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N Dascal

Publications and source records attributed to N Dascal.

At least 37 records · Page 2Linked to original sources
PubMed1996

A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel alpha 1C subunit.

The well-characterized enhancement of the cardiac Ca2+ L-type current by protein kinase A (PKA) is not observed when the corresponding channel is expressed in Xenopus oocytes, possibly because it is fully phosphorylated in the basal state. However, the activity of the expressed channel is reduced by PKA inhibitors. Using this paradigm as an assay to search for PKA sites relevant to channel modulation, we have found that mutation of serine 1928 of the alpha 1C subunit to alanine abolishes the modulation of the expressed channel by PKA inhibitors. This effect was independent of the presence of the beta subunit. Phosphorylation of serine 1928 of alpha 1C may mediate the modulatory effect of PKA on the cardiac voltage-dependent ca2+ channel.

Animals↗
PubMed1996

Serotonin and protein kinase C modulation of a rat brain inwardly rectifying K+ channel expressed in xenopus oocytes.

In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, perfusion with a high-K+ solution (96 mM KCl) generated an inward current (IHK) which was absent in water-injected oocytes. Part of IHK was blocked by low concentrations of Ba2+ (half-maximal inhibitory concentration, IC50: 4.2 +/- 0.5 microM). When serotonin (5-HT) was applied to these oocytes a transient inward oscillating Cl- current arising from activation of Ca2+ -dependent Cl- channels, ICl (Ca), was observed. When this response decayed, a 30% reduction of IHK could be detected. Electrophysiological characterization of the K+ channel down-modulated by 5-HT revealed that it is an inward rectifier. Anti-sense suppression experiments revealed that the 5-HT2C receptor mediates the down-modulatory effect of 5-HT. The nature of the modulatory pathway was investigated by application of phorbol esters and intracellular injection of protein kinase C (PKC) inhibitors, ethylenebis (oxonitrilo)tetraacetate (EGTA) and inositol 1,4,5-trisphosphate. The results demonstrate that PKC is responsible for the down-modulatory effect.

Animals↗
PubMed1995

Ca2+ current enhancement by alpha 2/delta and beta subunits in Xenopus oocytes: contribution of changes in channel gating and alpha 1 protein level.

1. A combined biochemical and electrophysiological approach was used to determine the mechanism by which the auxiliary subunits of Ca2+ channel enhance the macroscopic Ca2+ currents. Xenopus oocytes were injected with RNA of the main pore-forming subunit (cardiac: alpha 1C), and various combinations of RNAs of the auxiliary subunits (alpha 2/delta and beta 2A). 2. The single channel open probability (Po; measured at 0 mV) was increased approximately 3-, approximately 8- and approximately 35-fold by alpha 2/delta, beta 2A and alpha 2/delta+beta 2A, respectively. The whole-cell Ca2+ channel current was increased approximately 8- to 10-fold by either alpha 2/delta or beta 2A, and synergistically > 100-fold by alpha 2/delta+beta 2A. The amount of 35S-labelled alpha 1 protein in the plasma membrane was not changed by coexpression of beta 2A, but was tripled by coexpression of alpha 2/delta (either with or without beta). 3. We conclude that the increase in macroscopic current by alpha 2/delta is equally due to changes in amount of alpha 1 in the plasma membrane and an increase in Po, whereas all of the effect of beta 2A is due to an increase in Po. The synergy between alpha 2/delta and beta in increasing the macroscopic current is due mainly to synergistic changes in channel gating.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Amino Acid Sequence↗
PubMed1995

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes.

In many tissues, inwardly rectifying K channels are coupled to seven-helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.

Animals↗
PubMed1994

Phosphorylation by protein kinase A of RCK1 K+ channels expressed in Xenopus oocytes.

