Single calcium channels and their inactivation

Inactivation of single Ca channels in snail neurons was examined to test the idea that entering Ca ions react directly with the channel to produce this effect. Simulations of specific models were used for comparison with the experimental data. The Ca-dependent model predicts time-dependent changes in the single-channel events which were not found experimentally. It is possible that Ca-dependent inactivation is mediated by a Ca-binding protein that is associated with but not part of the channel itself.     

Calcium-dependent sodium currents inParamecium: Mutational manipulations and effects of hyper- and depolarization

Summary The membrane ofParamecium generates a Ca-dependent Na current upon depolarization. There is, however, also a Na current upon hyperpolarization in this membrane. The second Na current was analyzed under voltage clamp and found to have properties identical to those of the first. Both currents could be carried by Na and Li ions and not by K, Cs or choline ion. They were eliminated by either EGTA injection into the cell or Ca removal from the bath. Both currents were eliminated by a single-gene mutation,fast-2, that had no effect on Ca currents. These findings strongly suggest that these two currents are through the same Ca-dependent Na conductance. A hyperpolarization-induced Ca current was also identified, which served to activate the second Na current. These observations support a model that theParamecium membrane has two Ca channels with different voltage dependencies and only one Na channel, which is elicited by a rise of the itternal free Ca²⁺ concentration. The function of the Ca-dependent Na conductance is discussed.     

Multiple components of synaptosomal [3H]-gamma-aminobutyric acid release resolved by a rapid superfusion system

Release of [3H]-gamma-aminobutyric acid ([3H]GABA) from rat brain synaptosomes was studied with 60-ms time resolution, using a novel rapid superfusion method. Synaptosomes were prelabeled with [3H]GABA via an associated GABA uptake system. KCl depolarization stimulated at least three distinct components of GABA release: (1) a phasic Ca-dependent component, which develops rapidly and decays with a time constant of at most 60 ms; (2) a tonic Ca-dependent component that persists after KCl depolarization is ended; (3) a Ca-independent component. The three components of GABA release are pharmacologically distinct. The phasic component was selectively blocked by 50 microM Cd2+, while the tonic component was selectively blocked by 100 microM Ni2+. The Ca-independent component was selectively blocked by nipecotic acid (IC50 = 21 microM), a known inhibitor of Na+-dependent GABA uptake. The time course and amplitude of Ca-dependent GABA release evoked by the Ca2+ ionophore A23187 were nearly identical with Ca-dependent release evoked by depolarization. This result indicates that Ca-dependent GABA release depends primarily on Ca2+ entry into the nerve terminal, and not depolarization, per se. The properties of the phasic component suggest that it is normally initiated by a voltage-sensitive Ca2+ channel that is functionally and pharmacologically distinct from those previously described. The Ca-independent component of GABA release is probably mediated by reversal of the Na-dependent, electrogenic GABA uptake system. The ability to identify multiple components of GABA release on a physiologically relevant time scale may afford a more precise definition of the mechanism of action of drugs thought to affect neurotransmission in the brain.     

Inactivation of calcium channel current in the calf cardiac Purkinje fiber. Evidence for voltage- and calcium-mediated mechanisms

We have studied the influence of divalent cations on Ca channel current in the calf cardiac Purkinje fiber to determine whether this current inactivates by voltage- or Ca-mediated mechanisms, or by a combination of the two. We measured the reversal (or zero current) potential of the current when Ba, Sr, or Ca were the permeant divalent cations and determined that depletion of charge carrier does not account for time-dependent relaxation of Ca channel current in these preparations. Inactivation of Ca channel current persists when Ba or Sr replaces Ca as the permeant divalent cation, but the voltage dependence of the rate of inactivation is markedly changed. This effect cannot be explained by changes in external surface charge. Instead, we interpret the results as evidence that inactivation is both voltage and Ca dependent. Inactivation of Sr or Ba currents reflects a voltage-dependent process. When Ca is the divalent charge carrier, an additional effect is observed: the rate of inactivation is increased as Ca enters during depolarizing pulses, perhaps because of an additional Ca-dependent mechanism.     

