A concentration-clamp system allowing two-electrode voltage-clamp investigations in oocytes of Xenopus laevis

Madeja, Michael; Muβhoff, U.; Speckmann, E.‐J.

doi:10.1016/0165-0270(91)90178-3

Cited by 43 publications

(18 citation statements)

References 4 publications

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“…Propafenone (as hydrochloride; Sigma, Deisenhofen, Germany) in final concentrations of 0.01 µmol/l up to 300 µmol/l was added to the bath solution and applied at least 30 s before eliciting potassium currents. Solutions were applied with a concentration-clamp technique for oocytes (Madeja et al 1991). All experiments were performed at days 3 and 4 after injection of cRNA and were carried out at room temperature (22±1°C).…”

Section: Methodsmentioning

confidence: 99%

Blocking effects of the antiarrhythmic drug propafenone on the HERG potassium channel

Mergenthaler

Haverkamp

Hüttenhofer

et al. 2001

Naunyn-Schmiedeberg's Archives of Pharmacology

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Propafenone has been shown to affect the delayed-rectifier potassium currents in cardiomyocytes of different animal models. In this study we investigated effects and mechanisms of action of propafenone on HERG potassium channels in oocytes of Xenopus laevis with the two-electrode voltage-clamp technique. Propafenone decreased the currents during voltage steps and the tail currents. The block was voltage-dependent and increased with positive going potentials (from 18% block of tail current amplitude at -40 mV to 69% at +40 mV with 100 micromol/l propafenone). The voltage dependence of block could be fitted with the sum of a monoexponential and a linear function. The fractional electrical distance was estimated to be delta=0.20. The block of current during the voltage step increased with time starting from a level of 83% of the control current. Propafenone accelerated the increase of current during the voltage step as well as the decay of tail currents (time constants of monoexponential fits decreased by 65% for the currents during the voltage step and by 37% for the tail currents with 100 micromol/l propafenone). The threshold concentration of propafenone effect was around 1 micromol/l and the concentration of half-maximal block (IC50) ranged between 13 micromol/l and 15 micromol/l for both current components. With high extracellular potassium concentrations, the IC50 value rose to 80 degrees mol/l. Acidification of the extracellular solution to pH 6.0 increased the IC50 value to 123 micromol/l, alkalization to pH 8.0 reduced it to 10 micromol/l and coexpression of the beta-subunit minK had no statistically significant effect on the concentration dependence. In conclusion, propafenone has been found to block HERG potassium channels. The data suggest that propafenone affects the channels in the open state and give some hints for an intracellular site of action.

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Section: Methodsmentioning

confidence: 99%

Blocking effects of the antiarrhythmic drug propafenone on the HERG potassium channel

Mergenthaler

Haverkamp

Hüttenhofer

et al. 2001

Naunyn-Schmiedeberg's Archives of Pharmacology

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“…The osmolarity of solutions was measured with a cryoscopic osmometer (Gonotec, Berlin, Germany). Solutions were applied with a concentration-clamp technique (44) allowing an exchange of more than 90% of the extracellular solution within less than 10 ms. All experiments were performed at days 3 and 4 after injection of cRNA and were carried out at room temperature (22 5 1 C).…”

