Increase in Serum Ca2+/Mg2+ Ratio Promotes Proliferation of Prostate Cancer Cells by Activating TRPM7 Channels

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg²⁺ concentrations. Changes in Mg²⁺ concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca²⁺/Mg²⁺ ratio facilitated Ca²⁺ influx in both control and prostate cancer cells, a significantly higher Ca²⁺ influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca²⁺/Mg²⁺ ratio per se. Additionally, an increase in the extracellular Ca²⁺/Mg²⁺ ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca²⁺/Mg²⁺ ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca²⁺/Mg²⁺ ratio could be essential for the initiation/progression of prostate cancer.     

Molecular determinants of Mg2+ and Ca2+ permeability and pH sensitivity in TRPM6 and TRPM7

The channel kinases TRPM6 and TRPM7 have recently been discovered to play important roles in Mg2+ and Ca2+ homeostasis, which is critical to both human health and cell viability. However, the molecular basis underlying these channels' unique Mg2+ and Ca2+ permeability and pH sensitivity remains unknown. Here we have created a series of amino acid substitutions in the putative pore of TRPM7 to evaluate the origin of the permeability of the channel and its regulation by pH. Two mutants of TRPM7, E1047Q and E1052Q, produced dramatic changes in channel properties. The I-V relations of E1052Q and E1047Q were significantly different from WT TRPM7, with the inward currents of 8- and 12-fold larger than TRPM7, respectively. The binding affinity of Ca2+ and Mg2+ was decreased by 50- to 140-fold in E1052Q and E1047Q, respectively. Ca2+ and Mg2+ currents in E1052Q were 70% smaller than those of TRPM7. Strikingly, E1047Q largely abolished Ca2+ and Mg2+ permeation, rendering TRPM7 a monovalent selective channel. In addition, the ability of protons to potentiate inward currents was lost in E1047Q, indicating that E1047 is critical to Ca2+ and Mg2+ permeability of TRPM7, and its pH sensitivity. Mutation of the corresponding residues in the pore of TRPM6, E1024Q and E1029Q, produced nearly identical changes to the channel properties of TRPM6. Our results indicate that these two glutamates are key determinants of both channels' divalent selectivity and pH sensitivity. These findings reveal the molecular mechanisms underpinning physiological/pathological functions of TRPM6 and TRPM7, and will extend our understanding of the pore structures of TRPM channels.     

Regulation of the transient receptor potential channel TRPM2 by the Ca2+ sensor calmodulin

TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca(2+)-permeable channel activated by oxidative stress or tumor necrosis factoralpha involved in susceptibility to cell death. TRPM2 activation is dependent on the level of intracellular Ca(2+). We explored whether calmodulin (CaM) is the Ca(2+) sensor for TRPM2. HEK 293T cells were transfected with TRPM2 and wild type CaM or mutant CaM (CaM(MUT)) with substitutions of all four EF hands. Treatment of cells expressing TRPM2 with H(2)O(2) or tumor necrosis factor alpha resulted in a significant increase in intracellular calcium ([Ca(2+)](i)). This was not affected by coexpression of CaM, suggesting that endogenous CaM levels are sufficient for maximal response. Cotransfection of CaM(MUT) with TRPM2 dramatically inhibited the increase in [Ca(2+)](i), demonstrating the requirement for CaM in TRPM2 activation. Immunoprecipitation confirmed direct interaction of CaM and CaM(MUT) with TRPM2, and the Ca(2+) dependence of this association. CaM bound strongly to the TRPM2 N terminus (amino acids 1-730), but weakly to the C terminus (amino acids 1060-1503). CaM binding to an IQ-like motif (amino acids 406-416) in the TRPM2 N terminus was demonstrated utilizing gel shift, immunoprecipitation, biotinylated CaM overlay, and pull-down assays. A substitution mutant of the IQ-like motif of TRPM2 (TRPM2-IQ(MUT1)) reduced but did not eliminate CaM binding to TRPM2, suggesting the presence of at least one other CaM binding site. The functional importance of the TRPM2 IQ-like motif was demonstrated by treatment of TRPM2-IQ(MUT1)-expressing cells with H(2)O(2). The increase in [Ca(2+)](i) observed with wild type TRPM2 was absent and cell viability was preserved. These data demonstrate the requirement for CaM in TRPM2 activation. They suggest that Ca(2+) entering through TRPM2 enhances interaction of CaM with TRPM2 at the IQ-like motif in the N terminus, providing crucial positive feedback for channel activation.     

