Biomedical subjects

A E Busch

Publications and source records attributed to A E Busch.

At least 91 records · Page 5Linked to original sources

PubMed1994

Blockade of human IsK channels expressed in Xenopus oocytes by the novel class III antiarrhythmic NE-10064.

cRNA encoding the human IsK protein was injected into Xenopus oocytes and the expressed channels were investigated using the two-microelectrode voltage-clamp method. The novel class III antiarrhythmic NE-10064 (1-[[[5-(4-chlorophenyl)-2-furanyl]methylene]-amino]-3- [4-(4-methyl-1-piperazinyl)-butyl]-2,4-imidazolidinedione dihydrochloride) was tested for its ability to block these channels. The compound displayed potent inhibitory effects with an EC50 of 5.4 microM. The block caused by NE-10064 was use-dependent, i.e. channels had to be activated for the inhibition to occur. Further, the reversal of the inhibition during the wash-out period was use-dependent. Finally, the blockade of human IsK channels by NE-10064 appeared to be voltage-dependent, being more pronounced at depolarized potentials. We conclude that this novel class III antiarrhythmic is a potent inhibitor of human IsK channels and suggest that such effects could be involved in its antiarrhythmic action.

Animals↗

PubMed1994

The novel class III antiarrhythmics NE-10064 and NE-10133 inhibit IsK channels expressed in Xenopus oocytes and IKs in guinea pig cardiac myocytes.

Slowly activating, voltage-dependent IsK channels were expressed in Xenopus oocytes after injection of rat IsK protein cRNA and recorded with the two-microelectrode voltage-clamp technique. The IsK currents were inhibited by the new class III antiarrhythmic drugs NE-10064 and NE-10133. These compounds were equally potent in inhibiting a slowly activating potassium current (IKs) in guinea pig ventricular myocytes. No effects of these compounds could be observed on several other cloned delayed rectifier potassium channels, nor did they affect the inward rectifier current, IK1, in guinea pig cardiac myocytes at the concentrations tested. The blockade of IsK channels may contribute to the class III antiarrhythmic efficacy of these novel antiarrhythmics.

Animals↗

PubMed1994

Inhibition of human IsK channels expressed in Xenopus oocytes by calmodulin antagonists.

The calmodulin antagonists, trifluoperazine, chlorpromazine and W7 (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluomethyl)-10H-phen othiazine , 2-chloro-10-(dimethylaminopropyl)-phenothiazine and N-(6-aminohexyl)-5-chloro-1-naphtalen-sulfonamide, respectively), were tested for their effects on human IsK channels expressed in Xenopus oocytes and their interference with the previously described [Ca2+]i-mediated regulation of IsK. An increase in [Ca2+]i accelerated IsK activation and increased the current amplitude, as has been previously observed. Chlorpromazine, trifluoperazine and W7 inhibited depolarization-activated IsK channels with an EC50 between 70 and 100 microM. None of the calmodulin antagonists abolished the regulation of IsK by A23187 (calcimycin) or hypotonic extracellular fluid, although the inhibitory effects of these compounds were also obvious after enhancement of [Ca2+]i. In conclusion, the calmodulin antagonists inhibit IsK at both physiological and enhanced [Ca2+]i.

Animals↗

PubMed1994

Electrogenic cotransport of Na+ and sulfate in Xenopus oocytes expressing the cloned Na+SO4(2-) transport protein NaSi-1.

The Na+/sulfate cotransporter cloned from rat kidney cortex (NaSi-1) has been expressed in oocytes of Xenopus laevis and subjected to electrophysiological analysis by current and voltage clamp methods. In current-clamped oocytes, superfusion with 1 mM sulfate resulted in a 12-mV depolarization of the cell membrane. Accordingly, in voltage-clamped oocytes sulfate induced an inward current IS, which was dependent on both the concentration of Na+ and sulfate in the superfusate. Half-maximal IS was observed at about 0.1 mM sulfate and 70 mM Na+. The Hill coefficients were 1 and 2.8 for sulfate and Na+, respectively. Thiosulfate and selenate created similar currents as sulfate with a similar Km. At saturating concentrations of thiosulfate and selenate, addition of sulfate could not induce an additive current. Phosphate (1 mM) did not inhibit sulfate-induced currents. Finally, IS was dependent on the holding potential being larger at more negative potentials. The results of this study strongly suggest an electrogenic cotransport of sulfate and Na+ with a stoichiometry of 1:3.

