Search PubMed⌕ Search

Biomedical subjects

X Wei

Publications and source records attributed to X Wei.

At least 181 records · Page 10Linked to original sources
PubMed1994

Regulation of the cloned L-type cardiac calcium channel by cyclic-AMP-dependent protein kinase.

Hormones can regulate cardiac L-type Ca2+ channels via cAMP-dependent protein kinase (PKA) phosphorylation. However, regulation of the cloned L-type Ca2+ channel has been difficult to demonstrate conclusively. We stably transfected a human embryonic kidney (HEK-293) cell with the cardiac alpha 1 and beta 2 subunits, then examined PKA modulation of the Ca2+ current. Although forskolin did not increase basal Ca2+ current, the PKA inhibitors, H-89 and Rp-cAMPS, could inhibit basal current. We reversed H-89 inhibition with either forskolin or okadaic acid. We conclude that the channel was phosphorylated under basal conditions, and that inhibition of PKA allowed dephosphorylation. These studies demonstrate that reversible PKA regulation of cloned Ca2+ channels can be studied in HEK-293 cells.

Animals↗
PubMed1994

Modification of Ca2+ channel activity by deletions at the carboxyl terminus of the cardiac alpha 1 subunit.

Voltage-sensitive Ca2+ channels are multisubunit complexes that include, among others, a large alpha 1 subunit, which by itself is sufficient to form a channel. Several alpha 1 genes encoding L-, N-, and P-type Ca2+ channels have been cloned. These alpha 1 genes share a high degree of sequence homology in the putative transmembrane regions, but vary substantially in the putative intracellular loops and the flanking amino and carboxyl termini. In the present study, we investigated the functional roles of the 665-amino acid long carboxyl terminus of a cardiac alpha 1 by constructing deletion mutants. Expression in Xenopus oocytes of delta C1856, delta C1733, and delta C1700, which lack from 307 to 472 amino acids at the carboxyl terminus, led to inward Ba2+ currents that were 4- to 6-fold greater than observed with the 2171-amino acid long wild type alpha 1. Ionic currents increased without a change in the amount of charge moved during voltage-dependent gating, suggesting that the increase in ionic currents was not due to an increase in the number of channels that were expressed. Single channel analysis revealed an unaltered unitary conductance. Thus, removal of up to 70% of the carboxyl terminus increased current density by facilitating the coupling between the voltage-dependent gating and channel opening, leading to an increased opening probability of the channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

The amino terminus of a calcium channel beta subunit sets rates of channel inactivation independently of the subunit's effect on activation.

There is molecular diversity in both alpha 1 and beta subunits of voltage-gated Ca2+ channels. Coupling between voltage sensing and pore opening of the C-type alpha 1 (alpha 1c) is improved by the type 2 beta subunit (beta 2), and E-type alpha 1 beta complexes inactivate at different rates depending on the nature of beta. We compared the effects of type 1 and 2 beta subunits on activation of the human E-type alpha 1 (alpha 1E) with the effects they have on inactivation, as seen in Xenopus oocytes. The beta subtypes stimulated activation in similar fashion but affected inactivation differently, and even in opposing directions. beta subunits have a common central core but differ in their N- and C-termini and in a central region. N-terminal chimeras between beta 1 and beta 2 subunits that have opposing effects on inactivation resulted in the reciprocal transfer of their effects. We conclude that regulation of activation and inactivation of alpha 1 by beta are separable events and that the N-terminus of beta is one of the structural determinants important in setting the rate and voltage at which an alpha 1 inactivates.

Amino Acid Sequence↗
PubMed1994

T-type and N-type calcium channels of Xenopus oocytes: evidence for specific interactions with beta subunits.

We used amplifying effects of calcium channel beta subunits to identify endogenous calcium channels in Xenopus oocytes. Expression of rat brain beta 4 increased macroscopic endogenous current magnitude with a small effect on kinetics. In contrast, expression of rat brain/cardiac beta 2 produced a much larger increase in current magnitude and dramatically slowed current decay. Low concentrations of omega-conotoxin GVIA irreversibly blocked currents in both uninjected and beta 2-injected oocytes. Single channel recordings revealed both T- and N-type calcium channels with conductances of 9 and 18 pS, respectively, in uninjected oocytes and in oocytes expressing either beta subunit. Expression of either beta subunit slowed average current decay of T-type single channels. Slowing of T-type current decay by expression of beta 2 was due to reopening of the channels. N-type single channel average current decay showed little change with expression of beta 4, whereas expression of beta 2 slowed average current decay.