Phosphorylation-mediated regulation of voltage-gated K+ channels has been implicated in numerous electrophysiological studies; however, complementary biochemical studies have so far been hampered by the failure to isolate and characterize any K+ channel proteins of distinct molecular identity. We used the Xenopus oocyte expression system to study the biosynthesis and phosphorylation by protein kinase A (PKA) of rat brain RCK1 (Kv1.1) K+ channel protein. RCK1 protein was isolated by immunoprecipitation from oocytes injected with RCK1 cRNA and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The channel protein was expressed in the form of several polypeptides. The 57-kDa polypeptide, usually the major constituent, resided both in the cytosol and in the plasma membrane. Its levels were correlated with RCK1 current amplitudes (IRCK1) and upon incubation of the cRNA-injected oocytes with tunicamycin, its molecular weight was decreased and at the same time IRCK1 was reduced. These results suggest that the membranal 57-kDa polypeptides represent functional channels that are N-glycosylated. Furthermore, a study of the phosphorylation of the RCK1 polypeptides revealed that the 57-kDa polypeptide was specifically targeted for phosphorylation by PKA. It could be phosphorylated in vitro by the catalytic subunit of PKA (PKA-CS). In its native state in intact oocytes, the 57-kDa polypeptide was partially phosphorylated and could be further phosphorylated in vivo by addition of a membrane-permeant cAMP analog. Site-directed mutagenesis demonstrated that phosphorylation of a single site on the C-terminus of the channel molecule fully accounts for these phosphorylations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Distribution and localization of a G protein-coupled inwardly rectifying K+ channel in the rat.

The cellular distribution of the mRNA of the inwardly rectifying K+ channel KGA (GIRK1) was investigated in rat tissue by in situ hybridization. KGA was originally cloned from the heart and represents the first G protein-activated K+ channel identified. It is expressed in peripheral tissue solely in the atrium, but not in the ventricle, skeletal muscle, lung and kidney. In the central nervous system KGA is most prominently expressed in the Ammon's horn and dentate gyrus of the hippocampus, neocortical layers II-VI, cerebellar granular layer, olfactory bulb, anterior pituitary, thalamic nuclei and several distinct nuclei of the lower brainstem. The abundant expression of KGA in many CNS neurons supports its important role as a major target channel for G protein mediated receptor function.

Animals↗
PubMed1994

Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes.

Two-electrode voltage clamping of expressed ion channels in intact oocytes of the South African clawed frog Xenopus laevis has been refined to allow stable, low-resistance electrical access to the cytosol (50-800 k omega). Glass microelectrodes were filled with a cushion of 1% agarose at their tips to prevent KCl leakage (agarose-cushion electrodes). Insertion of these electrodes into X. laevis oocytes yielded stable preparations for periods of more than 1 h with a stable input resistance of 1-4 M omega. Furthermore, a simple modification of the voltage-clamp circuit (charging compensator) is described that increases the flexibility of arrangements for differential recording of the membrane potential in order to subtract voltage drops across a series resistance. The result is a considerable increase in the practically attainable speed of the voltage clamp with the conventional two-electrode arrangement. The performance of the charging compensator was tested on an equivalent circuit that simulates the oocyte and electrodes. In addition, the combination of agarose-cushion electrodes and the charging compensator was tested on oocytes expressing Shaker H4 currents. The fidelity of the voltage-clamp circuit was also verified by measuring the membrane potential with additional independent microelectrodes connected to a differential amplifier, independent of the two-electrode voltage clamp system. The system described here will be useful for ion channel studies in X. laevis oocytes requiring long-term recordings and/or measurements of large, fast ion currents.

Animals↗
PubMed1994

Beta-adrenergic modulation of currents produced by rat cardiac Na+ channels expressed in Xenopus laevis oocytes.

In Xenopus oocytes coexpressing beta 2-adrenergic receptors and the rat cardiac alpha SkM2 Na+ channel, superfusion with 10 microM isoproterenol led to modest (approximately 30%) increases in peak Na+ inward current. Intracellular injection of cAMP and of protein kinase A (PKA) catalytic subunit reproduced this increase, showing that the second messenger pathway involves PKA dependent phosphorylation. Coexpression of the Na+ channel beta 1 subunit had no influence on the modulation. The modulation had little or no effect upon Na+ current waveforms, steady-state activation, steady-state activation, steady-state inactivation, or recovery from both fast and slow inactivation; but maximum Na+ conductance was increased. Mutation of the five major consensus PKA phosphorylation sites on alpha SkM2 did not abolish the observed effect. In parallel experiments, beta-adrenergic stimulation of the neuronal alpha IIA Na+ channel subunit led to an attenuation of Na+ current. It is concluded that (i) the alpha SkM2 subunit might be directly phosphorylated by PKA, but at serine/threonine residue(s) in a cryptic phosphorylation site(s); or that (ii) the modulation might also be mediated by phosphorylation of another, as yet unknown protein(s). The divergent modulation of neuronal and cardiac Na+ channel alpha-subunits suggests that differential physiological modulation by identical second messenger pathways is the evolutionary basis for the isoform diversity within this protein family.