Regulation of type 1 inositol 1,4,5-trisphosphate-gated calcium channels by InsP3 and calcium: Simulation of single channel kinetics based on ligand binding and electrophysiological analysis.

Cytosolic calcium acts as both a coagonist and an inhibitor of the type 1 inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel, resulting in a bell-shaped Ca dependence of channel activity (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature. 351:751-754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science. 252: 443-446; Iino, M. 1990. J. Gen. Physiol. 95:1103-1122). The ability of Ca to inhibit channel activity, however, varies dramatically depending on InsP3 concentration (Combettes, L., Z. Hannaert-Merah, J.F. Coquil, C. Rousseau, M. Claret, S. Swillens, and P. Champeil. 1994. J. Biol. Chem. 269:17561-17571; Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). In the present report, we have extended the characterization of the effect of cytosolic Ca on both InsP3 binding and InsP3-gated channel kinetics, and incorporated these data into a mathematical model capable of simulating channel kinetics. We found that cytosolic Ca increased the Kd of InsP3 binding approximately 3.5-fold, but did not influence the maximal number of binding sites. The ability of Ca to decrease InsP3 binding is consistent with the rightward shift in the bell-shaped Ca dependence of InsP3-gated Ca channel activity. High InsP3 concentrations are able to overcome the Ca-dependent inhibition of channel activity, apparently due to a low affinity InsP3 binding site (Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). Constants from binding analyses and channel activity determinations were used to develop a mathematical model that fits the complex Ca-dependent regulation of the type 1 InsP3-gated Ca channel. This model accurately simulated both steady state data (channel open probability and InsP3 binding) and kinetic data (channel activity and open time distributions), and yielded testable predictions with regard to the regulation of this intracellular Ca channel. Information gained from these analyses, and our current molecular model of this Ca channel, will be important for understanding the basis and regulation of intracellular Ca waves and oscillations in intact cells.     

Interleukin 2-mediated events in gamma-interferon production are calcium dependent at more than one site

Both leukotrienes and dibutyryl cyclic GMP can replace the interleukin 2 (IL 2) requirement for gamma-interferon (gamma-IFN) production. In this study, the Ca dependence of the IL 2 help was demonstrated by blockage of gamma-IFN production by the Ca blocker Mn, and the competitive reversal of this block by Ca. Neither leukotriene C4 nor dibutyryl cyclic GMP could reverse the Mn block, which suggests that arachidonic acid release from phospholipids is not the only Ca-dependent event in IL 2 help for gamma-IFN production. A role for calmodulin or protein kinase C in the IL 2-mediated events was suggested by the blockage of gamma-IFN production by chlorpromazine. Relatively high concentrations of Ca were able to replace the IL 2 helper effects. Consistent with this were Ca influx experiments that showed that IL 2 helper signals for gamma-IFN production involved activation of a Ca channel.     

Cyclic AMP enhances calcium-dependent potassium current inAplysia neurons

The effect on the Ca-dependent potassium current, IK(Ca), of procedures that increase intracellular cAMP levels was studied in Aplysia neurons using three different pharmacological approaches. Exposure to cAMP analogues which were either resistant to or protected from phosphodiesterase hydrolysis caused an increase in IK(Ca) from 30 to 50% in 10 min. The degree of reversibility of this effect varied from complete with db cAMP to very little with pcpt cAMP. Exposure to cholera toxin, which stimulates the synthesis of endogenous cAMP, increased IK(Ca) 25% in 10 min and the effect was not reversible. Both approaches were effective in all seven neuron types studied. Application of serotonin plus phosphodiesterase inhibitor caused an increase in IK(Ca) in neuron R15 but not in the other neuron types. Application of pentylene tetrazole (PTZ) led to a decrease in IK(Ca). It is proposed that elevation of cyclic AMP mediates an increased sensitivity of the IK(Ca) channel to Ca ions.     