Animals↗
PubMed1994

Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field.

Alternative Splicing↗
PubMed1994

Molecular analysis and functional expression of the human type E neuronal Ca2+ channel alpha 1 subunit.

A human brain alpha 1 Ca2+ channel subunit was cloned and expressed in Xenopus laevis oocytes. The open reading frame, encoding 2,312 amino acids, has high homology to the marine ray doe-1, the rat E-type, and the rabbit brain BII alpha 1 subunits. The amino and carboxy termini of this human.E-type alpha 1 subunit (alpha 1E) are most similar to the rabbit BII-1 splice variant, the remainder being colinear with the BII alpha 1 with the exception of two insertions, one of 43 amino acids in the C-terminus and another of 7 amino acids, found also in the rat alpha 1E, between domains II and III. Two potential Ca2+ binding sites are predicted from its primary structure. The expression of inward Ba2+ currents reveals voltage-dependent activation and inactivation measured by the cut-open oocyte vaseline-gap technique, with kinetics that correspond to that of a high-voltage-activated neuronal Ca2+ channel, and pharmacologic properties that resemble those of some low-voltage-activated neuronal Ca2+ currents. The human alpha 1E currents are insensitive to omega-conotoxin-GVIA (1 microM), omega-agatoxin-IVA (200 nM), a synthetic funnel web spider toxin (FTX, 20 microM), and Bay-K8644 (0.5 microM); they are inhibited 20% by high concentrations of methoxyverapamil and diltiazem, 65% by 0.1% crude funnel web spider venom and 100% by Ni2+ (IC50 = 30 nM). Single-channel records show a complex activity pattern with several apparent conductance states, the largest having a conductance of 14 pS.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Pathogenesis of craniofacial and body wall malformations induced by ochratoxin A in mice.

Ochratoxin A (OA), a mycotoxin commonly found in soils and on moldy food such as cereal grains, is a potent teratogen. The present investigation was designed to examine the teratogenicity of OA administered acutely at early post-implantation stages in mice, with particular emphasis on the pathogenetic basis of induced malformations. Maternal OA administration on gestational day (GD) 7 or 8 resulted in excessive amounts of cell death in selected cell populations. After a single dose of 2-4 mg/kg, excessive cell death was notable within 6 hours, and persisted to 36 hours post-treatment. As observed in GD 14 or 18 fetuses, the spectrum of induced craniofacial malformations included exencephaly, midfacial clefting, cleft lip, as well as hypotelorism, and synophthalmia associated with holoprosencephaly. Body wall defects involved either the abdominal wall alone, or in combination with the thoracic wall, resulting in partial or complete exposure of the viscera. Potential mechanisms for OA-induced selective cell killing are discussed.

Abnormalities, Drug-Induced↗
PubMed1993

Potentiation by the beta subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel.

The voltage-activated rabbit cardiac calcium channel alpha 1 subunit was expressed in Xenopus oocytes. The charge movement of its voltage sensor was measured and related to the opening of the ion-conducting pore. The half-activation potential for charge movement was 35 millivolts more negative than that for pore opening. Coexpression of the cardiac calcium channel beta subunit reduced this difference without affecting charge movement. Thus, intramolecular coupling between the voltage sensor and the channel pore opening can be facilitated by a regulatory subunit.

Animals↗
PubMed1993

Cloning and expression of a neuronal calcium channel beta subunit.

Although pharmacological and electrophysiological studies have demonstrated the existence of multiple types of voltage-dependent calcium channels in neuronal tissue, the subunit composition of these channels is not well known. Here, we report the cloning and expression of a new rat brain beta subunit (beta 4). Northern blot analysis indicates that beta 4 mRNA is expressed almost exclusively in neuronal tissues, with the highest levels being found in the cerebellum. Coexpression studies indicate that rat beta 4 can interact with rabbit cardiac muscle alpha 1, rabbit skeletal muscle alpha 1, and calcium channels endogenous to Xenopus oocytes. beta 4 modulation of alpha 1 activity is similar to the modulation induced by beta 1, beta 2, or beta 3. The most striking effect of beta subunits is their ability to increase functional alpha 1 activity, which can be measured as either increased dihydropyridine binding to membranes from transfected COS cells or increased calcium channel activity in Xenopus oocytes.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Cloning and expression of a third calcium channel beta subunit.

