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N Dascal

Publications and source records attributed to N Dascal.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Calcium channel currents in Xenopus oocytes injected with rat skeletal muscle RNA.

1. Ba2+ currents (IBa) through voltage-dependent Ca2+ channels were studied in Xenopus laevis oocytes injected with heterologous RNA extracted from skeletal muscle (SkM) of young rats, using the two-electrode voltage clamp technique. 2. With 40 or 50 mM-extracellular Ba2+, native oocytes of most frogs displayed IBa between -5 and -20 nA at 0 mV. However, in 'variant' native oocytes of four frogs, IBa exceeded -30 nA and reached up to -100 nA. In oocytes injected with SkM RNA, IBa of up to -250 nA was observed. 3. In SkM RNA-injected oocytes and 'variant' native oocytes, the decay of IBa displayed two kinetic components. The faster component was selectively blocked by 40-100 microM-Ni2+ and thus was termed the Ni(2+)-sensitive IBa. The slower component was Ni2+ resistant, being inhibited only 10-20% by 100-200 microM-Ni2+. The half-activation and the half-inactivation voltages of the Ni(2+)-sensitive IBa were more negative (by 14.5 and 28.7 mV, respectively) than those of the Ni(2+)-resistant IBa. 4. Neither Ni(2+)-sensitive nor Ni(2+)-resistant IBa in native or SkM RNA-injected oocytes were affected by dihydropyridine antagonists nifedipine and (+) PN 200-110 (1-10 microM), by the dihydropyridine agonist (-)Bay K 8644 (0.01-2 microM), or by verapamil below 50 microM. IBa was blocked by diltiazem (half-block at about 500 microM). Thus, the pharmacology of IBa in SkM RNA-injected and in native oocytes was not characteristic of the L-type Ca2+ channel abundant in the skeletal muscle. 5. Destruction of the RNA coding for the channel-forming alpha 1-subunit of the SkM L-type Ca2+ channel using a hybrid arrest method failed to selectively suppress the appearance of either Ni(2+)-sensitive or Ni(2+)-resistant IBa in SkM RNA-injected oocytes. 6. Our results suggest that the appearance of large voltage-dependent Ba2+ currents in SkM RNA-injected oocytes is not due to the expression of the alpha 1-subunit of the SkM L-type Ca2+ channel. The possibility that the expression of a channel-forming subunit of another Ca2+ channel type underlies one of these currents cannot be rejected. However, since the Ba2+ currents in SkM RNA-injected oocytes resemble those observed in native oocytes, we suggest that their appearance may be the result of an enhanced activity of the native Ca2+ channels, possibly due to the expression of the 'auxiliary' subunits of the SkM Ca2+ channel that form complexes with a native alpha 1-subunit.

Animals↗
PubMed1992

NaCl-dependent expression of amiloride-blockable Na+ channel in Xenopus oocytes.

RNA was isolated from chicken lower intestine (both colon and coprodeum) and injected into Xenopus oocytes. 22Na+ fluxes measured after 1-4 days demonstrated the induction of an amiloride-blockable pathway. The Na+ transporter expressed by the exogenous RNA had a high affinity to amiloride (inhibitory constant less than 0.1 microM), but was insensitive to ethylisopropyl amiloride, i.e., it is likely to be the apical Na+ channel. Functional channels were readily expressed in oocytes injected with RNA derived from chickens fed a low-NaCl diet. On the other hand, no channel activity was detected in oocytes injected with RNA isolated from chickens fed a high-NaCl diet. Thus the previously reported regulation of transport by the dietary NaCl intake involves modulations in the level of mRNA that codes either for the Na+ channel or a posttranscriptional regulator of the channel.

Amiloride↗
PubMed1992

Protein kinase A reduces voltage-dependent Na+ current in Xenopus oocytes.

The voltage-dependent Na+ channel of the brain is a good substrate for phosphorylation by the cAMP-dependent protein kinase (protein kinase A, or PKA), but the physiological effects of PKA on Na+ channels are poorly documented. We studied modulation by PKA of voltage-dependent Na+ channels expressed in Xenopus oocytes injected with RNA coding for the alpha-subunit of the channel protein (rat brain type IIA and its variant VA200), using the two electrode voltage-clamp technique. Intracellularly injected cAMP or catalytic subunit of PKA, or extracellularly applied forskolin, inhibited the Na+ current by 20-30%. The effect of cAMP was attenuated by prior injection of PKA inhibitors. Injection of small doses of protein phosphatase 2A increased the Na+ current by 10%, whereas larger doses of protein phosphatase 1 and alkaline phosphatase were without effect. The inhibition by PKA showed little voltage dependence, being only slightly stronger at holding potentials at which the availability of the channels was reduced. The voltage dependence of activation and inactivation processes was not altered by cAMP. Similar effects were exerted by forskolin and cAMP on the Na+ channels expressed after the injection of heterologous (total) RNA from rat brain. Thus, PKA modulates the Na+ channel by a mechanism that does not involve major changes in the voltage dependency of the current and is exerted on the channel-forming alpha-subunit.

Animals↗
PubMed1991

Molecular mechanism of protein kinase C modulation of sodium channel alpha-subunits expressed in Xenopus oocytes.

The mechanism of modulation of sodium channel alpha-subunits (Type IIA) by a protein kinase C (PKC) activator was studied on single channel level. It was found that: (i) time constants for channel activation were prolonged; (ii) inactivation remained virtually unchanged; (iii) peak sodium inward current was reduced as evidenced by calculation of average sodium currents; and (iv) time constants for current activation and decay were prolonged. (i), (iii) and (iv) were voltage dependent, being most prominent at threshold potentials. The data show that a voltage dependent action on the activation gate can account for the observed reduction of peak inward sodium current and prolongation of current decay in macroscopic experiments.