Structural Regions of the Cardiac Ca Channel α1C Subunit Involved in Ca-dependent Inactivation

We investigated the molecular basis for Ca-dependent inactivation of the cardiac L-type Ca channel. Transfection of HEK293 cells with the wild-type α_1C or its 3′ deletion mutant (α_1C−3′del) produced channels that exhibited prominent Ca-dependent inactivation. To identify structural regions of α_1C involved in this process, we analyzed chimeric α₁ subunits in which one of the major intracellular domains of α_1C was replaced by the corresponding region from the skeletal muscle α_1S subunit (which lacks Ca-dependent inactivation). Replacing the NH₂ terminus or the III–IV loop of α_1C with its counterpart from α_1S had no appreciable effect on Ca channel inactivation. In contrast, replacing the I–II loop of α_1C with the corresponding region from α_1S dramatically slowed the inactivation of Ba currents while preserving Ca-dependent inactivation. A similar but less pronounced result was obtained with a II–III loop chimera. These results suggest that the I–II and II–III loops of α_1C may participate in the mechanism of Ca-dependent inactivation. Replacing the final 80% of the COOH terminus of α_1C with the corresponding region from α_1S completely eliminated Ca-dependent inactivation without affecting inactivation of Ba currents. Significantly, Ca-dependent inactivation was restored to this chimera by deleting a nonconserved, 211–amino acid segment from the end of the COOH terminus. These results suggest that the distal COOH terminus of α_1S can block Ca-dependent inactivation, possibly by interacting with other proteins or other regions of the Ca channel. Our findings suggest that structural determinants of Ca-dependent inactivation are distributed among several major cytoplasmic domains of α_1C.     

Ionophore A23187, Verapamil, Protonophores, and Veratridine Influence the Release of γ-Aminobutyric Acid from Synaptosomes by Modulation of the Plasma Membrane Potential Rather than the Cytosolic Calcium

The release of GABA induced by veratridine shows no correlation with the synaptosomal Ca content and is therefore not mediated by the release of mitochondrial Ca. Instead, with both Ca-repleted and -depleted synaptosomes, the extent of GABA efflux is correlated with the decrease in plasma membrane potential. The slow release of GABA induced by protonophores and the Ca-dependent release induced by ionophore A23187 are also consequences of the depolarization of the plasma membrane, rather than of elevated cytosolic Ca. Finally, the ability of verapamil to inhibit the release of GABA induced by low veratridine concentrations is due to the ability of the Ca channel inhibitor to antagonize the action of veratridine, rather than to inhibit Ca entry into the synaptosome. It is concluded that it is essential to monitor plasma membrane potentials in experiments in which amino acid efflux from synaptosomes is induced.     

Protein synthesis is required for the transition to Ca(2+)-dependent regulated secretion in progesterone-matured Xenopus oocytes

Calcium (Ca) ionophores trigger cortical granule exocytosis in progesterone-matured Xenopus oocytes (eggs), but not in immature oocytes. Prior work suggested that this secretory transition involved a Ca-dependent isoform of protein kinase C (PKC). To address this possibility, we treated eggs with several different inhibitors of Ca-dependent PKCs. Although these agents (eg., staurosporine, Ro31-8220) completely blocked cortical granule exocytosis that is triggered in eggs by phorbol esters, they had no impact on ionomycin-evoked secretion of cortical granule lectin. These data suggest that Ca-dependent PKCs do not mediate secretory triggering in eggs. Instead, further investigation revealed that protein synthesis (but not RNA synthesis) was required for eggs to secrete in response to ionomycin. Moreover, we observed that when oocytes were matured by injection of maturation promoting factor (MPF), they failed to secrete in response to ionomycin. Collectively, these results suggest that the progesterone-dependent maturation pathway induces these cells either to synthesize de novo, a protein that mediates Ca-dependent secretory triggering, or that intrinsic Ca-sensing machinery is modified in a protein-synthesis-dependent fashion. Initial efforts to distinguish between these possibilities (using Ca overlay, pharmacological and immunoblot strategies) revealed that such Ca-binding proteins as calmodulin, synaptotagmin1, CAPS, rabphilin-3A and calcineurin were unlikely to transduce the secretory effects of ionomycin in eggs. Thus, the cortical reaction in these cells may rely on a novel mechanism for initiating Ca-dependent exocytosis.     

