Microinjection of a conserved peptide sequence of p34cdc2 induces a Ca2+ transient in oocytes.

The product of the yeast cell cycle control gene cdc2, and its homologs in higher eukaryotes (p34cdc2), all contain a perfectly conserved sequence of 16 amino acids that has not been found in any other protein sequence. Microinjection of this peptide triggers a specific increase in the concentration of intracellular free Ca2+ that originates from intracellular stores in both starfish and Xenopus oocytes. Thus, p34cdc2 might interact through its conserved peptide domain with some component of the Ca2(+)-regulatory system.

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes.

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.

Transient down-regulation of L-type Ca(2+) channel and dystrophin expression after balloon injury in rat aortic cells.

OBJECTIVE: Migration and proliferation of arterial smooth muscle cells are critical responses during restenosis after balloon angioplasty. We investigated the changes in the expression of Ca(2+) channels and dystrophin, two determinants of contraction, after balloon injury of rat aortas. METHODS: Proliferation and migration of aortic myocytes were triggered in vivo by the passage of an inflated balloon catheter in the aortas of 12-week-old male Wistar rats. We used the whole-cell patch clamp technique to investigate Ba(2+) currents (I(Ba)) through Ca(2+) channels in single cells freshly isolated from media and neointima at various times after injury (days 2, 7, 15, 30 and 45). RESULTS: No T-type Ca(2+) channel current was recorded in any cell at any time. In contrast, a dihydropyridine (DHP)-sensitive L-type I(Ba)was recorded consistently in the media of intact aorta. After aortic injury, I(Ba) decreased dramatically (at days 2 and 7) but recovered over time to reach normal amplitude on days 30 and 45. In the neointima, I(Ba) was absent on day 15 but also increased gradually over time as observed at days 30 and 45. The use of a specific antibody directed against the L-type Ca(2+) channel alpha(1C) subunit showed, both by immunostaining and by Western blotting, no expression of the Ca(2+) channel protein on day 15. Parallel immunodetection of dystrophin showed that this marker of the contractile phenotype of SMCs was also not detectable at this stage in neointimal cells. Both proteins were re-expressed at days 45 and 63. Balloon injury induces a transient down-regulation of I(Ba) in arterial cells. CONCLUSIONS: Cell dedifferentiation and proliferation in vivo abolish the expression of L-type Ca(2+) channels and dystrophin in neointimal cells. These changes may be critical in the regulation of Ca(2+) homeostasis and, thereby, contraction of the arterial SMCs during restenosis following angioplasty.

Characterization of beta subunit modulation of a rabbit cardiac L-type Ca2+ channel alpha 1 subunit as expressed in mouse L cells.

Functional properties of a rabbit cardiac alpha 1 Ca2+ channel subunit (CARD alpha 1) were investigated using the patch-clamp technique in mouse L cells, a recipient cell line which is devoid of any Ca2+ channel subunits. Cell lines resulting from stable transfection of the CARD alpha 1 subunit as well as in coexpression with a beta subunit (CARD alpha 1 beta) derived from skeletal muscle (SKM beta) were characterized. The results show that while the CARD alpha 1-Ca2+ channel activity is negligible, the Ba2+ current density is dramatically increased in the presence of beta subunit (approximately 20-fold). CARD alpha 1- and CARD alpha 1 beta-Ba2+ currents were both sensitive to the 1,4-dihydropyridine (DHP) agonist, Bay K 8644 (5- to 8-fold increase). Activation kinetics of CARD alpha 1- and CARD alpha 1 beta-Ba2+ currents were comparable. The inactivation time-course was faster (3- to 4-fold) for CARD alpha 1 beta-Ba2+ currents. We conclude that the main role of the beta subunit in heart is to modulate the L-type current density and present several lines of evidence that SKM alpha 1 and CARD alpha 1 are differentially regulated by the beta subunit.

Electrophysiological expression of endothelin and angiotensin receptors in Xenopus oocytes injected with rat heart mRNA.

Functional endothelin and angiotensin receptors have been expressed in Xenopus oocyte following the microinjection of rat heart mRNA. Under voltage clamp conditions, application of these peptides clearly induced oscillatory Ca2+-activated chloride currents in a dose-dependent manner. In addition, no direct modulation of expressed or native cardiac Ca channels was observed.

Electrophysiological properties of the hypokalaemic periodic paralysis mutation (R528H) of the skeletal muscle alpha 1s subunit as expressed in mouse L cells.