Animals↗

PubMed1994

Positive regulation by chloride channel blockers of IsK channels expressed in Xenopus oocytes.

cRNA encoding the human IsK protein was injected into Xenopus oocytes and the induced IsK channels were investigated using the two-microelectrode voltage-clamp method. Niflumic acid, mefenamic acid, flufenamic acid, and 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid, which are commonly used in Xenopus oocytes to suppress endogenous Ca(2+)-activated Cl- channels, were tested for their effects on IsK channels. At low concentrations (10 microM) all compounds increased IsK amplitude and decreased the rate of IsK deactivation. At 100 microM these compounds further decreased the rate of IsK deactivation, resulting in persistent activation of IsK, similar to what has been previously described for the action of organic cross-linkers on IsK. However, at 100 microM niflumic acid and flufenamic acid decreased the time-dependent outward current, whereas 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and mefenamic acid caused an additional increase. When Cl- was completely substituted with gluconate, IsK had somewhat altered activation properties, but niflumic acid produced similar positive regulatory effects on IsK and shifted the voltage needed to evoke half-maximal IsK activation (V1/2) by about -20 mV. In summary, these compounds positively regulate IsK, presumably by stabilizing open IsK channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

Time dependent changes in biophysical properties of minK channels expressed in Xenopus oocytes.

Slowly activating, voltage-dependent minK channels cloned from rat kidney were expressed in Xenopus oocytes. The maximal conductance (gmax) through these channels increased from 11 microS at day two after mRNA injection to 22 microS after one week. This increase of gmax is presumably the result of an increase in the number of functional channels through protein synthesis of the oocyte. Concurrent with the increase of gmax the voltage needed to evoke a half-maximal conductance (V1/2) was shifted to more negative potentials, while the activation of minK channels was accelerated. These results suggest a relation between protein density and activation of minK channels. Twofold changes of gmax were also observed in oocytes expressing the Shaker related rat potassium channel RBK1; however, there was no relation between gmax, V1/2 and the activation kinetics of RBK1. These results are consistent with the hypothesis that minK channel-formation and activation might involve subunit assembly.

Animals↗

PubMed1993

The min K channel underlies the cardiac potassium current IKs and mediates species-specific responses to protein kinase C.

A clone encoding the guinea pig (gp) min K potassium channel was isolated and expressed in Xenopus oocytes. The currents, gpIsK, exhibit many of the electrophysiological and pharmacological properties characteristic of gpIKs, the slow component of the delayed rectifier potassium conductance in guinea pig cardiac myocytes. Depolarizing commands evoke outward potassium currents that activate slowly, with time constants on the order of seconds. The currents are blocked by the class III antiarrhythmic compound clofilium but not by the sotalol derivative E4031 or low concentrations of lanthanum. Like IKs in guinea pig myocytes, gpIsK is modulated by stimulation of protein kinase A and protein kinase C (PKC). In contrast to rat and mouse IsK, which are decreased upon stimulation of PKC, myocyte IK and gpIsK in oocytes are increased after PKC stimulation. Substitution of an asparagine residue at position 102 by serine (N102S), the residue found in the analogous position of the mouse and rat min K proteins, results in decreased gpIsK in response to PKC stimulation. These results support the hypothesis that the min K protein underlies the slow component of the delayed rectifier potassium current in ventricular myocytes and account for the species-specific responses to stimulation of PKC.

Amino Acid Sequence↗

PubMed1993

Effects of [Ca2+]i and temperature on minK channels expressed in Xenopus oocytes.

Slowly activating, voltage-dependent minK channels cloned from rat kidney were expressed in Xenopus oocytes. Increase in the bath temperature from 22 to 32 degrees C resulted in a dramatic acceleration of minK channel activation. The extraordinarily high Q10 of minK channel activation was voltage-dependent, being higher at more negative potentials (Q10 at -20 mV; 7.02; at 20 mV: 4.0). While activation of minK channels was highly voltage-dependent at 22 degrees C, voltage had only little effect on minK channel activation at 32 degrees C. Increase in [Ca2+]i which has recently been shown to increase the maximal conductance gmax at room temperature, did not affect gmax at 32 degrees C. However, increase of [Ca2+]i caused acceleration of minK channel activation at both temperatures. The interaction of [Ca2+]i and temperature on gmax and activation rate of minK channels described here is very similar to recent findings on Ca- and temperature-effects on the slowly activating potassium conductance IKs in guinea pig cardiac myocytes.

Animals↗

PubMed1992

Hypotonic solution increases the slowly activating potassium current IsK expressed in xenopus oocytes.

A slowly activating potassium current was expressed in Xenopus oocytes by injection of RNA transcribed from a rat kidney cDNA clone. Hypotonic solutions (160 mOsmol/l; control was 220 mOsmol/l) increased the current by increasing the rate of activation and by decreasing the depolarization needed to activate the current. This effect of hypotonicity was not observed in calcium-free solution, but was unaffected by staurosporine or the calmodulin antagonist W7. Cytochalasin D reduced the current and prevented the increase by hypotonic solution. The results suggest that the increase in this potassium current by hypotonic solution might result from calcium entry and changes in the actin network.