Separation and investigation of the regulatory properties of two forms of cyclic nucleotide phosphodiesterase from rabbit heart--sensitive and insensitive to Ca-dependent regulator protein]

Two forms of cyclic nucleotide phosphodiesterase (ES 3.1.4.17)--PDE-I and PDE-II--sensitive and resistant to Ca-dependent protein regulator, were isolated from the soluble fraction of rabbit heart by chromatography on DEAE-cellulose. Both forms of enzyme are inhibited by 30--50% by Ca2+ (10(-4) M). Addition of Ca-dependent protein regulator activates PDE-I and eliminates Ca2+-induced inhibition of PDE-II. In heart extract Ca2+ increases the phosphodiesterase activity 1.5-fold. The amount of PDE-I makes up to about 10% of total phosphodiesterase activity of the heart; that of PDE-II is about 90%. In the presence of Ca-dependent protein regulator the rate of 3', 5'-AMP hydrolysis by PDE-I is increased 5--15-fold, while that of 3', 5'-GMP hydrolysis only 2.5-fold. Both PDE-I and PDE-II have close Km values for substrates--(3.5--4.0).10(-6) M for 3', 5'-AMP and 14.10(-6) M for 3', 5'-GMP. Inhibition by Ca2+ and effect of Ca-dependent protein regulator manifest themselves in changes in V for cyclic nucleotide hydrolysis and do not alter the Km value for the enzyme.     

Inhibition of cardiac slow action potentials by 8-bromo-cyclic GMP occurs independent of changes in cyclic AMP levels

Cyclic GMP inhibits the slow inward Ca current of cardiac cells. This effect could be due to a cyclic GMP-mediated phosphorylation of the Ca channel (or some protein modifying Ca channel activity), or alternatively, to enhanced degradation of cyclic AMP owing to stimulation of a phosphodiesterase by cyclic GMP. To test the latter possibility, we examined the effect of extracellular 8-bromo-cyclic GMP on cyclic AMP levels in guinea pig papillary muscles, in parallel with electrophysiological experiments. Isoproterenol (10(-6) M) significantly increased the cyclic AMP levels and induced Ca-dependent slow action potentials. Superfusion with 8-bromo-cyclic GMP (10(-3) M) inhibited the slow action potentials induced by isoproterenol. However, muscles superfused with 8-bromo-cyclic GMP had cyclic AMP levels identical to those of muscles superfused with isoproterenol alone. Similarly, 8-bromo-cyclic GMP had no effect on the increase in cyclic AMP levels of muscles treated with forskolin (10(-6) M) or histamine (10(-6) M). We conclude that the inhibitory effect of cyclic GMP on slow Ca channels in guinea pig ventricular cells is not due to a decrease in the cyclic AMP levels. We hypothesize that a cyclic GMP-mediated phosphorylation is the most likely explanation for the Ca channel inhibition observed in this preparation.     

Acetylcholine suppresses calcium current in neurons of Aplysia californica

1. The left upper quadrant neurons L2-L6 in the abdominal ganglion of Aplysia californica were voltage clamped in order to examine effects of acetylcholine on voltage-dependent Ca and Ca-dependent K currents. 2. "Puffed" application of 10-100 microM acetylcholine reduced both the early inward and late outward phases of the current elicited by depolarizing voltage steps. An identical effect of the peptide FMRFamide was previously found to result from a suppression of the Ca and Ca-dependent K currents. 3. This effect of acetylcholine was obscured by the simultaneous activation of a previously described K current resembling the "S" current. Extracellular tetraethylammonium (TEA) and 4-aminopyridine could not be used to eliminate this current, because both compounds also appeared to block the acetylcholine receptor mediating the putative suppression of Ca and Ca-dependent K currents. 4. The acetylcholine-induced "S"-like and other K currents could, however, be reduced or eliminated by injection of TEA+ or Cs+ into the cell, replacement of extracellular Ca2+ with Ba2+, and by shifting the K+ equilibrium potential so as to null K currents at the potential used to record Ca current, revealing in each case a partial (10-40%) suppression of the Ca (or Ba) current by acetylcholine. 5. The reduction of the outward phase of depolarization-activated current was confirmed to represent suppression of the Ca-dependent K current by acetylcholine. This effect was indirect, secondary to the suppression of Ca current, since acetylcholine had no effect on Ca-dependent K current elicited by direct injection of Ca2+ into the cell. 6. Activation of the "S"-like K current and suppression of the Ca current by FMRFamide are likely to be important in its proposed role as an agent of presynaptic inhibition in Aplysia. Since acetylcholine has identical effects, it too may have such a function.     

