Enhancement Of Calcium Current Research Articles

β-Adrenoceptor activation enhances L-type calcium channel currents in anterior piriform cortex pyramidal cells of neonatal mice: implication for odor learning

Early odor preference learning occurs in one-week-old rodents when a novel odor is paired with a tactile stimulation mimicking maternal care. β-Adrenoceptors and L-type calcium channels (LTCCs) in the anterior piriform cortex (aPC) are critically involved in this learning. However, whether β-adrenoceptors interact directly with LTCCs in aPC pyramidal cells is unknown. Here we show that pyramidal cells expressed significant LTCC currents that declined with age. β-Adrenoceptor activation via isoproterenol age-dependently enhanced LTCC currents. Nifedipine-sensitive, isoproterenol enhancement of calcium currents was only observed in post-natal day 7–10 mice. APC β-adrenoceptor activation induced early odor preference learning was blocked by nifedipine coinfusion.

Involvement of protein kinase C and protein kinase A in the enhancement of L-type calcium current by GABA B receptor activation in neonatal hippocampus

In the early neonatal period activation of GABA B receptors attenuates calcium current through N-type calcium channels while enhancing current through L-type calcium channels in rat hippocampal neurons. The attenuation of N-type calcium current has been previously demonstrated to occur through direct interactions of the βγ subunits of G i/o G-proteins, but the signal transduction pathway for the enhancement of L-type calcium channels in mammalian neurons remains unknown. In the present study, calcium currents were elicited in acute cultures from postnatal day 6–8 rat hippocampi in the presence of various modulators of protein kinase A (PKA) and protein kinase C (PKC) pathways. Overnight treatment with an inhibitor of G i/o (pertussis toxin, 200 ng/ml) abolished the attenuation of calcium current by the GABA B agonist, baclofen (10 μM) with no effect on the enhancement of calcium current. These data indicate that while the attenuation of N-type calcium current is mediated by the G i/o subtype of G-protein, the enhancement of L-type calcium current requires activation of a different G-protein. The enhancement of the sustained component of calcium current by baclofen was blocked by PKC inhibitors, GF- 109203X (500 nM), chelerythrine chloride (5 μM), and PKC fragment 19–36 (2 μM) and mimicked by the PKC activator phorbol-12-myristate-13-acetate (1 μM). The enhancement of the sustained component of calcium current was blocked by PKA inhibitors H-89 (1 μM) and PKA fragment 6–22 (500 nM) but not Rp-cAMPS (30 μM) and it was not mimicked by the PKA activator, 8-Br-cAMP (500 μM–1 mM). The data suggest that activation of PKC alone is sufficient to enhance L-type calcium current but that PKA may also be involved in the GABA B receptor mediated effect.

Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway.

The small G-protein Ras, a critical component in the signalling pathways regulating cell growth, is involved in the tonic upregulation of voltage-dependent calcium channels (VDCCs) in rat sensory neurones. To investigate which downstream effector(s) of Ras is involved in this process, a series of Ras mutant cDNAs were co-expressed with green fluorescent protein (GFP) in primary cultured rat dorsal root ganglion neurones (DRGs). Constitutively active V12Ras (glycine 12 to valine) markedly increased basal calcium current density by 41 % compared with control cells (GFP alone). In contrast, a farnesylation-defective mutant, V12S186Ras (cysteine 186 to serine; activates no downstream effectors), significantly reduced calcium current density by 47 %. Ras effector region mutants V12C40 (tyrosine 40 to cysteine; activates the p110 alpha-subunit of phosphatidylinositol 3-kinase) and V12G37 (glutamic acid 37 to glycine; activates Ral guanine nucleotide dissociation stimulator) had no significant effect on VDCC current. However, V12S35Ras (threonine 35 to serine; activates Raf-1 and the mitogen-activated protein kinase (MAPK) pathway) markedly increased basal calcium current density by 67 %, suggesting that Raf-1 activation is sufficient for Ras enhancement of calcium current in these cells. Raf-1 activates MEK (MAPK kinase) in the MAPK pathway, and the MEK inhibitor U0126 reduced calcium current by 45 % after 10-15 min, whereas the inactive analogue U0124 had no effect. This rapid time course for MEK inhibition suggests direct modulation of VDCCs via the Ras-MAPK pathway rather than gene expression-mediated effects. The relative proportions of omega-conotoxin GVIA- and nicardipine-sensitive N- ( approximately 40 %) and L- ( approximately 40 %) type currents were unaffected by either V12S35Ras expression or U0126 pre-treatment, suggesting that all components of calcium current in DRGs, are enhanced via this pathway.