Animals↗
PubMed1994

Cardiac calcium channels expressed in Xenopus oocytes are modulated by dephosphorylation but not by cAMP-dependent phosphorylation.

Enhancement of cardiac L-type Ca2+ channel activity by norepinephrine via phosphorylation by protein kinase A (PKA) underlines the positive inotropic effect of this transmitter and is a classical example of an ion channel modulation. However, it is not clear whether the channel protein itself (and which subunit) is a substrate for PKA. We have expressed various combinations of the cardiac Ca2+ channel subunits in Xenopus oocytes by injecting subunit mR-NAs. Expression of beta or alpha 2/delta + beta subunits potentiated the native (endogenous) Ca2+ channel currents in the oocyte (similar to T or N but not L-type). This potentiated endogenous current was enhanced by intracellular injection of cAMP or of the catalytic subunit of PKA, and this effect was reversed by the injection of a PKA inhibitor suggesting the presence of basal phosphatase activity. When a cardiac channel of alpha 1 + beta, alpha 1 + alpha 2/delta or alpha 1 + alpha 2/delta + beta composition was expressed at levels high enough that the contribution of the endogenous current became negligible, cAMP and PKA failed to increase the Ca2+ channel current, whereas PKA inhibitors and the catalytic subunit of protein phosphatase 1 reduced the amplitude of the current. Reduction of the current by PKA inhibitors was observed regardless of the presence of the beta subunit, suggesting a major role for the alpha 1 subunit in this process. These results suggest that, like in the heart, when expressed in Xenopus oocytes, the cardiac L-type Ca2+ channels are phosphorylated in basal state and dephosphorylation reduces their activity. However, unlike the situation in the heart, the activity of the channel cannot be enhanced by PKA-catalyzed phosphorylation, suggesting that the channel is already fully phosphorylated in its basal state.

Animals↗
PubMed1993

Atrial G protein-activated K+ channel: expression cloning and molecular properties.

Activity of several ion channels is controlled by heterotrimeric GTP-binding proteins (G proteins) via a membrane-delimited pathway that does not involve cytoplasmic intermediates. The best studied example is the K+ channel activated by muscarinic agonists in the atrium, which plays a crucial role in regulating the heartbeat. To enable studies of the molecular mechanisms of activation, this channel, denoted KGA, was cloned from a rat atrium cDNA library by functional coupling to coexpressed serotonin type 1A receptors in Xenopus oocytes. KGA displays regions of sequence homology to other inwardly rectifying channels as well as unique regions that may govern G-protein interaction. The expressed KGA channel is activated by serotonin 1A, muscarinic m2, and delta-opioid receptors via G proteins. KGA is activated by guanosine 5'-[gamma-thio]triphosphate in excised patches, confirming activation by a membrane-delimited pathway, and displays a conductance equal to that of the endogenous channel in atrial cells. The hypothesis that similar channels play a role in neuronal inhibition is supported by the cloning of a nearly identical channel (KGB1) from a rat brain cDNA library.

Amino Acid Sequence↗
PubMed1993

Primary structure and functional expression of a cyclic nucleotide-gated channel from rabbit aorta.

Sequences specific for cyclic nucleotide-gated channels (CNG channels) have been amplified by PCR from cDNA of heart, aorta, sino-atrial node, cerebellum, C-cells and kidney. The complete amino acid sequence of a CNG channel from rabbit aorta has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CNG channel in Xenopus oocytes.

Amino Acid Sequence↗
PubMed1993

Expression of an atrial G-protein-activated potassium channel in Xenopus oocytes.