Glutamate 90 at the Luminal Ion Gate of Sarcoplasmic Reticulum Ca2+-ATPase Is Critical for Ca2+ Binding on Both Sides of the Membrane

The roles of Ser⁷², Glu⁹⁰, and Lys²⁹⁷ at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca²⁺-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca²⁺ concentration of phosphorylation by P_i (“Ca²⁺ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca²⁺ in mutants with alanine or leucine replacement of Glu⁹⁰, whereas arginine replacement of Glu⁹⁰ or Ser⁷² allowed E2P formation from P_i even at luminal Ca²⁺ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF₃⁻ complex to luminal Ca²⁺. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu⁹⁰ or Ser⁷², because the positive charge of the arginine side chain mimics Ca²⁺ occupying a luminally exposed low affinity Ca²⁺ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca²⁺ exit pathway. Mutants with alanine or leucine replacement of Glu⁹⁰ further displayed a marked slowing of the Ca²⁺ binding transition as well as slowing of the dissociation of Ca²⁺ from Ca₂E1 back toward the cytoplasm, thus demonstrating that Glu⁹⁰ is also critical for the function of the cytoplasmically exposed Ca²⁺ sites on the opposite side of the membrane relative to where Glu⁹⁰ is located.     

Voltage dependence of the Ca2+-activated cation channel TRPM4

TRPM4 is a Ca2+-activated but Ca2+-impermeable cation channel. An increase of [Ca2+]i induces activation and subsequent reduction of currents through TRPM4 channels. This inactivation is strikingly decreased in cell-free patches. In whole cell and cell-free configuration, currents through TRPM4 deactivate rapidly at negative potentials. At positive potentials, currents are much larger and activate slowly. This voltage-dependent behavior induces a striking outward rectification of the steady state currents. The instantaneous current-voltage relationship, derived from the amplitude of tail currents following a prepulse to positive potentials, is linear. Currents show a Boltzmann type of activation; the fraction of open channels increases at positive potentials and is low at negative potentials. Voltage dependence is not due to block by divalent cations or to voltage-dependent binding of intracellular Ca2+ to an activator site, indicating that TRPM4 is a transient receptor potential channel with an intrinsic voltage-sensing mechanism. Voltage dependence of TRPM4 may be functionally important, especially in excitable tissues generating plateau-like or bursting action potentials.     

Blocking of Cloned Low-Threshold Ca2+ Channels by Neuroleptic Drugs

We studied the blocking action of neuroleptic drugs, haloperidol, pimozide, and fluspirilene, on three types of cloned low voltage-activated (T-type) Ca²⁺ channels, 1G, 1H, and 1I, functionally expressed in Xenopus oocytes. Fluspirilene and pimozide (members of the diphenylbutylpiperidine group) and haloperidol (belonging to butyrophenones) inhibited Ca²⁺ currents with different values of the K _d constant and maximum intensity of blocking. Effects of the neuroleptics were voltage-dependent and were accompanied by slowing-down of the kinetics of the currents. The mechanism of blocking is probably based on interaction between the neuroleptics and the channels in the activated and inactivated states. The difference in efficiency and specificity of blockade of various T-channel subtypes by neuroleptics should be considered when estimating the therapeutic significance of the tested pharmacological agents.     

