Voltage Dependence Of Current Inactivation Research Articles

Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes.

The effects of changing cytosolic [Mg(2+)] ([Mg(2+)](i)) on L-type Ca(2+) currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg(2+)] and [Ca(2+)]. To control [Mg(2+)](i) and cytosolic [Ca(2+)] ([Ca(2+)](i)), the pipette solution included 30 mM citrate and 10 mM ATP along with 5 mM EGTA (slow Ca(2+) buffer) or 15 mM EGTA plus 5 mM BAPTA (fast Ca(2+) buffer). With pipette [Ca(2+)] ([Ca(2+)](p)) set at 100 nM using a slow Ca(2+) buffer and pipette [Mg(2+)] ([Mg(2+)](p)) set at 0.2 mM, peak l-type Ca(2+) current density (I(Ca)) was 17.0 +/- 2.2 pA pF(-1). Under the same conditions, but with [Mg(2+)](p) set to 1.8 mM, I(Ca) was 5.6 +/- 1.0 pA pF(-1), a 64 +/- 2.8% decrease in amplitude. This decrease in I(Ca) was accompanied by an acceleration and a -8 mV shift in the voltage dependence of current inactivation. The [Mg(2+)](p)-dependent decrease in I(Ca) was not significantly different when myocytes were preincubated with 10 microM forskolin and 300 microM 3-isobutyl-L-methylxanthine and voltage-clamped with pipettes containing 50 microM okadaic acid, to maximize Ca(2+) channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, I(Ca) decreased only 25 +/- 3.4% on changing [Mg(2+)](p) from 0.2 to 1.8 mM. In the presence of 0.2 mM[Mg(2+)](p), changing channel phosphorylation conditions altered I(Ca) over a 4-fold range; however, with 1.8 mM[Mg(2+)](p), these same manoeuvres had a much smaller effect on I(Ca). These data suggest that [Mg(2+)](i) can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca(2+)](p) to 1, 100 or 300 nM also showed that the [Mg(2+)](p)-induced reduction of I(Ca) was smaller at the lowest [Ca(2+)](p), irrespective of channel phosphorylation conditions. This interaction between [Ca(2+)](i) and [Mg(2+)](i) to modulate I(Ca) was not significantly affected by ryanodine, fast Ca(2+) buffers or inhibitors of calmodulin, calmodulin-dependent kinase and calcineurin. Thus, physiologically relevant [Mg(2+)](i) modulates I(Ca) by counteracting the effects of Ca(2+) channel phosphorylation and by an unknown [Ca(2+)](i)-dependent mechanism. The magnitude of these effects suggests that changes in [Mg(2+)](i) could be critical in regulating L-type channel gating.

Absence of modulation of the expressed calcium channel alpha1G subunit by alpha2delta subunits.

1. The modulatory action of the alpha2delta subunit on various high-voltage-activated calcium channels has been demonstrated previously. However, very little is known about auxiliary subunit modulation of low-voltage-activated (LVA) calcium channels. We have examined the modulation of the alpha1G subunit corresponding to the neuronal T-type calcium channel by the ubiquitously expressed alpha2delta-1 and brain-specific alpha2delta-3 subunits. 2. The alpha1G subunit was expressed alone or in combination with either the alpha2delta-1 or alpha2delta-3 subunit in human embryonic kidney (HEK 293) cells and whole-cell barium currents were measured. The current density-voltage relationships for peak and sustained current, kinetics of current activation and inactivation, voltage dependence of current inactivation and time course of the recovery from inactivation were analysed for each type of expressed channel. No significant difference was found for any of the examined parameters. 3. These results suggest that the LVA alpha1G channel is not regulated by known auxiliary alpha2delta subunits.

Effect of alkaloid toxins from tropical marine sponges on membrane sodium currents

Dibromosceptrin and clathrodin are alkaloid compounds purified from tropical marine sponges of the genus Agelas. Experiments done using the whole cell configuration of the patch clamp technique revealed that these compounds have neurotoxic activity. Both compounds decreased by 27–40% the average maximum amplitudes of pharmacologically isolated inward sodium currents in cells isolated from chick embryo sympathetic ganglia. Current-voltage data, fitted using Boltzmann's equation, did not show any effect of these agents on the voltage dependence of current activation. However, the voltage dependence of current inactivation was shifted toward more negative potentials by dibromosceptrin, changing by an average of 20 mV the voltage for 50% inactivation. In contrast, clathrodin shifted this voltage dependence of inactivation toward more positive potentials and changed the voltage for 50% inactivation by 14 mV. Time for current reactivation was not altered by clathrodin but was slightly prolonged by dibromosceptrin. Similarly, dibromosceptrin was more effective than clathrodin in delaying the time course of current decay. Thus, these two alkaloids appear to be new sodium channel neurotoxins acting through different mechanisms, dibromosceptrin modifying the channel inactivation characteristics and clathrodin probably influencing channel ionic conductance.