Animals↗
PubMed1991

The roles of the subunits in the function of the calcium channel.

Dihydropyridine-sensitive voltage-dependent L-type calcium channels are critical to excitation-secretion and excitation-contraction coupling. The channel molecule is a complex of the main, pore-forming subunit alpha 1 and four additional subunits: alpha 2, delta, beta, and gamma (alpha 2 and delta are encoded by a single messenger RNA). The alpha 1 subunit messenger RNA alone directs expression of functional calcium channels in Xenopus oocytes, and coexpression of the alpha 2/delta and beta subunits enhances the amplitude of the current. The alpha 2, delta, and gamma subunits also have pronounced effects on its macroscopic characteristics, such as kinetics, voltage dependence of activation and inactivation, and enhancement by a dihydropyridine agonist. In some cases, specific modulatory functions can be assigned to individual subunits, whereas in other cases the different subunits appear to act in concert to modulate the properties of the channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

Tissue-specific expression of high-voltage-activated dihydropyridine-sensitive L-type calcium channels.

The cloning of the cDNA for the alpha 1 subunit of L-type calcium channels revealed that at least two genes (CaCh1 and CaCh2) exist which give rise to several splice variants. The expression of mRNA for these alpha 1 subunits and the skeletal muscle alpha 2/delta, beta and gamma subunits was studied in rabbit tissues and BC3H1 cells. Nucleic-acid-hybridization studies showed that the mRNA of all subunits are expressed in skeletal muscle, brain, heart and aorta. However, the alpha 1-, beta- and gamma-specific transcripts had different sizes in these tissues. Smooth muscle and heart contain different splice variants of the CaCh2 gene. The alpha 1, beta and gamma mRNA are expressed together in differentiated but not in proliferating BC3H1 cells. A probe specific for the skeletal muscle alpha 2/delta subunit did not hybridize to poly(A)-rich RNA from BC3H1 cells. These results suggest that different splice variants of the genes for the alpha 1, beta and gamma subunits exist in tissues containing L-type calcium channels, and that their expression is regulated in a coordinate manner.

Amino Acid Sequence↗
PubMed1991

Modulation of a Shaker potassium A-channel by protein kinase C activation.

Brain fast transient K+ channel (A channel) is known to be modulated by PKC activation. We studied, by two-electrode voltage clamp, the molecular mechanism of modulation by PKC activation of A-channels expressed in Xenopus oocytes from the Shaker H4 clone. The modulation is inhibitory affecting primarily the maximal conductance of the channels. A secondary effect is a small change in the voltage-dependence of activation and inactivation of the channel.

Animals↗
PubMed1991

Recording of voltage and Ca(2+)-dependent currents in Xenopus oocytes using an intracellular perfusion method.

We describe a method for internal perfusion of Xenopus laevis oocytes that allows control of the composition of intracellular and extracellular solutions, including the possibility of sequential introduction of different substances inside and outside the cell. Using this method, it was possible to record Ca2+ dependent Cl- current and to inhibit it by intracellular perfusion of EGTA-containing solution. With a high BA2+ solution at the external surface of the perfused oocyte, Ba2+ currents through voltage-dependent Ca2+ channels were observed in native and in cardiac RNA-injected oocytes. Finally, a delayed rectifier K+ current was recorded and blocked by internally perfused Cs+ in oocytes injected with mRNA of a cloned (MBK1) K+ channel. The method is expected to be useful for the study of function and modulation of ion channels and transporters in the oocyte, which is an important and widely used model system.

Animals↗
PubMed1991

Activation of protein kinase C alters voltage dependence of a Na+ channel.

Phorbol esters and purified protein kinase C (PKC) have been shown to down-modulate the voltage-dependent Na+ channels expressed in Xenopus oocytes injected with chick brain RNA. We used the two-electrode voltage-clamp technique to demonstrate that a Na+ channel expressed in oocytes injected with RNA coding for the alpha subunit of the channel alone (VA200, a variant of rat brain type IIA) is also inhibited by PKC activation. The inhibition of Na+ currents, expressed in oocytes injected with either alpha subunit RNA (rat) or total brain RNA (chick), is voltage-dependent, being stronger at negative potentials. It appears to result mainly from a shift in the activation curve to the right and possibly a decrease in the steepness of the voltage dependence of activation. There is little effect on the inactivation process and maximal Na+ conductance. Thus, PKC modulates the Na+ channel by a mechanism involving changes in voltage-dependent properties of its main, channel-forming alpha subunit.

Animals↗
PubMed1990

Interaction between injected Ca2+ and intracellular Ca2+ stores in Xenopus oocytes.

Upon two repetitive deep injections of Ca2+ into Xenopus oocyte (200-300 microns under the membrane), the amplitude of the transient Cl- current induced by the second injection is several-fold higher than that of the first one. This 'potentiation' persists even at 60-90 min intervals between injections. However, in oocytes permeabilized to Ca2+ by the ionophore A23187 in a Ca2(+)-free solution, the potentiation completely disappears after 30 min. It is proposed that the injected Ca2+ is largely taken up by the stores, whereas following the second injection, a higher proportion of Ca2+ reaches the membrane, since the stores are already loaded. In ionophore-treated oocytes, the stores lose the accumulated Ca2+ over several minutes and are then ready to take up Ca2+ again, hindering its arrival at the membrane.

Animals↗