Electrical Polarization of Tobacco Cells by Ca2+ Ion Channels

It has been suggested that neighbouring cells in higher plantsco-ordinate their direction of growth by sensing the electricalpolarities of their neighbours. The present work sets out toexamine the role played by calcium in the response of theirindividual cells to externally-applied fields. The transcellular currents of cultured tobacco cells were investigatedwith a vibrating probe before and after the application of anartificial electric current with a density of 250 µA cm^–2,giving a potential difference of approximately 3 mV across thecell. When calcium was omitted from the experimental medium,the externally-applied current had little effect on either thedirection or magnitude of the cells' own transcellular currents.When 01 mM calcium was present, the external current repolarizedthe cells so that their own currents tended to flow in the samedirection as the current applied. This was due to a large localizedincrease in inward current in the region nearest the positiveelectrode, with the outward current being more evenly spread. Adding cobalt ions (a Ca²⁺ -channel blocker) in the presenceof external calcium had little immediate effect on the transcellularcurrents themselves, but they lost their ability to change inresponse to the artificially applied current. This suggeststhat the cells may have detected the applied current by enhancedcalcium ingress through calcium channels in the most hyperpolarizedregion of the membrane. An hypothesis is presented which proposesthat asymmetric calcium entry results in the electrical polarizationof cells by a mechanism involving both the opening of calcium-gatedion channels and the lateral movement of electrophoreticallymobile channels. Key words: Cell-culture, cobalt, plant electrophysiology, polarity, vibrating-probe     

Influence of Ca2+ Ions on the Activity of Lantibiotics Containing a Mersacidin-Like Lipid II Binding Motif