Section: Electrophysiological Techniquesmentioning

confidence: 99%

Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1

Elinder

Madeja

Zeberg

et al. 2016

Biophysical Journal

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The transmembrane voltage needed to open different voltage-gated K (Kv) channels differs by up to 50 mV from each other. In this study we test the hypothesis that the channels' voltage dependences to a large extent are set by charged amino-acid residues of the extracellular linkers of the Kv channels, which electrostatically affect the charged amino-acid residues of the voltage sensor S4. Extracellular cations shift the conductance-versus-voltage curve, G(V), by interfering with these extracellular charges. We have explored these issues by analyzing the effects of the divalent strontium ion (Sr 2þ ) on the voltage dependence of the G(V) curves of wild-type and chimeric Kv channels expressed in Xenopus oocytes, using the voltage-clamp technique. Out of seven Kv channels, Kv1.2 was found to be most sensitive to Sr 2þ (50 mM shifted G(V) by þ21.7 mV), and Kv2.1 to be the least sensitive (þ7.8 mV). Experiments on 25 chimeras, constructed from Kv1.2 and Kv2.1, showed that the large Sr 2þ -induced G(V) shift of Kv1.2 can be transferred to Kv2.1 by exchanging the extracellular linker between S3 and S4 (L3/4) in combination with either the extracellular linker between S5 and the pore (L5/P) or that between the pore and S6 (LP/6). The effects of the linker substitutions were nonadditive, suggesting specific structural interactions. The free energy of these interactions was~20 kJ/mol, suggesting involvement of hydrophobic interactions and/or hydrogen bonds. Using principles from double-layer theory we derived an approximate linear equation (relating the voltage shifts to altered ionic strength), which proved to well match experimental data, suggesting that Sr 2þ acts on these channels mainly by screening surface charges. Taken together, these results highlight the extracellular surface potential at the voltage sensor as an important determinant of the channels' voltage dependence, making the extracellular linkers essential targets for evolutionary selection.

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“…Membrane currents were measured at a holding potential of Ϫ70 mV. The solutions were administered to the oocytes using a concentration clamp technique (Madeja et al, 1991). The control bath fluid was a salt solution composed of (in mmol/l): NaCl 115, KCl 2, CaCl 2 1.8, HEPES 10, pH 7.2.…”

Section: Electrophysiological Studies On Xenopus Oocytesmentioning

confidence: 99%

Melatonin receptors in rat hippocampus: molecular and functional investigations

et al. 2002

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Since binding sites for melatonin have been found in the hippocampus of several mammals, it has been suggested that the pineal hormone melatonin is able to modulate neuronal functions of hippocampal cells. In order to get more insight into the role of melatonin for the functions of hippocampal cells, the following experiments were performed: male rats, maintained under a 12/12-h light-dark cycle, were sacrificed by decapitation at zeitgeber times (h) ZT2, ZT8, and ZT15 (ZT0 = lights on); for experiment 1, gene expression for melatonin receptors was detected in the hippocampus and in hippocampal subfields by means of the RT-PCR technique; for experiment 2, electrophysiological and pharmacological properties of melatonin receptors heterologously expressed in Xenopus oocytes after injection of mRNA from the hippocampus were analyzed by means of voltage clamp technique; and for experiment 3, effects of melatonin on the spontaneous firing rate of action potentials in the CA1 regions of hippocampal slices were analyzed by means of extracellular recordings. The RT-PCR data revealed that transcripts for both the MT1 and MT2 melatonin receptors are present in the dentate gyrus, CA3, and CA1 regions, and the subiculum of the hippocampus. Injection of mRNA from rat hippocampus into the Xenopus oocytes led to the functional reconstitution of melatonin-sensitive receptors, which activates calcium-dependent chloride inward currents. The melatonin responses were abolished by simultaneous administration of the antagonists 2-phenylmelatonin and luzindole, and were unaffected by the MT2 antagonist 4-phenyl-2-propionamidotetralin. Bath-applied melatonin (1 micromol/l) enhances the firing rate of neurons in the CA1 region. The effect was small in experiments performed at ZT8 (<2 times the initial level) and large in experiments performed at ZT15 (>6 times). The changes of neuronal firing rate induced by melatonin were completely suppressed with simultaneous administration of the melatonin receptor antagonist luzindole (10 micromol/l). The results indicate that melatonin may play an important role in modulating neuronal excitability in the hippocampus.

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A concentration-clamp system allowing two-electrode voltage-clamp investigations in oocytes of Xenopus laevis

Cited by 43 publications

References 4 publications

Blocking effects of the antiarrhythmic drug propafenone on the HERG potassium channel

Blocking effects of the antiarrhythmic drug propafenone on the HERG potassium channel

Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1

Melatonin receptors in rat hippocampus: molecular and functional investigations

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