Injection of rat atrial RNA into Xenopus oocytes resulted in the expression of guanine nucleotide binding (G) protein-activated K+ channel. Current through the channel could be activated by acetylcholine or, if RNA encoding a neuronal 5HT1A receptor was coinjected with atrial RNA, by serotonin (5HT). A 5HT-evoked current (I5HT) was observed in oocytes injected with ventricle RNA fractions (of 2.5-5.5 kb) and 5HT1A receptor RNA. I5HT displayed strong inward rectification with very little conductance above the K+ equilibrium potential, was highly selective for K+ over Na+, and was blocked by 5-300 microM Ba2+. I5HT was suppressed by intracellular injection of the nonhydrolyzable analog of GDP, guanosine 5'-[beta-thio]diphosphate, but not by treatment with pertussis toxin (PTX), suggesting coupling of the receptor to the G-protein-activated K+ channel via a PTX-insensitive G protein, possibly endogenously present in the oocyte. Coexpression of the alpha subunit of a PTX-sensitive G protein, G(i2), rendered I5HT sensitive to PTX inhibition. Native oocytes displayed a constitutively active inwardly rectifying K+ current with a lower sensitivity to Ba2+ block; expression of a similar current was also directed by atrial or ventricle RNA of 1.5-3 kb. Xenopus oocytes may be employed for cloning of the G-protein-activated K+ channel cDNA and for studying the coupling between this channel and G proteins.

Acetylcholine↗
PubMed1992

Evidence for the existence of RNA of Ca(2+)-channel alpha 2/delta subunit in Xenopus oocytes.

Ba(2+)-currents (IBa) through voltage-dependent Ca(2+)-channels were studied in Xenopus oocytes injected with RNA from several excitable tissues, using the two-electrode voltage-clamp technique. Previous studies have shown that the expression of cardiac Ca(2+)-channels can be suppressed by an hybrid-arrest procedure that includes co-injection of the tissue-derived RNA with an 'antisense' oligonucleotide complementary to a part of RNA coding for the Ca(2+)-channel alpha 1 subunit. In this study, this method was used to investigate the role of the alpha 2/delta subunit. Co-injection of RNA extracted from either rabbit heart, rat brain or rat skeletal muscle (SkM) with 'antisense' oligonucleotides complementary to the alpha 2/delta subunit RNA did not substantially affect the expression of IBa in the oocytes. Using the Northern blot hybridization method, it was shown that native oocytes contain large amounts of alpha 2/delta subunit RNA of Ca(2+)-channel. It is proposed that te oligonucleotide treatment fails to eliminate the alpha 2/delta RNA because of the vast excess of endogenous alpha 2/delta RNA. These results impose a limit on the use of the hybrid-arrest method.

Animals↗
PubMed1992

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L-Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Ca2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4 beta-phorbol 12-myristate 13-acetate) were studied. Currents through channels composed of the main (alpha 1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the alpha 1 subunit was expressed in combination with alpha 2/delta, beta and/or gamma subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. beta subunit moderate the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L-type Ca2+ currents by PKC are mediated via an effect on the alpha 1 subunit, while the beta subunit may play a mild modulatory role.

Alkaloids↗
PubMed1992

Level of expression controls modes of gating of a K+ channel.

Several distinct subfamilies of K+ channel genes have been discovered by molecular cloning, however, in some cases the structural differences among them do not account for the diversity of K+ current types, ranging from transient A-type to slowly inactivating delayed rectifier-type, as members within each subfamily have been shown to code for K+ channels of different inactivation kinetics and pharmacological properties. We show that a single K+ channel cDNA of the Shaker subfamily (ShH4) can express in Xenopus oocytes not only a transient A-type K+ current but also, upon increased level of expression, slowly inactivating K+ currents with markedly reduced sensitivity to tetraethylammonium. In correlation with the macroscopic currents there are single-channel gating modes ranging from the fast-inactivation mode which underlies the transient A-type current, to slow-inactivation modes characterized by bursts of longer openings, and corresponding to the slowly inactivating macroscopic currents.

Animals↗
PubMed1992

Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain.

Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L-type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.

Amino Acid Sequence↗