T-type ICa Research Articles

Inhibition of human recombinant T-type calcium channels by phytocannabinoids in vitro.

T-type Ca channels (ICa ) regulate neuronal excitability and contribute to neurotransmitter release. The phytocannabinoids Δ9 -tetrahydrocannabinol and cannabidiol effectively modulate T-type ICa , but effects of other biologically active phytocannabinoids on these channels are unknown. We thus investigated the modulation of T-type ICa by low abundance phytocannabinoids. A fluorometric (fluorescence imaging plate reader [FLIPR]) assay was used to investigate modulation of human T-type ICa (CaV 3.1, 3.2 and 3.3) stably expressed in FlpIn-TREx HEK293 cells. The biophysical effects of some compounds were examined using whole-cell patch clamp recordings. In the FLIPR assay, all 11 phytocannabinoids tested modulated T-type ICa , with most inhibiting CaV 3.1 and CaV 3.2 more effectively than CaV 3.3. Cannabigerolic acid was the most potent inhibitor of CaV 3.1 (pIC50 6.1±0.6) and CaV 3.2 (pIC50 6.4±0.4); in all cases, phytocannabinoid acids were more potent than their corresponding neutral forms. In patch clamp recordings, cannabigerolic acid inhibited CaV 3.1 and 3.2 with similar potency to the FLIPR assay; the inhibition was associated with significant hyperpolarizing shift in activation and steady-state inactivation of these channels. In contrast, cannabidiol, cannabidivarin, and cannabigerol only affected channel inactivation. Modulation of T-type calcium channels is a common property of phytocannabinoids, which all increase steady-state inactivation at physiological membrane potentials, with some also affecting channel activation. Thus, T-type ICa may be a common site of action for phytocannabinoids, and the diverse actions of phytocannabinoids on channel gating may provide insight into structural requirement for selective T-type ICa modulators.

Modulation of Recombinant Human T-Type Calcium Channels by Δ9-Tetrahydrocannabinolic Acid In Vitro.

Introduction: Low voltage-activated T-type calcium channels (T-type ICa), CaV3.1, CaV3.2, and CaV3.3, are opened by small depolarizations from the resting membrane potential in many cells and have been associated with neurological disorders, including absence epilepsy and pain. Δ9-tetrahydrocannabinol (THC) is the principal psychoactive compound in Cannabis and also directly modulates T-type ICa; however, there is no information about functional activity of most phytocannabinoids on T-type calcium channels, including Δ9-tetrahydrocannabinolic acid (THCA), the natural nonpsychoactive precursor of THC. The aim of this work was to characterize THCA effects on T-type calcium channels. Materials and Methods: We used HEK293 Flp-In-TREx cells stably expressing CaV3.1, 3.2, or 3.3. Whole-cell patch clamp recordings were made to investigate cannabinoid modulation of ICa. Results: THCA and THC inhibited the peak current amplitude CaV3.1 with pEC50s of 6.0±0.7 and 5.6±0.4, respectively. THC (1 μM) or THC produced a significant negative shift in half activation and inactivation of CaV3.1, and both drugs prolonged CaV3.1 deactivation kinetics. THCA (10 μM) inhibited CaV3.2 by 53%±4%, and both THCA and THC produced a substantial negative shift in the voltage for half inactivation and modest negative shift in half activation of CaV3.2. THC prolonged the deactivation time of CaV3.2, while THCA did not. THCA inhibited the peak current of CaV3.3 by 43%±2% (10 μM) but did not notably affect CaV3.3 channel activation or inactivation; however, THC caused significant hyperpolarizing shift in CaV3.3 steady-state inactivation. Discussion: THCA modulated T-type ICa currents in vitro, with significant modulation of kinetics and voltage dependence at low μM concentrations. This study suggests that THCA may have potential for therapeutic use in pain and epilepsy through T-type calcium channel modulation without the unwanted psychoactive effects associated with THC.

Electrical excitability of the heart in a Chondrostei fish, the Siberian sturgeon (Acipenser baerii).