Potassium currents in isolated CA1 neurons of the rat after kindling epileptogenesis

Daily tetanic stimulation of the Schaffer collaterals generates an epileptogenic focus in area CA1 of the rat hippocampus, ultimately leading to generalized tonic-clonic convulsions (kindling). Potassium currents were measured under voltage-clamp conditions in pyramidal neurons, acutely dissociated from the focus of fully kindled rats, one day and six weeks after the last generalized seizure. Their amplitude, kinetics, voltage dependence and calcium dependence were compared with controls. With Ca2+ influx blocked by 0.5 mM Ni2+, the sustained current (delayed rectifier) and the transient current (A-current) were not different after kindling. Calcium influx evoked an additional fast transient current component. This transient calcium-dependent current component was increased by 154%, but only immediately after the seizure. A second, slow calcium-dependent potassium current component was dependent on the intracellular calcium level, set by the pipette as well as on calcium influx. The peak amplitude of this slow calcium-dependent current was under optimal calcium conditions not different after kindling, but we found indications that either calcium homeostasis or the calcium sensitivity of the potassium channels was affected by the kindling process. In contrast to the previously described enhancement of calcium current, kindling epileptogenesis did not change the total potassium current amplitude. The minor changes that were observed can be related either to changes in calcium current or to changes in intracellular calcium homeostasis.

Kindling-induced long-lasting enhancement of calcium current in hippocampal CA1 area of the rat: Relation to calcium-dependent inactivation

Daily tetanization of the Schaffer collaterals (kindling) in the rat hippocampus induces a persistent epileptogenic focus in area CA1. Neurons were enzymatically isolated from the focal region one day or six weeks after seven class V generalized seizures had been evoked. Calcium currents were measured under voltage-clamp conditions in the whole-cell patch configuration. One day after kindling, as well as six weeks later, the amplitudes of a slow-inactivating (τ = 90ms) and a non-inactivating calcium current component were, in comparison to controls, enhanced by 30 and 40%, respectively. This enhancement was therefore related to the kindled state of enhanced excitability. The enhancement of the calcium current was independent of the steady-state intracellular calcium concentration. Fast calcium-dependent inactivation was provoked with double-pulse protocols that conditioned the neuron with a defined calcium-influx in the first pulse. Despite the larger calcium current during the conditioning pulse, the relative calcium-dependent inactivation of the sustained current component was reduced in neurons from the kindled focus. Repetitive depolarizations, once every second, evoked a cumulative calcium-dependent inactivation. Nothwithstanding the larger calcium current, kindling also persistently reduced this slow inactivation of both transient and sustained high threshold calcium current. The reduction in calcium-dependent inactivation cannot be responsible for the increased current, but can certainly enhance the calcium influx during prolonged activation or seizures. The changes can be explained by assuming that additional calcium channels are recruited at a location that prevents calcium-dependent inactivation.

Characterization of adenylate cyclase toxin from a mutant of Bordetella pertussis defective in the activator gene, cyaC.

Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin which is able to bind to target cells but unable to elicit cAMP accumulation. In that AC toxin from BPDE386 is able to function normally when injected artificially to an intracellular site, we conclude that the disruption of cyaC produces a defect in insertion and transmembrane delivery of the catalytic domain.

Pertussis toxin blocks dynorphin A(DYN)-mediated reduction and CGRP-mediated enhancement of calcium currents in rat dorsal root ganglion neurons: Evidence that DYN-mediated reduction involves Go subtype of G-protein

Pertussis toxin blocks dynorphin A(DYN)-mediated reduction and CGRP-mediated enhancement of calcium currents in rat dorsal root ganglion neurons: Evidence that DYN-mediated reduction involves Go subtype of G-protein

The peptide CGRP increases a high-threshold Ca2+ current in rat nodose neurones via a pertussis toxin-sensitive pathway.

