is translated into changes in biochemical signaling systems that alter cellular function and gene expression. Green et al. show that, beyond the acute Ca 2+ -dependent inhibition of LTC conductance by calmodulin, there is an additional mechanism providing a longer-term negative feedback--Ca 2+ -dependent internalization of the channel. The authors modified the LTC Ca v 1.2 with tags in an extracellular loop and on the intracellular N terminus, which allowed them to monitor the fraction of the channel protein that was present on the surface of individual cultured cortical neurons. Electrical depolarization of the neurons decreased the fraction of the channels that was present on the cell surface. Total internal reflection microscopy showed that the channels became internalized in endosomal vesicles. The authors identified a fragment of the channel that produced a dominant-negative inhibition of internalization and, assuming it might be sequestering a protein that regulated the channel, used it in a two-hybrid screen to find an unexpected interacting protein, eIF3e. The eIF3e protein gets its name from its association with the elongation initiation complex, but it also interacts with the proteasome and the COP9 signalosome, complexes that function to degrade or regulate proteins modified by ubiquitination. At least when overexpressed, eIF3e interacted with Ca v 1.2 in cells, and increased amounts of eIF3e were associated with immunoprecipitated Ca v 1.2 when cells had been depolarized. In commentary on the paper, Varela and Zamponi note that a C-terminal fragment of Ca v 1.2 is reported to move to the nucleus and function as a transcription factor. Might eIF3e help route the channel to a compartment where proteolytic release of the C terminus would be activated for control of gene expression? Even if this intriguing scenario does not pan out, it seems likely that alterations in the density of LTCs at the cell surface is a mechanism that will affect a range of physiological processes, from cardiac function to neuronal plasticity. E. M. Green, C. F. Barrett, G. Bultynck, S. M. Shamah, R. E. Dolmetsch, The tumor suppressor eIF3e mediates calcium-dependent internalization of the L-type calcium channel Ca v 1.2. Neuron 55 , 615-632 (2007). [Online Journal] D. Varela, G. W. Zamponi, Use ‘em and lose ‘em--Activity-induced removal of calcium channels from the plasma membrane. Neuron 55 , 539-541 (2007). [Online Journal]

The effect of inflammation on ligand binding and density of L-type calcium and erg K-channels in rat myocardium

The purpose of this study was to investigate the effect of inflammation on ligand binding and density of L-type calcium channels (LCC) and erg potassium channels (EKC) in the rat heart. Acute and chronic inflammation were induced, hearts were removed and either their myocytes were isolated or they were homogenized to provide cardiac cell membranes. Equilibrium ligand binding of 3H-nitrendipine (NIT) to LCC and 3H-dofetilide (DOF) to EKC was performed. Direct effect of TNF on ligand binding was examined using normal rat cardiac membranes. Western blotting of LCC was performed to estimate the density of the target protein. The results indicate that inflammation had no significant effects on dissociation constants (KD). However, the maximum binding (Bmax) for NIT was significantly reduced in chronic (38.7 ± 6.8%) and acute (26.1 ± 8.1%) inflammation. Binding of DOF to EKC in isolated myocytes was not affected by inflammation. The presence of TNF did not affect the binding parameters of NIT to LCC. Western blotting revealed that inflammation had no effect on the density of α1C subunit of LCC. In summary, decrease in efficacy of cardiovascular drug seen in inflamed states could be mainly due to reduced binding to their ligands. Reduced binding may explain, in part, the reduced pharmacological response to calcium channel blockers such as verapamil, in spite of higher plasma concentrations, in the presence of inflammatory diseases such as rheumatoid arthritis. Supported by CIHR. Clinical Pharmacology & Therapeutics (2004) 75, P48–P48; doi: 10.1016/j.clpt.2003.11.179

Regulation of tyrosine hydroxylase expression in tottering mouse Purkinje cells.

Tottering (tg) mice inherit a missense mutation in the Alpha1A subunit of P/Q-type calcium channels. This mutation results in an increased density of L-type calcium channels in the cerebellum and abnormal regulation of tyrosine hydroxylase (TH) gene expression in a subset of cerebellar Purkinje cells, a cell type that does not normally express TH. The behavioral phenotype includes attacks of dyskinesia, which can be blocked by L-type calcium channel antagonists. To test the hypothesis that cerebellar TH mRNA expression can be manipulated in vivo by L-type calcium channel blockade, control and tottering mice were chronically treated with the L-type calcium channel antagonist nimodipine. Chronic nimodipine treatment significantly reduced the expression of TH mRNA in tottering mouse Purkinje cells. This effect was observed without altering the increased density of L-type calcium channels in tottering mouse cerebella. Chronic nimodipine treatment had no effect on TH mRNA expression in tottering mouse catecholaminergic neurons, including those of the locus coeruleus and substantia nigra. However, a small reduction in TH mRNA expression in the substantia nigra of control mice was observed after drug treatment. These data suggest that the abnormal expression of TH in tottering mouse Purkinje cells is regulated by Purkinje cell excitability.