Sturgeon (family Acipenseridae) are regarded as living fossils due to their ancient origin and exceptionally slow evolution. To extend our knowledge of fish cardiac excitability to a Chondrostei fish, we examined electrophysiological phenotype of the Siberian sturgeon (Acipenser baerii) heart with recordings of epicardial ECG, intracellular action potentials (APs), and sarcolemmal ion currents. Epicardial ECG of A. baerii had the typical waveform of the vertebrate ECG with Q-T interval (average duration of ventricular AP) of 650±30 ms and an intrinsic heart rate of 45.5±5 beats min(-1) at 20°C. Similar to other fish species, atrial AP was shorter in duration (402±33 ms) than ventricular AP (585±40) (P<0.05) at 20°C. Densities of atrial and ventricular Na+ currents were similar (-47.6±4.5 and -53.2±5.1 pA/pF, respectively) and close to the typical values of teleost hearts. Two major K+ currents, the inward rectifier K+ current (IK1), and the delayed rectifier K+ current (IKr) were found under basal conditions in sturgeon cardiomyocytes. The atrial IKr (3.3±0.2 pA/pF) was about twice as large as the ventricular IKr (1.3±0.4 pA/pF) (P<0.05) conforming to the typical pattern of teleost cardiac IKr. Divergent from other fishes, the ventricular IK1 was remarkably small (-2.5±0.07 pA/pF) and not different from that of the atrial myocytes (-1.9±0.06 pA/pF) (P>0.05). Two ligand-gated K+ currents were also found: ACh-activated inward rectifier (IKACh) was present only in atrial cells, while ATP-sensitive K+ current (IKATP) was activated by a mitochondrial blocker, CCCP, in both atrial and ventricular cells. The most striking difference to other fishes appeared in Ca2+ currents (ICa). In atrial myocytes, ICa was predominated by nickel-sensitive and nifedipine-resistant T-type ICa, while ventricular myocytes had mainly nifedipine-sensitive and nickel-resistant L-type ICa. ICaT/ICaL ratio of the sturgeon atrial myocytes (2.42) is the highest value ever measured for a vertebrate species. In ventricular myocytes, ICaT/ICaL ratio was 0.09. With the exception of the large atrial ICaT and small ventricular IK1, electrical excitability of A. baerii heart is similar to that of teleost hearts.

Distinct inhibition of voltage-activated Ca2+ channels by δ-opioid agonists in dorsal root ganglion neurons devoid of functional T-type Ca2+ currents

Both μ- and δ-opioid agonists selectively inhibit nociception but have little effect on other sensory modalities. Voltage-activated Ca2+ channels in the primary sensory neurons are important for the regulation of nociceptive transmission. In this study, we determined the effect of δ-opioid agonists on voltage-activated Ca2+ channel currents (ICa) in small-diameter rat dorsal root ganglion (DRG) neurons that do and do not bind isolectin B4 (IB4). The δ-opioid agonists [d-Pen2,d-Pen5]-enkephalin (DPDPE) and deltorphin II produced a greater inhibition of high voltage–activated ICa in IB4-negative than IB4-positive neurons. Furthermore, DPDPE produced a greater inhibition of N-, P/Q-, and L-type ICa in IB4-negative than IB4-positive neurons. However, DPDPE had no significant effect on the R-type ICa in either type of cells. We were surprised to find that DPDPE failed to inhibit either the T-type or high voltage–activated ICa in all the DRG neurons with T-type ICa. Double immunofluorescence labeling showed that the majority of the δ-opioid receptor-immunoreactive DRG neurons had IB4 labeling, while all DRG neurons immunoreactive to δ-opioid receptors exhibited Cav3.2 immunoreactivity. Additionally, DPDPE significantly inhibited high voltage–activated ICa in Tyrode's or N-methyl-d-glucamine solution but not in tetraethylammonium solution. This study provides new information that δ-opioid agonists have a distinct effect on voltage-activated Ca2+ channels in different phenotypes of primary sensory neurons. High voltage–activated Ca2+ channels are more sensitive to inhibition by δ-opioid agonists in IB4-negative than IB4-positive neurons, and this opioid effect is restricted to DRG neurons devoid of functional T-type Ca2+ currents.

Sarco-Endoplasmic ATPase Blocker 2,5-Di(tert-butyl)-1,4-benzohydroquinone Inhibits N-, P-, and Q- but Not T-, L-, or R-Type Calcium Currents in Central and Peripheral Neurons

The effects of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBHQ), a synthetic phenolic antioxidant and a blocker of the sarco-endoplasmic ATPase, were evaluated on low and high voltage-activated Ca(2+) currents (ICas) with rodent dorsal root ganglion, hippocampal, and motor neurons. In all cell types tested, tBHQ (IC(50) = 35 microM) blocked ICa at concentrations used to inhibit sarco-endoplasmic ATPase. This effect was specific to tBHQ because the other sarco-endoplasmic reticulum calcium ATPase pump inhibitors (thapsigargin and cyclopiazonic acid) had no effect. Selective blockade of the N-type current with omega-conotoxin GVIA and of P- (motoneuron) or Q-type currents (hippocampal neuron) with omega-agatoxin IVA indicated that tBHQ inhibited N, P, and Q types of ICa. tBHQ had no effect on nitrendipine-sensitive (L-type) and residual drug-resistant (R-type) ICa, nor on the low voltage-activated T-type ICa. Contrary to neuronal cells, the L-type ICa was inhibited by tBHQ in a differentiated mouse neuroblastoma and rat glioma hybrid cell line. Injection of cDNAs encoding the alpha1A, alpha1B, alpha1C, and alpha1E subunits into oocytes showed that tBHQ blocked ICas at the level of the pore-forming protein. This effect of tBHQ on ICa should be considered when interpreting results obtained with tBHQ used on neuronal preparations. It also may be useful for developing new strategies for the generation of more potent intracellular calcium transient inhibitors.