1. The whole-cell variation of the patch clamp technique was used to study the effect of calcitonin gene-related peptide (CGRP) on voltage-gated calcium currents in acutely dissociated rat nodose ganglion neurones and to determine if its effects were mediated via a guanine nucleotide binding (G) protein. 2. Both low- and high-threshold calcium current components were present in nodose ganglion neurones. CGRP had no effect on the isolated low-threshold current component. However, CGRP (1-1000 nM, ED50 = 50 nM) caused a concentration-dependent increase in high-threshold calcium currents. CGRP (1 microM) increased the peak of these calcium currents 21 +/- 4% over controls. 3. CGRP enhanced a transient high-threshold calcium current evoked from a holding potential of -80 mV but did not affect the slowly inactivating high-threshold current evoked from -40 mV. Multiple high-threshold calcium currents have been reported in sensory neurones. We cannot state unequivocally which high-threshold calcium current component was enhanced by CGRP. However, based on the observation that CGRP increased a transient but not the slowly inactivating high-threshold calcium current, we believe the peptide enhanced primarily the N-type calcium current component. 4. CGRP increased the maximal peak current and caused a modest negative shift of < or = 10 mV in the peak of the current-voltage (I-V) relation in three of six neurones. In the remaining three neurones the peptide increased the maximal peak current without a detectable shift in the peak of the I-V relation. 5. To determine if the CGRP-induced enhancement in calcium current was associated with an increase in calcium conductance, we studied the effect of the peptide on the instantaneous current-voltage (I-V) relation when currents were evoked at a clamp potential (Vc) of +30 mV, positive to the observed maximal current (Vc = 0 to +10 mV). CGRP increased the maximal conductance 23 +/- 4%. 6. The enhancement of calcium current by CGRP was not due to a shift in the voltage dependency of steady-state inactivation of the calcium channels. The stimulatory effect of CGRP on calcium current was evaluated by evoking currents from different holding potentials (Vh) at the same Vc (+10 mV). CGRP-induced increases in calcium currents were similar over the range of (Vh) from -60 to -110 mV, suggesting that the peptide did not alter voltage-dependent steady-state inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of calcium currents in rat hippocampal CA1 neurons induced by kindling epileptogenesis

Kindling of the Schaffer collaterals in the dorsal hippocampus of the rat induced an epileptogenic focus in area CA1. Pyramidal neurons were acutely isolated from this area in fully kindled rats one day after the last class five generalized seizure. Calcium currents were measured in these cells under the whole-cell patch voltage-clamp condition after blockade of sodium and potassium currents. Voltagedependent calcium currents were activated by depolarizing voltage steps from different prepulse potentials. Calcium currents activated at 0 mV consisted of a sustained component and two voltage-dependent inactivating components. Current inactivation was fitted with two exponentials (time-constants of 13 and 72 ms) and a constant. When cells from kindled rats were compared with those from controls, the amplitudes of the slow-inactivating and the sustained component were significantly enhanced by 36% and 39%, respectively; the fast inactivating current showed only a small enhancement. Inactivation kinetics, time-to-peak and voltage dependency of activation and steady-state inactivation were unchanged. Shape and size of the analysed cells from kindled rats were not different from those in controls. We concluded that an increased specific calcium conductance of as yet unknown origin underlies the larger current. The magnitude of the observed changes is such that it will considerably increase calcium influx and consequently raise intracellular calcium concentration during tetanic stimulation and subsequent periods of paroxysmal activity. This increase will modulate calcium-dependent factors that regulate neuronal excitability and may lead to the enhanced excitability found in kindled tissue.

Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II.

Calcium entry through voltage-activated Ca2+ channels is important in regulating many cellular functions. Activation of these channels in many cell types results in feedback regulation of channel activity. Mechanisms linking Ca2+ channel activity with its downregulation have been described, but little is known of the events responsible for the enhancement of Ca2+ current that in many cells follows Ca2+ channel activation and an increase in cytoplasmic Ca2+ concentration. Here we investigate how this positive feedback is achieved in single smooth muscle cells. We find that in these cells voltage-activated calcium current is persistently but reversibly enhanced after periods of activation. This persistent enhancement of the Ca2+ current is mediated by activation of calmodulin-dependent protein kinase II because it is blocked when either the rise in cytoplasmic Ca2+ is inhibited or activation of calmodulin-dependent protein kinase II is prevented by specific peptide inhibitors of calcium-calmodulin or calmodulin-dependent protein kinase II itself. This mechanism may be important in different forms of Ca2+ current potentiation, such as those that depend on prior Ca2+ channel activation or are a result of agonist-induced release of Ca2+ from internal stores.