L-type calcium channels contribute to the tottering mouse dystonic episodes.

Tottering mice inherit a recessive mutation of the calcium channel alpha1A subunit that causes ataxia, polyspike discharges, and intermittent dystonic episodes. The calcium channel alpha1A subunit gene encodes the pore-forming protein of P/Q-type voltage-dependent calcium channels and is predominantly expressed in cerebellar granule and Purkinje neurons with moderate expression in hippocampus and inferior colliculus. Because calcium misregulation likely underlies the tottering mouse phenotype, calcium channel blockers were tested for their ability to block the motor episodes. Pharmacologic agents that specifically block L-type voltage-dependent calcium channels, but not P/Q-type calcium channels, prevented the inducible dystonia of tottering mutant mice. Specifically, the dihydropyridines nimodipine, nifedipine, and nitrendipine, the benzothiazepine diltiazem, and the phenylalkylamine verapamil all prevented restraint-induced tottering mouse motor episodes. Conversely, the L-type calcium channel agonist Bay K8644 induced stereotypic tottering mouse dystonic at concentrations significantly below those required to induce seizures in control mice. In situ hybridization demonstrated that L-type calcium channel alpha1C subunit mRNA expression was up-regulated in the Purkinje cells of tottering mice. Radioligand binding with [3H]nitrendipine also revealed a significant increase in the density of L-type calcium channels in tottering mouse cerebellum. These data suggest that although a P/Q-type calcium channel mutation is the primary defect in tottering mice, L-type calcium channels may contribute to the generation of the intermittent dystonia observed in these mice. The susceptibility of L-type calcium channels to voltage-dependent facilitation may promote this abnormal motor phenotype.

Carbon Centered Radicals Reduce the Density of L-Type Calcium Channels in Rat Cardiac Membranes

The carbon centered radicals generated by preincubation of cumene hydroperoxide with rat cardiac membranes dose dependently reduced the Bmaxof [3H]nitrendipine binding, while KDwas unchanged. The reduction induced by cumene hydroperoxide was prevented by 2,6-di-t-butlyl-4-methyl-phenol. The generation of ·OH did not influence the [3H]nitrendipine binding; also other oxidative agents (H2O2and HClO4) did not modify this binding. Therefore the reduction in [3H]nitrendipine binding sites is not attributable to generic oxidative stress but to the formation of carbon centered radicals.

Evidence for uneven distribution of L-type calcium channels in rat pulmonary circulation.

Experiments were performed on isolated, perfused rat lungs to determine the segmental sites of vasoconstriction in response to factors that open voltage-sensitive, L-type calcium channels on vascular smooth muscle cells. Lungs from male Sprague-Dawley rats were perfused at constant flow with a physiological saline solution (PSS) containing albumin. Measurements were made of pulmonary arterial and venous pressure, whereas capillary pressure was estimated by the double-occlusion technique. After equilibration, lungs were constricted with depolarizing PSS containing high K+ (35 or 45 mM). With both stimuli, approximately 80% of the observed increase in vascular resistance occurred on the arterial side of the circulation. Both nifedipine and verapamil reversed this response; however, reversal was more consistent in the arterial segment. In additional experiments, the L-type channel activator (-)BAY K 8644 caused increased resistance in the arterial but not the venous segment. Another group of lungs constricted with the thromboxane mimetic U-46619 demonstrated equal arterial and venous vasoconstriction. In U-46619-constricted lungs, nifedipine caused a 28% reversal of the agonist-induced increase in arterial resistance but was without effect on the venous circulation. These data suggest that a greater density of L-type calcium channels may exist within the arterial segment of the pulmonary circulation than in the veins.