Hypokalaemic periodic paralysis (HypoPP) is an autosomal dominant muscle disease which has been linked to point mutations in the skeletal muscle L-type calcium channel alpha 1 subunit (alpha 1s). Here, we have introduced one of the point mutations causing HypoPP (R528H) into cDNA of the rabbit alpha 1s. Expression of either the wild-type alpha 1s or the mutant R528H alpha 1s (alpha 1s-R528H) subunits was obtained in mouse Ltk- cells using a selectable expression vector. The alpha 1s-R528H subunit led to the expression of functional L-type Ca2+ channels. Corresponding whole-cell Ba2+ currents exhibit very slow activation and inactivation kinetics, typical for recombinant skeletal Ca2+ channel currents. Voltage-dependent activation and inactivation properties were similar for alpha 1s- and alpha 1s-R528H, as well as their sensitivity to the dihydropyridine agonist Bay K 8644. Differences in alpha 1s- and alpha 1s-R528H-directed channels reside in the Ba2+ current density, which was significantly reduced 3.2 fold in cells expressing alpha 1s-R528H. It was concluded that the R528H mutation af alpha 1s results in minor differences in the electrophysiological properties but significantly reduces the whole-cell Ca2+ channel current in its amplitude.

Functional expression of Ca2(+)-mobilizing alpha-thrombin receptors in mRNA-injected Xenopus oocytes.

alpha-Thrombin (TH) initiates a program of intracellular events that lead to DNA replication in quiescent CCL39 Chinese hamster lung fibroblasts via membrane receptors that have yet to be characterized at a molecular level. Functional TH receptors were expressed in Xenopus laevis oocytes following injection of poly(A)+ RNA from TH-responsive CCL39 cells; their presence was demonstrated by TH-stimulated 45Ca2+ efflux or Ca2(+)-dependent Cl- channel activation. In voltage clamp experiments on microinjected oocytes a Ca2(+)-activated Cl- current was detected in response to TH (0.2-10 U/ml). The TH response was blocked by a specific TH inhibitor, and potentiated by addition of FGF or intracellular injection of GTP-gamma-S.

Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation.

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.

Endogenous cardiac Ca2+ channels do not overcome the E-C coupling defect in immortalized dysgenic muscle cells: evidence for a missing link.

The expression of subunit genes of the Ca2+ channel complex was studied in differentiating, immortalized mouse mdg cells. These cells expressed alpha 1 and alpha 2/delta transcripts of the skeletal muscle Ca2+ channel genes, a cardiac Ca2+ channel alpha 1 subunit gene and several known transcript variants of skeletal, cardiac and brain beta genes. The mdg mutation is retained in the 129DA3 cell line and occurs exclusively at nucleotide position 4010 in the skeletal alpha 1 transcript in which a cytosine residue is deleted. In early stages of differentiation and fusion, Ba2+ currents were detected in dysgenic myotubes the same as the cardiac L-type Ca2+ channel. These data provide specific structural evidence [Chaudhari, N. (1992) J. Biol. Chem. 267, 25636-25639] for the major genetic defect in mouse muscular dysgenesis and show a change in the expression levels of alpha 1S and alpha 1C. The upregulation of the expression of alpha 1C results in functional Ca2+ channel activity, however, presumably not sufficient for excitation-contraction coupling.

Cell cycle dependent toxicity of an amphiphilic synthetic peptide.

The cytotoxic properties of an amphiphilic synthetic peptide are presented. Comparative analysis of proliferating, differentiated and confluent H9C2 adherent cells and L1210 cells in suspension shows a correlation between toxicity and cell stage (proliferating cells). Electrophysiological measurements on Xenopus laevis oocytes bathed in the peptide also demonstrated the induction of cationic currents, which is voltage and phosphate dependent. These results allow us to hypothesize that the observed toxicity is related to membrane hyperpolarization of proliferating cells at the G1/S cell cycle phase transition.

cAMP-dependent phosphorylation of the cardiac L-type Ca channel: a missing link?

Cardiac inotropic effects of beta adrenergic agonists occur mainly through an increase in L-type (class C) calcium channel activity. This response has been attributed to phosphorylation of the L-type Ca channel, or a closely associated protein, by the cAMP-dependent protein kinase A (PKA). Among the three subunits forming the cardiac L-type Ca channel (alpha 1, beta and alpha 2-delta), biochemical studies have revealed that two subunits, alpha 1 and beta, are phosphorylated in vitro by protein kinase A, the alpha 1 subunit being the primary target. However, attempts to reconstitute the cAMP-dependent regulation of the expressed class C Ca channel, either in Xenopus oocytes or in cell lines, have provided contradictory results. We were unable to detect cAMP-dependent modulation of class C alpha 1 subunit Ca channels expressed in Xenopus oocytes, even when coinjected with auxiliary subunits beta and alpha 2-delta. Nevertheless, activity of Ca channels recorded from cardiac-mRNA injected oocytes was potentiated by injection of cAMP or PKA, even when expression of the beta subunit was suppressed using antisense oligonucleotide. Taken together, these results indicate that cAMP-dependent regulation does not exclusively involve the alpha 1 and the beta subunits of the Ca channel and suggest that unidentified protein(s), expressed in cardiac tissue, are most likely necessary.