Early Enhancement of Calcium Currents by H- ras Oncoproteins Injected into Hermissenda Neurons

Influx of calcium through membrane channels is an important initial step in signal transduction of growth signals. Therefore, the effects of Ras protein injection on calcium currents across the soma membrane of an identified neuron of the snail Hermissenda were examined. With the use of these post-mitotic cells, a voltage-sensitive, inward calcium current was increased 10 to 20 minutes after Harvey-ras oncoproteins were injected. The effects of oncogenic Harvey ras p21 protein (v-Ras) occurred quickly and were sustained, whereas the effects of proto-oncogenic ras protein (c-Ras) were transient. This relative potency is consistent with the activities of these oncoproteins in stimulating cell proliferation. Thus, this calcium channel may be a target for Ras action.

Inhibitors of protein kinase C prevent enhancement of calcium current and action potentials in peptidergic neurons of Aplysia

Following brief stimulation of an afferent pathway, the bag cell neurons of Aplysia undergo a dramatic change in excitability, resulting in a prolonged discharge of spontaneous action potentials. During the discharge, the action potentials of the bag cell neurons become enhanced in height and width. The afterdischarge triggers release of neuroactive peptides that initiate egg-laying behavior in this animal. Evidence suggests that changes in excitability of the bag cell neurons may be mediated by activation of protein kinase C (PKC) and cAMP-dependent protein kinase (cAMP-PK). PKC activators, such as the phorbol ester TPA (12-O-tetradecanoyl-13-phorbol acetate), enhance the amplitude of action potentials in isolated bag cell neurons in cell culture. These agents act by unmasking a previously covert species of voltage-dependent calcium channel resulting in an increase in calcium current. In the accompanying paper (Conn et al., 1989), we showed that H-7, a protein kinase inhibitor, inhibits the effect of TPA, and is a selective inhibitor of PKC relative to cAMP-PK in these cells. We now report that another PKC inhibitor, sphinganine, also inhibits the effect of TPA on action potential height and calcium current in cultured bag cell neurons, and that N-acetylsphinganine, an inactive sphinganine analog, fails to inhibit the effects of PKC activators. Although both H-7 and sphinganine prevent the effects of TPA when added prior to TPA addition, neither compound reverses the effects of TPA when added after the action potentials have already become enhanced by TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase inhibitors selectively block phorbol ester- or forskolin- induced changes in excitability of Aplysia neurons

Exposure of the bag cell neurons of Aplysia to activators of protein kinase C, such as phorbol esters, enhances electrically evoked action potentials by increasing the voltage-dependent calcium current. We have hypothesized that this effect is mediated by the activation of protein kinase C (PKC). An important prediction of this hypothesis is that inhibitors of PKC should inhibit these phorbol ester-induced changes in bag cell neuronal excitability. We have now found that treatment of bag cell neurons with the protein kinase inhibitor 1-[5-isoquinolinesulfonyl]-2-methyl piperazine (H-7) inhibits the phorbol ester-induced enhancement of bag cell action potentials and prevents the enhancement of calcium current by phorbol esters. The height and width of electrically evoked action potentials in bag cell neurons can also be enhanced by cAMP analogs or agents that elevate cAMP. These agents do not influence the major voltage-dependent calcium current in the bag cell neurons but may act by modulating potassium currents and other voltage-dependent currents. We have found that microinjection of a protein inhibitor of cAMP-PK (PKA-I) into isolated bag cell neurons prevents and reverses the effect of the adenylate cyclase activator forskolin on action potentials of these cells. In contrast, H-7 does not inhibit the effects of forskolin on a variety of responses in these cells, including its effects on action potentials, granule movement, and 32P incorporation into phosphoproteins. This suggests that H-7 is selective for PKC relative to cAMP-PK in intact bag cell neurons.

Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons

The modulation of voltage-activated calcium currents by protein kinases provides excitable cells with a mechanism for regulating their electrical behaviour. At the single channel level, modulation of calcium current has, to date, been characterized only in cardiac muscle, where beta-adrenergic agonists, acting through cyclic AMP-dependent protein kinase, enhance the calcium current by increasing channel availability and opening. We now report that enhancement of calcium current in the peptidergic bag cell neurons of Aplysia by protein kinase C occurs through a different mechanism, the recruitment of a previously covert class of calcium channel. Under control conditions, bag cell neurons contain only one class of voltage-activated calcium channel with a conductance of approximately 12 pS. After exposure to agents that activate protein kinase C, these neurons also express a second class of calcium channel with a different unitary conductance (approximately 24 pS) that is never seen in untreated cells.