The effects of dihydropyridine derivatives on force and Ca2+ current in frog skeletal muscle fibres.

1. The effects of dihydropyridine (DHP) derivatives on current through the slow Ca2+ channel and on isometric force were investigated in short toe muscle fibres of the frog (Rana temporaria). The experiments were performed under voltage-clamp conditions with two flexible internal microelectrodes. 2. The non-chiral DHP derivative nifedipine was used mainly because it allowed control measurements after the inactivation of the drug with UV light. 3. In a TEA sulphate solution containing 4 mM-free Ca2+, nifedipine (1 microM) caused no relevant alterations in the time course of successive contractures induced by depolarizing steps to 0 mV of 3.5 min duration followed by a restoration time at -90 mV of 1.5 min. 4. When external Ca2+ was replaced by Mg2+, nifedipine caused a dose-dependent shortening of contractures. The effect reached saturation at about 50% of shortening with 1-5 microM-nifedipine. In the absence of divalent cations and with Na+ being the only metallic cation in the solution, shortening became more pronounced and maximum force decreased. 5. The application of 2 microM-nifedipine to a Ca2(+)-free, Mg2(+)-containing solution shifted the voltage dependence of force inactivation by 5-10 mV to more negative potentials. 6. Force activation was facilitated by nifedipine. In the presence of 2 microM-nifedipine in a Ca2(+)-containing solution, threshold potentials (rheobase) as negative as -75 mV were measured under microscopical observation. UV irradiation shifted the threshold potential back to the normal value of about -50 mV. 7. The slow Ca2+ inward current was blocked almost completely by approximately 5 microM-nifedipine, even when induced from negative holding potentials (-90 to -120 mV), i.e. under conditions where normal phasic contractures could still be observed. 8. Nifedipine (0.8 microM) caused a shift of the voltage dependence of current inactivation (V0.5) by 4 mV from -26 to -30 mV and at negative holding potentials (-90 mV), a reduction of maximum current by 35%. 9. The voltage dependence of current activation was not significantly altered by nifedipine (2 microM). 10. It is assumed that nifedipine binds with low affinity to the resting state of the DHP receptor (KD 0.8 microM) and with high affinity (KD 1 nM) to the inactivated and the active state (or to a precursor of this state). These assumptions could explain the relatively small shift of the inactivation curves (points 5 and 8) to more negative potentials and the facilitation of force activation (point 6).

Voltage- and use-dependent modulation of cardiac calcium channels by the dihydropyridine (+)-202-791.

The modulation of L-type voltage sensitive calcium channels in isolated guinea pig ventricular myocytes by the dihydropyridine (+)-202-791 was examined with the whole-cell voltage-clamp technique with 1.8 mM Ba or Ca as the charge carrier. Striking voltage- and use-dependent effects of the dihydropyridine calcium channel "agonist" (+)-202-791 were revealed. From a holding potential of -60 mV, depolarizing test pulses in the presence of (+)-202-791 demonstrated a concentration-dependent (EC50, 177 nM) increase in the measured peak inward barium current compared to control. In contrast, more depolarized holding potentials (greater than or equal to -30 mV) (+)-202-791 caused a biphasic effect on the peak inward current resulting in a transient enhancement followed by a steady-state block. A saturable, concentration-dependent hyperpolarizing shift in the voltage dependence of current inactivation was observed in the presence of (+)-202-791 with an EC50 of 10.2 nM. The voltage dependence of current activation was also shifted in the hyperpolarizing direction in the presence of (+)-202-791. A use-dependent relative block by (+)-202-791 was observed after repetitive depolarizing test pulses at a frequency of 2 Hz. Thus, the single enantiomer (+)-202-791 can result in either an increase in the whole cell calcium channel current (favored by hyperpolarized holding potentials and low rates of stimulation) or block of calcium channel current (favored by depolarized holding potentials and high rates of stimulation). Various combinations of (-)-202-791, a reported calcium channel antagonist, and (+)-202-791 resulted in intermediate effects on voltage sensitive calcium or barium currents compared with the presence of either enantiomer alone, and no clear cooperative interactions between the enantiomers were observed in contrast to a previous single channel study (Kokuban S, Prod'ham B, Becker C, Porzig H, Reuter H: Studies on Ca channels in intact cardiac cells: Voltage-dependent effects and cooperative interaction of dihydropyridine enantiomers. Mol Pharmacol 1986;30:571-584). The results are discussed in relation to the possible presence of multiple dihydropyridine receptors associated with the voltage sensitive calcium channel.