Mersacidin binds to lipid II and thus blocks the transglycosylation step of the cell wall biosynthesis. Binding of lipid II involves a special motif, the so-called mersacidin-lipid II binding motif, which is conserved in a major subgroup of lantibiotics. We analyzed the role of Ca²⁺ ions in the mode of action of mersacidin and some related peptides containing a mersacidin-like lipid II binding motif. We found that the stimulating effect of Ca²⁺ ions on the antimicrobial activity known for mersacidin also applies to plantaricin C and lacticin 3147. Ca²⁺ ions appear to facilitate the interaction of the lantibiotics with the bacterial membrane and with lipid II rather than being an essential part of a peptide-lipid II complex. In the case of lacticin 481, both the interaction with lipid II and the antimicrobial activity were Ca²⁺ independent.Bacteriocins are a heterogeneous group of ribosomally synthesized antibiotic peptides and proteins which were proposed to fall into three classes, the lanthionine-containing bacteriocins (class I), the non-lanthionine-containing bacteriocins (class II), and the bacteriolysins, respectively (for a review, see reference 12).The lanthionine-containing bacteriocins (lantibiotics) are produced by and are effective against a broad spectrum of gram-positive bacteria. They are small, posttranslationally modified antimicrobial peptides containing characteristic thioether ring structures (lanthionine and 3-methyllanthionine) and other unusual amino acids, e.g., d-Ala (3, 49).Mersacidin was the first lantibiotic shown to interact with a defined target molecule, the ultimate cell wall precursor lipid II (6) (Fig. ​(Fig.1).1). Further studies revealed that this molecule is also the target of nisin and many other lantibiotics (19). Lipid II is synthesized on the cytoplasmic side of the membrane and translocated to the outside of the bacterial cell membrane, where the disaccharide pentapeptide part of lipid II is incorporated into the growing peptidoglycan network by the cell wall biosynthesis machinery (for reviews, see references 5 and 45).Open in a separate windowFIG. 1.Primary structure of lantibiotics containing the mersacidin-lipid II binding motif (A) and the structure of the cell wall precursor lipid II (B). The binding motif of mersacidin and identical amino acids in the mersacidin-like lantibiotics are highlighted in gray. Dha, dehydroalanine; Dhb, dehydrobutyrine; Ala-S-Ala, lanthionine; Abu-S-Ala, methyllanthionine; DAla, d-alanine.To date, two different lipid II binding motifs in lantibiotics have been identified, referred to as the nisin-lipid II and mersacidin-lipid II binding motifs, and a classification regarding their interaction with the cell wall precursor was recently proposed by Bierbaum and Sahl (3).The nisin-lipid II binding motif is also found in related lantibiotics, e.g., gallidermin, epidermin (4), mutacin 1140 (40), and subtilin (30). Nisin displays a dual mode of action by binding to lipid II. It prevents lipid II incorporation into the growing murein layer, thereby blocking cell wall biosynthesis (8), and it uses lipid II as an anchor molecule for subsequent pore formation (48). The nisin/lipid II interaction was analyzed by nuclear magnetic resonance spectroscopy and it was shown that the N-terminal part of the peptide forms a cage-like structure encompassing the pyrophosphate group of the lipid II molecule, leading to the formation of five intermolecular hydrogen bonds between the backbone amids of the lantibiotic and pyrophosphate groups (22).The second binding motif occurs in mersacidin and related lantibiotics (Fig. ​(Fig.1).1). The interaction of mersacidin with lipid II leads to inhibition of the peptidoglycan biosynthesis at the level of transglycosylation (7). In contrast to nisin, the activity of mersacidin is influenced by Ca²⁺ ions, since its antimicrobial activity increased twofold in Ca²⁺-containing medium (2). When a Ca²⁺ binding pocket was identified in the mersacidin-like lantibiotic actagardine by crystal structure determination, it was suggested that the deprotonated Glu17 in the mersacidin-lipid II binding motif (Fig. ​(Fig.1)1) is involved in Ca²⁺ binding (24). Furthermore, nuclear magnetic resonance studies revealed that, upon binding of lipid II, mersacidin effectively alters its overall backbone geometry with Ala-12 and Abu-13, acting as a hinge region. The conformational change exposes the amino group of Lys1 and the carboxyl group of Glu17 to the lipid II molecule (21). It was speculated that Ca²⁺ is needed to bridge the mersacidin Glu17 side chain to the negatively charged groups of lipid II; alternatively, a direct salt bridge with the positively charged side chain of Lys3 in lipid II is formed (21). This hypothesis is in good agreement with the observation that replacement of Glu17 by Ala abolished the antimicrobial activity of mersacidin (43).To analyze the impact of Ca²⁺ on the activity of mersacidin-like lantibiotics, we selected four peptides which possess the respective lipid II-binding motif, yet show significant differences in primary structures (Fig. ​(Fig.1).1). Like mersacidin, plantaricin C and the two-component lantibiotic lacticin 3147 have been shown to inhibit cell wall biosynthesis at the level of transglycosylation (46, 47). Additionally, lacticin 3147 shows a dual mode of action and is able to form lipid II-dependent pores (28, 47). The mode of action of lacticin 481 so far has not been characterized in sufficient detail.We found that Ca²⁺ increases the antimicrobial activity of all peptides containing the mersacidin-lipid II binding motif, except for lacticin 481, however, which was also found to bind to lipid II.     

NAD+ levels control Ca2+ store replenishment and mitogen-induced increase of cytosolic Ca2+ by Cyclic ADP-ribose-dependent TRPM2 channel gating in human T lymphocytes

Intracellular NAD(+) levels ([NAD(+)](i)) are important in regulating human T lymphocyte survival, cytokine secretion, and the capacity to respond to antigenic stimuli. NAD(+)-derived Ca(2+)-mobilizing second messengers, produced by CD38, play a pivotal role in T cell activation. Here we demonstrate that [NAD(+)](i) modifications in T lymphocytes affect intracellular Ca(2+) homeostasis both in terms of mitogen-induced [Ca(2+)](i) increase and of endoplasmic reticulum Ca(2+) store replenishment. Lowering [NAD(+)](i) by FK866-mediated nicotinamide phosphoribosyltransferase inhibition decreased the mitogen-induced [Ca(2+)](i) rise in Jurkat cells and in activated T lymphocytes. Accordingly, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores was greatly reduced in these cells in the presence of FK866. When NAD(+) levels were increased by supplementing peripheral blood lymphocytes with the NAD(+) precursors nicotinamide, nicotinic acid, or nicotinamide mononucleotide, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores as well as cell responsiveness to mitogens in terms of [Ca(2+)](i) elevation were up-regulated. The use of specific siRNA showed that the changes of Ca(2+) homeostasis induced by NAD(+) precursors are mediated by CD38 and the consequent ADPR-mediated TRPM2 gating. Finally, the presence of NAD(+) precursors up-regulated important T cell functions, such as proliferation and IL-2 release in response to mitogens.     