Animals↗

PubMed1992

An amino acid mutation in a potassium channel that prevents inhibition by protein kinase C.

A slowly activating, voltage-dependent potassium channel protein cloned from rat kidney was expressed in Xenopus oocytes. Two activators of protein kinase C, 1-oleoyl-2-acetyl-rac-glycerol and phorbol 12,13-didecanoate, inhibited the current. This inhibition was blocked by the kinase inhibitor staurosporine. Inhibition of the current was not seen in channels in which Ser103 was replaced by Ala, although other properties of the current were unchanged. These results indicate that inhibition of the potassium current results from direct phosphorylation of the channel subunit protein at Ser103.

Amino Acid Sequence↗

PubMed1992

Regulation by second messengers of the slowly activating, voltage-dependent potassium current expressed in Xenopus oocytes.

1. Voltage-clamp recordings of membrane current were made from Xenopus oocytes that had been injected with RNA which had been transcribed in vitro from a cloned complementary DNA. 2. Depolarization from -80 mV evoked outward potassium currents that developed very slowly. At -20 mV the time constant for activation was about 50 s, and at +40 mV about 6 s. 3. The potassium current was increased by the calcium ionophore A23187 or by intracellular injection of inositol 1,4,5-trisphosphate (IP3), each of which should increase the intracellular calcium concentration ([Ca2+]i). The current was decreased by injection of BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). The current was also reduced by phorbol esters; this effect was blocked by staurosporine. 4. In oocytes that had also been injected with RNA encoding the 5-hydroxytryptamine (5-HT2) receptor, 5-HT increased the potassium current. After caffeine pretreatment, to block the release of intracellular calcium, 5-HT decreased the current; this decrease was prevented by staurosporine. 5. It is concluded that the slowly activating, voltage-dependent potassium current expressed in Xenopus oocytes is increased by increases in [Ca2+]i and is decreased by activation of protein kinase C. Stimulation of 5-HT2 receptors can have both these effects, but the former normally predominates.

Animals↗

PubMed1991

Current inactivation involves a histidine residue in the pore of the rat lymphocyte potassium channel RGK5.

RGK5 is a rat genomic DNA clone that encodes the n-type potassium channel found in T-lymphocytes and other cells. Current through this channel declines (inactivates) over a period of hundreds of milliseconds during a maintained depolarizing pulse, whether in lymphocytes or when expressed in Xenopus oocytes. Here we demonstrate that an amino acid residue near the outer pore of the channel, histidine401, is involved in the inactivation process. Replacement of this residue by tyrosine, the amino acid found in the equivalent position of the homologous but non-inactivating channel RBK1, reduced inactivation of RGK5 over a 5 s depolarizing pulse from 84.3 +/- 1.9% to 18.3 +/- 1.1%. Conversely, replacement of this tyrosine in RBK1 (Tyr379) by histidine increased its inactivation from 21.6 +/- 1.1% to 42.3 +/- 1.5%. These results suggest a mechanism of channel inactivation distinct from that previously described for the A-type potassium channel.

Cloning, Molecular↗

PubMed1991

Transmitter regulation of voltage-dependent K+ channels expressed in Xenopus oocytes.

Voltage-dependent K+ channels (RBK1, RBK2 and RGK5) were co-expressed in Xenopus oocytes with 5-hydroxytryptamine (5-HT2) receptors. K+ currents measured 2-4 days later were inhibited by 5-HT (100 nM-10 microM, 20-30 s application) by up to 90%. The effect of 5-HT was mimicked by intracellular injection of Ins(1,4,5)P3. Increasing the Ca2+ concentration at the inner surface of excised membrane patches did not decrease the K+ current.

Animals↗

PubMed1991

Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels.

Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (KD 0.3 mM), RGK5 (KD 11 mM), and RBK2 (KD greater than 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between trans-membrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr379 of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 (Val381) and RGK5 (His401) made these channels as sensitive to TEA as RBK1. Nonionic forces are involved in TEA binding because (i) substitution of the Phe for Tyr379 in RBK1 increased its affinity, (ii) protonation of His401 in RGK5 selectively reduced its affinity, and (iii) the affinity of TEA was unaffected by changes in ionic strength. The results suggest an explanation for the marked differences in TEA sensitivity that have been observed among naturally occurring and cloned potassium channels and indicate that the amino acid corresponding to residue 379 in RBK1 lies within the external mouth of the ion channel.

Amino Acid Sequence↗

PubMed1991

In vitro testing of triamterene derivatives for antiarrhythmic activity.