Calcium/phosphatidylserine/diacylglycerol-dependent protein phosphorylation in the Aplysia nervous system

It has been shown that intracellular injection of protein kinase C (calcium/phosphatidylserine/diacylglycerol-dependent protein kinase), purified from mammalian brain, or application of the tumor-promoting phorbol diester, 12-O-tetradecanoyl-13-phorbol acetate (TPA), leads to an enhancement of calcium currents in the bag cell neurons of Aplysia. We now present evidence of an endogenous enzyme in bag cell neurons which is activated by TPA and which has properties similar to those of mammalian protein kinase C. Calcium/phosphatidylserine/diacylglycerol-dependent protein kinase activity was found in both cytosolic and particulate fractions prepared from isolated clusters of bag cell neurons. This endogenous enzyme phosphorylated an 87,000-dalton protein from bovine brain, which appears to be a specific substrate for protein kinase C, as well as several substrates present in cytosolic fractions prepared from isolated bag cell clusters. Similar results were obtained using preparations made from pooled head ganglia from Aplysia. The pharmacological properties of the calcium/phosphatidylserine/diacylglycerol-dependent protein kinase activity in the Aplysia nervous system were similar to those of protein kinase C from mammalian tissues. Thus, the same group of endogenous substrate proteins were phosphorylated when diacylglycerol was replaced by TPA in cytosolic fractions prepared from isolated bag cell clusters. Non-tumor-promoting phorbols (4-alpha-phorbol, 4-alpha-phorbol-12,13-didecanoate, and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate) did not stimulate protein phosphorylation in these preparations. Phosphorylation by the Aplysia calcium/phosphatidylserine/diacylglycerol-dependent protein kinase was inhibited by polymixin B sulfate, by calmodulin, and by the "calmodulin antagonists" trifluoperazine, calmidazolium and W7.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of calcium current in Aplysia neurones by phorbol ester and protein kinase C.

One of the molecular mechanisms capable of regulating the physiological properties of neurones is the phosphorylation of ion channels and other cellular components by cyclic AMP-dependent protein kinase. Another protein kinase present in high concentrations in the mammalian brain is protein kinase C (a calcium/phosphatidylserine/diacylglycerol-dependent protein kinase), but there is no direct evidence, as yet, for the involvement of this enzyme in the control of neuronal excitability. We now present evidence that activation of endogenous protein kinase C by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl- phorbol-13-acetate), or intracellular injection of the purified enzyme, enhances the voltage-sensitive calcium current in bag cell neurones of the mollusc Aplysia.

Enhancement of calcium current during digitalis inotropy in mammalian heart: positive feed-back regulation by intracellular calcium?

1. Effects of digitalis compounds on slow inward Ca current I(si)) and contractile force were examined in ferret ventricular muscle (single sucrose-gap voltage clamp) and calf Purkinje fibres (two micro-electrode voltage clamp).2. In ventricular muscle, ouabain increased I(si) and inward current tails associated with I(si) conductance. The enhancement of I(si) followed a time course similar to the development of the positive inotropic effect, and it could be observed in the absence of aftercontractions or other signs of toxicit.3. The response of myocardial I(si) and twitch force to ouabain depended strongly on a previous history of driven action potentials.4. Veratridine, a toxin that promotes Na entry through tetrodotoxin-sensitive channels, also increased I(si) and twitch force in driven ventricular muscle preparations.5. The effects of ouabain, action potential stimulation and veratridine are consistent with reported effects of K-poor solutions in indicating that elevation of intracellular Na can lead to enhancement of I(si). Additional experiments suggest that the link between Na(i) and I(si) involves intracellular Ca.6. When Cs-loaded Purkinje fibres were bathed in solutions containing Sr instead of Ca, enhancement of I(si) by strophanthidin was abolished even though a positive inotropic response persisted.7. After intracellular injection of Purkinje fibres with EGTA, I(si) no longer increased with strophanthidin, although it remained responsive to adrenaline.8. Clear-cut increases in I(si) were seen in Cs-loaded Purkinje fibres even at very low concentrations of strophanthidin (20-50 nM), where the occurence of Na pump inhibition has been questioned.9. Positive regulation of Ca entry by intracellular Ca may act as a facilitory mechanism that amplifies myocardial responsiveness to digitalis and other inotropic interventions. Through changes in I(si), small rises in diastolic free Ca might lead to large increases in the activator Ca transient during contraction.