Imaging of L-type Ca 2+ channels in olfactory bulb neurones using fluorescent dihydropyridine and a styryl dye

We have imaged the fluorescence of dihydropyridine-Bodipy (fDHP) in cultured olfactory bulb neurones in order to investigate the subcellular distribution of L-type calcium channels in these neurones. The neurones were stained with both fDHP and the voltage-sensitive styryl dye RH414. The fluorescence emission maxima of these dyes were in the green and red ranges of the spectrum, respectively, and were recorded by the 2 photomultiplier channels of a laser scanning microscope. The fDHP images were ratioed with the RH414 images taken simultaneously. The resulting ratio images revealed the spatial distribution of the surface density of L-type calcium channels. This density was highest on somata, in particular at the base of dendrites, and decreased with the distance from this maximum. Two classes of dendritic L-type channel distributions were observed: one with a homogeneously low density and another one with a characteristic gradient of the L-type channel density along proximal dendrites. This subcellular localization of L-type calcium channels is discussed in the light of specific functional roles.

In vivo labelling of L-type Ca 2+ channels by fluorescent dihydropyridine: correlation between ontogenesis of the channels and the acquisition of neural competence in ectoderm cells from Pleurodeles waltl embryos

The ability of the ectodermal cells to be induced and to differentiate toward neural tissue, called neural competence, is acquired shortly before gastrulation and lost during late gastrula stages in Pleurodeles walfl embryos. We have examined ectodermal cells' neural competence in relation to the evolution of the density of L-type calcium channels using the fluorescent labelled dihydropyridine probe (STBodipy-DHP). We find that the appearance of dihydropyridine sensitive calcium channels (L-type Ca 2+ channels) is correlated with the acquisition of neural competence by the ectoderm cells. The highest density of these channels is reached when competence of the ectoderm is optimal. Conversely, the decrease of L-type Ca 2+ channel density occurs simultaneously with the normal loss of competence. In addition, we show that these channels are functional since stimulation by S(−)-Bay K 8644 triggered an increase in [Ca 2+] i revealed by fluorescence measurements using FIuo-3. This increase in [Ca 2+] i is a function of the L-type Ca 2+ channels' density. We propose that the molecular basis of the gain and loss of neural competence is linked to the presence of L-type Ca 2+ channels in ectodermal cell membranes of Pleurodeles waltl embryos.

Cerebrovascular, neuronal, and behavioral effects of long-term Ca2+ channel blockade in aging normotensive and hypertensive rat strains.

The pathogenesis of essential hypertension is not fully understood, but most of the cardiovascular, metabolic, neurogenic, and humoral abnormalities are explained by dysfunctions in the control of intracellular Ca2+ concentrations in the cells of the vascular wall. Most theories of disturbed calcium regulation focus on the calcium concentration within vascular smooth muscle cells. The implications of hypertension for the increased calcium content of aging arteries seem to be clear, but were only studied in the peripheral circulation; hypertension prominently augments the aging-related accumulation of calcium in the vessel wall. Although the contribution of calcium overload in hypertensive cerebrovascular damage is well documented, it is not clear yet if hypertension per se is the main cause of hypertension-associated calcium-dependent cerebral damage. Thus far, the hypotensive effects of most calcium antagonists were extensively described, and their efficacy in stroke prevention was proven. Earlier studies indicated that chronic administration of nimodipine revealed a protective effect in the occurrence of strokes in SHR-SP rats, yielding a decreased mortality rate. Because nimodipine did not lower the extremely high blood pressure of these animals, the mechanisms behind such nimodipine-induced stroke prevention may be attributed to a direct cerebrovascular and/or neuronal action of nimodipine. Hypertension is generally considered a vascular pathologic condition, and most research has been directed towards the influences of hypertension on large peripheral arteries such as the aorta and coronary artery. The influence of the CNS on the regulation of cardiovascular system and blood pressure regulation was described in detail, and the role of the CNS in hypertension also was the subject of study. The increased risk of stroke in hypertensive subjects generated numerous studies on the precise nature of compromised cerebrovascular functioning under hypertensive conditions. Few data are available on Ca2+ alterations in cerebral neurons during hypertension. Honda et al. demonstrated that voltage-dependent Ca2+ uptake was higher in cortical synaptosomes from SHR than form normotensive animals and suggested that an important alteration in Ca2+ channel characteristics may occur in SHR brain synaptosomes. Although the density of L-type calcium channels was shown to be higher in the hippocampus of SHR rats, others reported that the number of L-type calcium channels was significantly lower in the brain of SHR rats than WKY normotensive controls. The latter data suggest that hypertension may be associated with similar alterations in neuronal calcium homeostasis as demonstrated for aging in normotensive subjects.(ABSTRACT TRUNCATED AT 400 WORDS)