Hypokalemic periodic paralysis: an autosomal dominant muscle disorder caused by mutations in a voltage-gated calcium channel.

Hypokalemic periodic paralysis (hypoPP) is an autosomal dominant disorder characterized by acute attacks of muscle weakness concomitant to a drop in blood potassium levels. Recent molecular work has shown that hypoPP is due to mutations in a skeletal muscle voltage-gated calcium channel: the dihydropyridine receptor (DHP receptor). Mutations affect segments S4 of domains II and IV, changing an arginine in position 528 and 1239 into an histidine, or an histidine or a glycine respectively. Surprisingly, expressing in vitro mutants channels in a non-muscular environment resulted in functional calcium channels with minor modifications in electrophysiological properties. Expressing mutant channels in a muscular environment or transgenic mice might help to bridge the gap between the knowledge of the molecular defect and the understanding of the pathophysiology of the disease.

Antisense depletion of beta-subunits fails to affect T-type calcium channels properties in a neuroblastoma cell line.

Voltage-gated calcium channels can be classified into high voltage activated (HVA) and low voltage activated (LVA or T-type) subtypes. The molecular diversity of HVA channels primarily results from different genes encoding their pore-forming alpha1 subunits. These channels share a common structure with an alpha1 subunit associated with at least two regulatory subunits (beta, alpha2-delta). Any of the six alpha1-related channels identified to date are regulated in their functional properties through an interaction with the ancillary beta-subunit. By contrast, the diversity and the molecular identity of LVA or T-type calcium channels have yet to be defined. Whether LVA channels are modulated by a beta-subunit, like HVA channels, is unknown. To address this issue, we have used an antisense strategy to inhibit beta-subunit expression in the NG 108-15 neuroblastoma cell line. Differentiated NG 108-15 cells express both LVA and HVA channels. We found that LVA currents were unaffected when cells were incubated with beta-antisense, while HVA currents were drastically decreased. Since LVA Ca channel currents in NG 108-15 cells are not regulated by beta-subunits, it is reasonable to postulate that the pore-forming subunit(s) of these channels lacks an interaction domain with a beta-subunit (AID). This molecular feature, which is common to various T-type channels, indicates further that LVA calcium channels belong to a channel family structurally distant from HVA channels.

A specific quisqualate agonist inhibits kainate responses induced in Xenopus oocytes injected with rat brain RNA.

Electrophysiological recording was used to study non-N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors after injection of rat brain ribonucleic acid (RNA) in Xenopus laevis oocytes. Quisqualate (QA) induced two types of responses, a smooth one and an oscillatory one. These responses are probably mediated by the ionotropic (QAi, a cationic channel) and the metabotropic (QAp, a newly discovered receptor coupled to phospholipase C) QA receptors respectively. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) only induced a smooth inward current suggesting that it acts only on QAi. Kainate (KA) also induced a smooth inward current, the maximal KA response being 10-fold higher than the maximal AMPA. AMPA inhibited the KA response in a dose-dependent and competitive manner. Amongst various complex hypotheses the simplest to explain these results would be that KA and AMPA both activate the same receptor-channel complex, AMPA inducing a smaller response than KA.

[Molecular genetics of cardiovascular calcium channels]. / Génétique moléculaire des canaux calciques cardiovasculaires.

Cardiac and vascular myocytes exhibit L type calcium channel currents with slightly different properties. The structural bases of this concept of functional diversity of cardiovascular calcium channels are now known. Firstly, there are multiple isoforms of the alpha 1 pore subunit. In addition, the beta subunit should be presented as an endogenous regulator of the calcium channel. Like the alpha 1 subunit, there are many isoforms of this regulatory subunit. A series of recent experiments has changed our understanding of the mechanisms which govern the expression of a functional diversity of calcium channels in the cardiovascular cells. Several pharmacological sites, such as the dihydropyridine, phenylalkylamine and benzothiazepine receptors, have been identified. The recent developments in the field of molecular genetics of the calcium channels are many and open up new perspectives. Mutations within these channels could be the cause of certain cardiovascular genetic diseases.

[Genetic diversity of voltage-gated calcium channels]. / Diversité génétique des canaux calciques activés par la dépolarisation membranaire.