Development of ionic conductances in neurons of the inferior olive in the rat: an in vitro study

Neurons of the inferior olive of the rat were studied at different stages of their postnatal (PN) development by using the current clamp technique in slices maintained in vitro. Antidromic and synaptic activation of inferior olivary neurons could be achieved in preparations as young as PN day 2. Neurons at this age already exhibited a variety of ionic conductances which included fast sodium-dependent spikes, high-threshold and low-threshold calcium spikes, potassium-dependent currents, Ca-dependent after-hyperpolarizing potentials (AHPS), and both instantaneous and time-dependent inward rectification at hyperpolarized levels of membrane potential. The two types of Ca-dependent responses recorded in olivary neurons during the first postnatal week were graded with the magnitude of the depolarization imposed on the cells. Furthermore, the high-threshold Ca spikes were only clearly observed during this early period when K conductances were depressed by the injection of caesium into the cells or by bath application of 4-aminopyridine. In contrast, the high-threshold Ca spikes could be obtained without suppression of K currents and were all-or-none in character in some neurons after PN day 8 and in all neurons after PN day 11. The observations suggest that the balance between K and Ca currents changes throughout maturation and is largely in favour of the K current until about the end of the first PN week. At all ages studied, the low-threshold Ca spikes were much less sensitive to the Ca channel blocker cadmium than were the high-threshold Ca spikes. Finally, spontaneous, regular oscillations of the membrane potential were observed for the first time at PN day 16 and were only commonly observed after PN day 19, suggesting a late development of electrotonic coupling between olivary neurons.     

2 types of neurons differing in their plastic properties: study of ionic mechanisms

Plasticity ionic mechanisms of 2 types of neurones identified in the brain of snail Helix pomatia: habituating and non-habituating to rhythmic intracellular stimulation by DC pulses were studied. It has been shown that the development of habituation is due to entering of Ca2+ into the cell and by its activation of Ca-dependent K-conductivity of the membrane, leading to hyperpolarization, decrease of input resistance and elimination of spike response to stimulation. Depression of Ca-dependent K-conductivity by quinine totally blocks the ability of the neurons to habituate to stimulation. The data obtained on non-habituating neurons testify that their non-habituation to intracellular stimulation results from their absence or weak manifestation of Ca-dependent K-conductivity in their membrane. Ca2+ entering non-habituating cells during electrical stimulation may have depolarizing and facilitating effects.     

Role of calcineurin in regulation of high voltage-activated calcium channel activity

In the Review, personal data of the Author and the data of other experimenters on calcium/calmodulin-dependent protein phosphatase-2B (calcineurin) are summarized and analyzed; the role of this enzyme in regulation of the calcium channel activity in the membrane of excitable cells is discussed. A two-phase mechanism of Ca-dependent suppression of the activity of Ca channels in the molluscan neurons is described in details. Special attention is paid to the analysis of changes in the activity of Ca channels in the transfected hybrid cells overexpressing calcineurin.     

Regulation of nerve terminal calcium channel selectivity by a weak acid site.