Highly Ca2+-selective TRPM channels regulate IP3-dependent oscillatory Ca2+ signaling in the C. elegans intestine

Posterior body wall muscle contraction (pBoc) in the nematode Caenorhabditis elegans occurs rhythmically every 45-50 s and mediates defecation. pBoc is controlled by inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in the intestine. The intestinal epithelium can be studied by patch clamp electrophysiology, Ca2+ imaging, genome-wide reverse genetic analysis, forward genetics, and molecular biology and thus provides a powerful model to develop an integrated systems level understanding of a nonexcitable cell oscillatory Ca2+ signaling pathway. Intestinal cells express an outwardly rectifying Ca2+ (ORCa) current with biophysical properties resembling those of TRPM channels. Two TRPM homologues, GON-2 and GTL-1, are expressed in the intestine. Using deletion and severe loss-of-function alleles of the gtl-1 and gon-2 genes, we demonstrate here that GON-2 and GTL-1 are both required for maintaining rhythmic pBoc and intestinal Ca2+ oscillations. Loss of GTL-l and GON-2 function inhibits I(ORCa) approximately 70% and approximately 90%, respectively. I(ORCa) is undetectable in gon-2;gtl-1 double mutant cells. These results demonstrate that (a) both gon-2 and gtl-1 are required for ORCa channel function, and (b) GON-2 and GTL-1 can function independently as ion channels, but that their functions in mediating I(ORCa) are interdependent. I(ORCa), I(GON-2), and I(GTL-1) have nearly identical biophysical properties. Importantly, all three channels are at least 60-fold more permeable to Ca2+ than Na+. Epistasis analysis suggests that GON-2 and GTL-1 function in the IP3 signaling pathway to regulate intestinal Ca2+ oscillations. We postulate that GON-2 and GTL-1 form heteromeric ORCa channels that mediate selective Ca2+ influx and function to regulate IP3 receptor activity and possibly to refill ER Ca2+ stores.     

Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice

Non-selective cation (NSC) channels activated by intracellular Ca2+ ([Ca2+]i) play an important role in Ca2+ signaling and membrane excitability in many cell types. TRPM4 and TRPM5, two Ca2+-activated cation channels of the TRP superfamily, are potential molecular correlates of NSC channels. We compared the functional properties of mouse TRPM4 and TRPM5 heterologously expressed in HEK 293 cells. Dialyzing cells with different Ca2+ concentrations revealed a difference in Ca2+ sensitivity between TRPM4 and TRPM5, with EC50 values of 20.2+/-4.0 microM and 0.70+/-0.1 microM, respectively. Similarly, TRPM5 activated at lower Ca2+ concentration than TRPM4 when [Ca2+]i was raised by UV uncaging of the Ca2+-cage DMNP-EDTA. Current amplitudes of TRPM4 and TRPM5 were not correlated to the rate of changes in [Ca2+]i. The Ca2+ sensitivity of both channels was strongly reduced in inside-out patches, resulting in approximately 10-30 times higher EC50 values than under whole-cell conditions. Currents through TRPM4 and TRPM5 deactivated at negative and activated at positive potentials with similar kinetics. Both channels were equally sensitive to block by intracellular spermine. TRPM4 displayed a 10-fold higher affinity for block by flufenamic acid. Importantly, ATP4- blocked TRPM4 with high affinity (IC50 of 0.8+/-0.1 microM), whereas TRPM5 is insensitive to ATP4- at concentrations up to 1 mM.     

Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4

TRPM4, a Ca(2+)-activated cation channel of the transient receptor potential superfamily, undergoes a fast desensitization to Ca(2+). The mechanisms underlying the alterations in Ca(2+) sensitivity are unknown. Here we show that cytoplasmic ATP reversed Ca(2+) sensitivity after desensitization, whereas mutations to putative ATP binding sites resulted in faster and more complete desensitization. Phorbol ester-induced activation of protein kinase C (PKC) increased the Ca(2+) sensitivity of wild-type TRPM4 but not of two mutants mutated at putative PKC phosphorylation sites. Overexpression of a calmodulin mutant unable to bind Ca(2+) dramatically reduced TRPM4 activation. We identified five Ca(2+)-calmodulin binding sites in TRPM4 and showed that deletion of any of the three C-terminal sites strongly impaired current activation by reducing Ca(2+) sensitivity and shifting the voltage dependence of activation to very positive potentials. Thus, the Ca(2+) sensitivity of TRPM4 is regulated by ATP, PKC-dependent phosphorylation, and calmodulin binding at the C terminus.     

Crystal Structures of Progressive Ca2+ Binding States of the Ca2+ Sensor Ca2+ Binding Domain 1 (CBD1) from the CALX Na+/Ca2+ Exchanger Reveal Incremental Conformational Transitions

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.     

T-type Ca2+ Channels Promote Oxygenation-induced Closure of the Rat Ductus Arteriosus Not Only by Vasoconstriction but Also by Neointima Formation

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca²⁺ influx through several membrane Ca²⁺ channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca²⁺ channel (VDCC) in DA closure remains unclear. Here we found that the expression of α1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that α1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by α1G overexpression. Moreover, it was decreased by adding α1G-specific small interfering RNAs or using R(−)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca²⁺ concentration of DA SMCs was significantly decreased by adding α1G-specific siRNAs or using R(−)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(−)-efonidipine (10⁻⁶ m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(−)-efonidipine (10⁻⁶ m) significantly attenuated oxygenation-induced vasoconstriction by ∼27% using a vascular ring of fetal DA at term. Finally, R(−)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially α1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.The ductus arteriosus (DA)² is an essential vascular shunt between the aortic arch and the pulmonary trunk during a fetal period (1). After birth, the DA closes immediately in accordance with its smooth muscle contraction and vascular remodeling, whereas the connecting vessels such as the aorta and pulmonary arteries remain open. When the DA fails to close after birth, the condition is known as patent DA, which is a common form of congenital heart defect. Patent DA is also a frequent problem with significant morbidity and mortality in premature infants. Investigating the molecular mechanism of DA closure is important not only for vascular biology but also for clinical problems in pediatrics.Voltage-dependent Ca²⁺ channels (VDCCs) consist of multiple subtypes, named L-, N-, P/Q-, R-, and T-type. L-type VDCCs are known to play a primary role in regulating Ca²⁺ influx and thus vascular tone in the development of arterial smooth muscle including the DA (2–4). Our previous study demonstrated that all T-type VDCCs were expressed in the rat DA (5). α1G subunit, especially, was the most dominant isoform among T-type VDCCs. The abundant expression of α1G subunit suggests that it plays a role in the vasoconstriction and vascular remodeling of the DA. In this regard, Nakanishi et al. (6) demonstrated that 0.5 mm nickel, which blocks T-type VDCC, inhibited oxygen-induced vasoconstriction of the rabbit DA. On the other hand, Tristani-Firouzi et al. (7) demonstrated that T-type VDCCs exhibited little effect on oxygen-sensitive vasoconstriction of the rabbit DA. Thus, the role of T-type VDCCs in DA vasoconstriction has remained controversial.In addition to their role in determining the contractile state, a growing body of evidence has demonstrated that T-type VDCCs play an important role in regulating differentiation (8, 9), proliferation (10–12), migration (13, 14), and gene expression (15) in vascular smooth muscle cells (SMCs). Hollenbeck et al. (16) and Patel et al. (17) demonstrated that nickel inhibited platelet-derived growth factor-BB-induced SMC migration. Rodman et al. (18) demonstrated that α1G promoted SMC proliferation in the pulmonary artery. The DA dramatically changes its morphology during development. Intimal cushion formation, a characteristic feature of vascular remodeling of the DA (19–21), involves many cellular processes: an increase in SMC migration and proliferation, production of hyaluronic acid under the endothelial layer, impaired elastin fiber assembly, and so on (1, 19, 21–23). Although our previous study demonstrated that T-type VDCCs are involved in smooth muscle cell proliferation in the DA (5), the role of T-type VDCCs in vascular remodeling of the DA has remained poorly understood.In the present study, we hypothesized that T-type VDCCs, especially α1G subunit, associate with vascular remodeling and vasoconstriction in the DA. To test our hypothesis, we took full advantage of recent molecular and pharmacological developments. We chose the recently developed, highly selective T-type VDCC blocker R(−)-efonidipine instead of low dose nickel for our study. Selective inhibition or activation of α1G subunit was also obtained using small interfering RNA (siRNA) technology or by overexpression of the α1G subunit gene, respectively. We found that Ca²⁺ influx through T-type VDCCs promoted oxygenation-induced DA closure through SMC migration and vasoconstriction.     