The skeletal muscle dihydropyridine receptor/Ca2+ channel consists of five distinct subunits (alpha 1, alpha 2 delta, beta 1, and gamma). Homologous alpha 1, alpha 2 delta, and beta 2 subunits are expressed in heart and brain. The present study reports the cloning and expression of a third beta subunit, beta 3, which is expressed predominantly in brain. Its open reading frame encodes a protein with 484 amino acids with a predicted molecular mass of 54,571 Da. Coexpression of beta 3 with a cardiac alpha 1 in Xenopus oocytes induces similar changes in Ca2+ channel activity as beta 1 and beta 2, that is, it increases peak currents, modulates the voltage dependence of activation, and accelerates activation. In addition, beta 3 accelerates the rate of inactivation at positive test potentials.

Amino Acid Sequence↗
PubMed1993

Application of cDNA selection techniques to regions of the human MHC.

Identification of transcribed sequences by cDNA selection is a potentially rapid and efficient way of scanning large genomic DNA fragments for the presence of genes. To evaluate this approach further, we have applied it to three yeast artificial chromosomes (YACs) and examined the products obtained from a total of about 1100 kb from two regions of the human major histocompatibility complex (MHC). One YAC was derived from an extensively studied portion of the Class II region of the MHC. The cDNAs recovered from this YAC included representatives of the previously described genes as well as one or more cDNA clones not described in the databases. A second YAC spanned about 330 kb of DNA surrounding the Class I gene HLA-A. In addition to Class I clones, 10 distinct cDNA products were identified from this YAC. A third YAC contained about 700 kb of human DNA, including 260 kb of overlap with the second YAC, and recovered an additional cDNA complementary to YAC B30 H3 DNA. Overall, the method is shown to be able to detect very scarce cDNAs and to detect a large fraction of coding sequences in YAC clones. Advantages and limitations of the approach are discussed.

Chromosome Mapping↗
PubMed1993

The effect of tocainide on theophylline metabolism.

The effect of 5 days of oral tocainide (400 mg every 8 h) on the kinetics of theophylline given as a single 5 mg kg-1 i.v. infusion over 30 min was investigated in eight healthy male nonsmokers. Treatment with tocainide decreased the plasma clearance of theophylline from 37.5 +/- 6.9 (mean +/- s.d.) to 33.7 +/- 5.0 ml kg-1 h-1 (difference -3.8, 95% CI, -1.7 to -5.9; P = 0.004) and increased its terminal elimination half-life from 9.7 +/- 2.5 to 10.4 +/- 2.1 h (difference 0.7, 95% CI, 0.2 to 1.2; P = 0.011). Tocainide decreased the formation clearances of 3-methylxanthine and 1-methyluric acid, but the formation clearance of 1,3-dimethyluric acid was unaltered. These data indicate that tocainide exerts a modest inhibitory effect on theophylline metabolism. The magnitude of this change is substantially smaller than that reported to be produced by mexiletine.

Adult↗
PubMed1993

The locomotion, shape and pseudopodial dynamics of unstimulated Dictyostelium cells are not random.

The dynamic periphery of unstimulated, preaggregation, hunger-stage Dictyostelium discoideum amoebae was investigated by time-lapse videomicroscopy and digital image processing. Circular maps (i.e. of each of 360 radii around the cell transformed upon Cartesian coordinates) were constructed around the centroid of individual cell images and analysed in time series. This novel technique generated spatiotemporal structures of various degrees of order in the maps, which resemble classical wave interference patterns. The patterns thus demonstrate that cell movement is not random and that cells are intrinsically vibrating bodies, transited by self-organized, superpositioned, harmonic modes of rotating oscillatory waves (ROWS). These waves appear to depend upon spatiotemporal oscillations in the physicochemical reactions associated with actin polymerization, and they govern pseudopodial movements, cell shape and locomotion generally. ROWS in this case are unrelated to the cyclic-AMP-regulated oscillations, which characterize later, aggregative populations of Dictyostelium. However, the exposure of aggregation-stage cells to a pulse of the chemoattractant cyclic-AMP induces a characteristic sequence of changes in the global cellular concentration and spatiotemporal distribution of fibrillar (F-)actin. This reaction begins with what appears to be a phase resetting of ROWS and it may, therefore, underlie the cellular perception of and response to chemotactic signals. We also develop here an analytical mathematical description of ROWS, and use it to simulate cell movements accurately.

Actins↗