The effects of low pH, and of alkaline earth cations, were examined on calcium uptake by pinched-off nerve terminals (synaptosomes). This uptake appears to be mediated by voltage-sensitive Ca channels (J. Physiol. 247:617, 1975). Ca uptake was measured in low (5 mM) or high (77 mM) potassium media. The extra uptake promoted by depolarizing (K-rich) media was almost maximal at pH 7.5, and decreased as the pH was lowered. Data relating depolarization-induced 45Ca uptake to pH fit a titration curve with a pKa approximately 6. Experiments in which Ca concentration and pH were both varied indicated that Ca2+ and H+ compete for a common binding site. Inhibition of depolarization-induced 45Ca uptake by the alkaline earth cations was studied to determine the apparent binding sequence for these cations in the Ca channels: Ca greater than Sr greater than Ba greater than Mg. This sequence resembles that observed for block of Ca channels in other preparations. The apparent binding sequence of the alkaline earth cations and the apparent pKa (approximately 6) of the Ca-binding site indicate that the Ca channel is a "high field strength" system. Protonation of a Ca channel binding site could explain the inhibitory effect of low pH on Ca-dependent neurotransmitter release (cf. Del Castillo et al., J. Cell. Comp. Physiol. 59:35, 1962).     

Calcium currents recorded from a neuronal α1C L-type calcium channel in Xenopus oocytes

Xenopus oocytes expressing neuronal α_1C, α₂ and β_1b calcium channel subunit cDNAs were used in this study. During two-electric voltage clamp recording the oocyte was injected with 10–20 nl of a 100 mM BAPTA solution. Under these conditions, the endogenous Ca-activated Cl current was completely suppressed resulting in an α_1C Ba current free from Cl current contamination. BAPTA injection also allowed α_1C currents with different permeating ions, including Ca, to be examined. Compared to Ba and Sr, α_1C whole cell Ca currents were smaller in magnitude and showed kinetic and voltage-dependent properties more similar to those for L-type Ca currents recorded in native cells. That Ca-dependent inactivation occurs in BAPTA-buffered cells suggests that the Ca-binding site involved in this type of inactivation is very close to the pore of the channel.     

Inactivation of calcium channels in single vascular and visceral smooth muscle cells of the guinea-pig.

Inactivation of currents carried through calcium channels by calcium (ICa), barium (IBa) and monovalent cations (In.s.) was studied in single smooth muscle cell (SMC) of the guinea-pig coronary artery and taenia caeci by the whole-cell patch-clamp method. The rate of ICa inactivation in the coronary artery SMC was correlated with ICa amplitude, and acceleration was observed with the increasing ICa peak amplitude. The availability curve of ICa in double-pulse experiments was found to be U-shaped, however, no complete restoration of ICa availability was observed. Inactivation of IBa was considerably slower than that of ICa. These findings may indicate that inactivation of calcium channels in the membrane of coronary artery SMC is, at least partially, a Ca-dependent process. However, some facts observed contradict the validity of this hypothesis for coronary artery SMC in contrast to taenia caeci: 1) elevation of external Ca2+ concentration did not affect the time course of ICa inactivation; 2) inactivation of In.s., i.e. without calcium entry into the cell, was faster than that of ICa. It was concluded that the characteristics of Ca channel inactivation were changed by the removal of divalent cations from extracellular solution. Differences and similarities in Ca channel inactivation between coronary artery and taenia caeci SMC are discussed.     

The effect of ethanol on the passive Ca permeability of human red cell ghosts measured by means of arsenazo III

Ethanol in the range of 0.76-2.40 M caused an immediate increase in the Ca permeability of the plasma membrane of resealed human red blood cell ghosts in which intracellular free Ca could be continuously monitored by means of the Ca chromophore arsenazo III. At a given concentration of ethanol, the Ca permeability increased markedly a few minutes following the mixing of the ghosts and the ethanol, and continued to increase over at least the next 30 min. Preincubating the ghosts in ethanol for 15, 60 and 120 min before measuring the rate of free Ca accumulation, progressively increased the effect of a given concentration of ethanol. These results indicate that the effect of a given concentration of ethanol is a complex function of concentration and exposure time. The effects of ethanol in this concentration range were completely reversible. The resealed ghosts used in these experiments were depleted of ATP to avoid interference from the Ca pump and all experiments were carried out with 150 mM KCl on both sides of the membrane to minimize changes in either the volume or membrane potential associated with activation of the Ca-dependent K channel.