Modulation of Ca2+ entry and plasma membrane potential by human TRPM4b

TRPM4b is a Ca(2+)-activated, voltage-dependent monovalent cation channel that has been shown to act as a negative regulator of Ca(2+) entry and to be involved in the generation of oscillations of Ca(2+) influx in Jurkat T-lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca(2+)-dependent inward and outward currents in whole cell patch clamp recordings. HEK-293 cells stably overexpressing TRPM4b showed higher ionomycin-activated Ca(2+) influx than wild-type cells. In addition, analysis of the membrane potential using the potentiometric dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca(2+) entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca(2+) influx. In conclusion, in cells with a small negative membrane potential, such as HEK-293 cells, TRPM4b acts as a positive regulator of Ca(2+) entry.     

Cholesterol depletion affects the Ca2+ influx but not the Ca2+ pump in human erythrocytes

Control and cholesterol-depleted human erythrocytes were loaded with permeant Ca2+ chelators (Benz2-AM or Quin2-AM) in order to increase their exchangeable Ca2+ pool and to measure both Ca2+ fluxes and [Ca]i (free cytoplasmic calcium concentration). The fluxes were independent of the concentration and of the nature of the intracellular chelator. The ATP content was not decreased by more than 50% under our experimental conditions. Cholesterol depletion (up to 28%) induced a decrease in both Ca2+ fluxes and [Ca]i which was proportional to the extent of the depletion. It is shown that cholesterol depletion primarily altered the properties of the system responsible for Ca2+ entry causing a diminution of the [Ca]i. This, in turn, induced a diminution of the activity of the Ca2+ pump without affecting the properties of this pump.     

Large Conductance Ca2+-activated K+ Channels in the Soma of Rat Motoneurones

Properties of large conductance Ca²⁺-activated K⁺ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K⁺ _o //155 mm K⁺ _i ) and 231 ± 4 pS in external high-K _o solution (155 mm K⁺ _o //155 mm K⁺ _i ). The channels were activated by depolarization and by an increase in internal Ca²⁺ concentration. Potentials of half-maximum channel activation (E₅₀) were −13, −34, −64 and −85 mV in the presence of 10⁻⁶, 10⁻⁵, 10⁻⁴ and 10⁻³ m internal Ca²⁺, respectively. Using an internal solution containing 10⁻⁴ m Ca²⁺, averaged K_Ca currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged K_Ca currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC₅₀) of 0.17 ± 0.02 mm. K_Ca channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that K_Ca channels activated by Ca²⁺ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997