A series of para-substituted triamterene derivatives (Table 1) were evaluated for their antiarrhythmic properties in vitro. Pharmacological evaluation of the compounds and some class-I- (quinidine, lidocaine, and propafenone) as well as class-III-antiarrhythmic drugs ((+/-)-sotalol and amiodarone) was carried out by measuring the functional refractory period (FRP), the maximal driving frequency (MDF) and the force of myocardial contractions (FC) of electrically stimulated guinea pig atria. The increase in FRP and the decrease in MDF was most pronounced with the class-I-antiarrhythmic drugs, but these compounds showed the typical negative inotropic effects, too. For the class-III-antiarrhythmics only a weak influence on FRP and MDF could be demonstrated, while FC was not altered in the presence of (+/-)-sotalol and amiodarone. Neutral substituted triamterenes like compounds 2-5 as well as most of the benzyltriamterene derivatives showed similar or stronger effects on FRP and MDF as (+/-)-sotalol and amiodarone. With the exception of 4 and 5, these effects were combined with an increase of FC. Compounds 6 and 7, well known-potent diuretics, showed no influence to FRP, MDF and FC. Therefore, we conclude different mechanisms for the antikaliuretic and cardiac activity.

Animals↗

PubMed1991

Antiarrhythmic properties of triamterene derivatives in the coronary artery ligated rat model.

Triamterene and several triamterene derivatives were tested for antiarrhythmic activity in the coronary artery ligated and reperfused (CAL-R) rat. The class-III antiarrhythmic drugs (+/-)-sotalol and amiodarone, the class-I antiarrhythmics lidocaine and quinidine as well as the potassium sparing diuretic amiloride were used as reference drugs. Triamterene at the highest dose (30 mumol/kg) revealed a 100% protection against ventricular fibrillation (VF), whereas at 10 mumol/kg no antiarrhythmic activity for triamterene could be found. For compound 4 (10 mumol/kg) a 75% protection against VF could be demonstrated, while 2, 3, and 5 revealed only a 25% protection. Compared to the reference drugs, triamterene and the derivatives 2-5 are more potent than (+/-)-sotalol, but less potent than lidocaine, quinidine and amiodarone. For amiloride as well as for the potent potassium retaining triamterene derivative 6 no antiarrhythmic activity could be shown. Therefore, we conclude different mechanisms responsible for the potassium sparing and antiarrhythmic properties of triamterene and its derivatives.

Animals↗

PubMed1991

Identification of amino acid residues involved in dendrotoxin block of rat voltage-dependent potassium channels.

alpha-Dendrotoxin (DTX) is a 60-amino acid peptide belonging to the family of mamba snake neurotoxins; it is a potent blocker of some but not all voltage-gated potassium currents. Potassium currents recorded from oocytes injected with cloned potassium channel RNAs also vary in sensitivity to DTX. Expression of channels that were chimeras of the DTX-sensitive channel RBK2 and the DTX-insensitive channel RGK5 showed that the putative extracellular loop between transmembrane domains S5 and S6 contributes strongly to DTX sensitivity. Mutation of two residues (Ala352Glu353) in this region of RBK1 to conform to those at equivalent positions in RGK5 (Pro374Ser375) reduced the potency of DTX about 70-fold, and the substitution of Tyr379 in RBK1 by its counterpart in RGK5 (His401) caused an additional 2.5-fold decrease in sensitivity. Converse substitutions in RGK5 significantly increased sensitivity to DTX. The results suggest that these residues contribute significantly to the channel-toxin interaction, providing further evidence that the S5-S6 loop lies at or near the external mouth of the channel, where DTX binding leads to channel occlusion. They offer a molecular explanation for the differences in DTX sensitivity observed among native potassium channels.

Amino Acid Sequence↗

PubMed1991

Antiarrhythmic properties of benzyl-triamterene derivatives in the coronary artery ligated and reperfused rat.

Triamterene (CAS 396-01-0) and a series of benzyl-triamterene derivatives were evaluated for their antiarrhythmic properties in the coronary artery ligated and reperfused (CAL-R) rat. The effects were compared with the antiarrhythmic activity of the potassium sparing diuretic amiloride and drugs out of the class-I (lidocaine) and class-III (amiodarone and sotalol). Triamterene and sotalol revealed at high doses antifibrillator activity, while the benzyl-triamterenes 2, 3, 5 and 6 could also depress ventricular extrasystoles (VES) and ventricular tachycardia (VT). At low doses the most benzyltriamterenes protected significantly against ventricular fibrillation (VF) and so they were equieffective or more effective than amiodarone or lidocaine. Amiloride showed in the CAL-R rat no antiarrhythmic activity, so that we conclude different mechanisms responsible for antikaliuretic and antiarrhythmic properties of amiloride and triamterenes. Taking into account the results of recently reported in vitro studies, where we could demonstrate antiarrhythmic activity combined with positive inotropic properties for triamterenes, the antiarrhythmic profile of these compounds may offer new possibilities for the treatment of ventricular arrhythmias.

Animals↗