Understanding of the properties of normal and diseased voltage-dependent calcium channels has greatly improved these last Years after the extensive development of the patch-clamp and molecular biology studies and the functional expression strategies. The calcium channel diversity is based on the expression of numerous genes that encode pore channel subunits (10 genes) and auxiliary/regulatory subunits (16 genes). In addition, most of these genes are subject to alternative splicing. The study of calcium channels has also benefited from the discovery of genetic diseases linked to calcium channel mutations: the calcium channelopathies. The review describes the recent data and working hypothesis that address the challenging question of how the calcium channel diversity occurs and how alterations in channel function lead to selective cellular dysfunction.

[Molecular diversity of calcium channel activities by depolarization]. / Diversité moléculaire des canaux calciques activés par la dépolarisation.

Voltage-gated calcium channels are involved in a large variety of cellular functions such as excitation-contraction coupling, hormone secretion, firing and pacemaker activity, gene activation and proliferation. Cloning of complementary DNAs encoding for calcium channel subunits has challenged the study of the functional properties of calcium channels and has allowed analysis of the molecular basis of calcium channel diversity. Recently, pore-forming subunits of T-type calcium channels have been cloned. Recent data describing the genes encoding calcium channels, their molecular and pharmacological studies, as well as their linkage to human genetic diseases are reviewed in this article.

Myogenic tone is impaired at low arterial pressure in mice deficient in the low-voltage-activated CaV 3.1 T-type Ca(2+) channel.

AIM: Using mice deficient in the CaV 3.1 T-type Ca(2+) channel, the aim of the present study was to elucidate the molecular identity of non-L-type channels involved in vascular tone regulation in mesenteric arteries and arterioles. METHODS: We used immunofluorescence microscopy to localize CaV 3.1 channels, patch clamp electrophysiology to test the effects of a putative T-type channel blocker NNC 55-0396 on whole-cell Ca(2+) currents, pressure myography and Ca(2+) imaging to test diameter and Ca(2+) responses of the applied vasoconstrictors, and Q-PCR to check mRNA expression levels of several Ca(2+) handling proteins in wild-type and CaV 3.1(-/-) mice. RESULTS: Our data indicated that CaV 3.1 channels are important for the maintenance of myogenic tone at low pressures (40-80 mm Hg), whereas they are not involved in high-voltage-activated Ca(2+) currents, Ca(2+) entry or vasoconstriction to high KCl in mesenteric arteries and arterioles. Furthermore, we show that NNC 55-0396 is not a specific T-type channel inhibitor, as it potently blocks L-type and non-L-type high-voltage-activated Ca(2+) currents in mouse mesenteric vascular smooth muscle cell. CONCLUSION: Our data using mice deficient in the CaV 3.1 T-type channel represent new evidence for the involvement of non-L-type channels in arteriolar tone regulation. We showed that CaV 3.1 channels are important for the myogenic tone at low arterial pressure, which is potentially relevant under resting conditions in vivo. Moreover, CaV 3.1 channels are not involved in Ca(2+) entry and vasoconstriction to large depolarization with, for example, high KCl. Finally, we caution against using NNC 55-0396 as a specific T-type channel blocker in native cells expressing high-voltage-activated Ca(2+) channels.

Dehydroepiandrosterone (DHEA) inhibits voltage-gated T-type calcium channels.

BACKGROUND AND PURPOSE: Dehydroepiandrosterone (DHEA) and its sulfated form, DHEAS, are the most abundant steroid hormones in the mammalian blood flow. DHEA may have beneficial effects in various pathophysiological conditions such as cardiovascular diseases or deterioration of the sense of well-being. However to date, the cellular mechanism underlying DHEA action remains elusive and may involve ion channel modulation. In this study, we have characterized the effect of DHEA on T-type voltage-activated calcium channels (T-channels), which are involved in several cardiovascular and neuronal diseases. KEY RESULTS: Using the whole-cell patch-clamp technique, we demonstrate that DHEA inhibits the three recombinant T-channels (Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3) expressed in NG108-15 cell line, as well as native T-channels in pulmonary artery smooth muscle cells. This effect of DHEA is both concentration (IC(50) between 2 and 7µM) and voltage-dependent and results in a significant shift of the steady-state inactivation curves toward hyperpolarized potentials. Consequently, DHEA reduces window T-current and inhibits membrane potential oscillations induced by Ca(V)3 channels. DHEA inhibition is not dependent on the activation of nuclear androgen or estrogen receptors and implicates a PTX-sensitive Gi protein pathway. Functionally, DHEA and the T-type inhibitor NNC 55-0396 inhibited KCl-induced contraction of pulmonary artery rings and their effect was not cumulative. CONCLUSIONS: Altogether, the present data demonstrate that DHEA inhibits T-channels by a Gi protein dependent pathway. DHEA-induced alteration in T-channel activity could thus account for its therapeutic action and/or physiological effects.