Three types of voltage-dependent calcium currents developing in cultured human neuroblastoma cells.

Voltage-dependent calcium currents (ICa), which developed in cultured human neuroblastoma cells (NB-I), were studied using a whole-cell recording technique. Three types (T-, N- and L-type) of ICa were identified based on their biological and pharmacological properties. The T-type ICa was observed in about 60% of the cells from day 2 to day 12 and in about 20% after day 14. Likewise, the amplitude gradually decreased from -61.7 +/- 10.1 pA on day 2 to -18.3 +/- 9.1 pA on day 18. The N-type ICa appeared on day 6, with the number of cells exhibiting this current increasing up to 90.9% (-73.4 +/- 16.0 pA) on day 14 and immediately decreasing thereafter. The L-type ICa was observed in 50.0% of the cells on day 2, increasing to 84.6% of the cells on day 6 and remaining thereafter, while the amplitude gradually increased from -37.8 +/- 14.0 pA on day 2 to -158.8 +/- 22.8 pA on day 18, and decreased thereafter. These findings indicate that the expression of the voltage-dependent Ca2+ channels is strongly regulated by the developmental stage of the cell.

Two types of calcium currents of the mouse bipolar cells recorded in the retinal slice preparation.

In the vertebrate retina, the bipolar cell makes reciprocal synapses with amacrine cells at the axon terminal. It has been postulated that amacrine cells may control the transmitter release from bipolar cells by modulating their calcium currents (ICa). To clarify this possibility calcium currents were studied in bipolar cells of the mouse retina using a slice preparation. ICa was identified by voltage clamp protocols, ionic substitution and pharmacological tools. Depolarization to -30 mV from a holding voltage of -80 mV induced an inward current consisting of an initial transient and a long-lasting sustained component. The transient component was inactivated by holding the membrane at more positive voltages. Addition of 100 microM nifedipine suppressed the sustained component, leaving the transient component almost intact. The sustained component was enhanced when external solution contained 0.1 microM Bay K 8644 or when the external Ca2+ was substituted by equimolar Ba2+. Omega-conotoxin (10 microM omega-ctxn GVIA) did not alter either component. We concluded that the transient component is a low-voltage activated T-type ICa, while the sustained component is a high-voltage activated L-type ICa. T-type ICa was recorded in all cells tested, while L-type ICa was found only in cells that retained axon terminals ramifying in the inner plexiform layer. Thus, it is highly likely that L-type ICa is generated at the axon terminal and contributes to the transmitter release from the bipolar cell. The present results confirm that in addition to the T-type ICa that had been previously described, bipolar cells of the mammalian retina also contain L-type ICa similar to the one that has been reported in bipolar cells of the goldfish. The use of retinal slice preparation allowed us to record this current that was not seen previously in the dissociated mouse bipolar cells.

Mechanisms of T-type calcium channel blockade by zonisamide

We investigated the effects of zonisamide, a new antiepileptic drug, on voltage-dependent T-type calcium current (ICa) in cultured neuroblastoma cells of human origin (NB-I). Zonisamide reduced T-type ICa in a concentration-dependent manner without evoking any change in its inactivation kinetics or voltage dependence of action. The mean percent reduction was 38.3 +/- 5.8% at 50 microM. Further, zonisamide shifted the inactivation curve approximately 20 mV negative compared to the control. These resting blocking actions suggest that zonisamide shifts the channel population toward the inactivation state, allowing fewer channels to open during membrane depolarization. The blockade of T-type calcium channels by zonisamide could suppress an important component of inward current that underlies epileptiform cellular bursting, thereby inhibiting the spread of seizure activity.

Antiepileptic drugs—calcium current interaction in cultured human neuroblastoma cells

The voltage-dependent calcium channel current (ICa) in the neuroblastoma cell line of human origin (NB-I) was studied by the whole-cell clamp recording. Three types of ICa were identified in NB-I cells. Our electrophysiological and pharmacological findings have suggested that these three types of ICa are consistent with the T-, N- and L-type ICa, respectively. Phenytoin (PHT) inhibited T-type ICa by 13.0% at a concentration of 5 microM, and L-type ICa by 6.3% at a concentration of 100 microM. At a concentration of 100 microM, carbamazepine (CBZ) inhibited T- and L-type ICa by 6.0% and 5.9%, respectively. At a concentration of 50 microM, sodium valproate (VPA) blocked T- and L-type ICa by 6.1% and 47.5%, respectively. At a concentration of 50 microM, zomisamide (ZNS) inhibited T- and L-type ICa by 38.3% and 41.9%, respectively. Na+ channel blockade has been reported to be responsible for the clinical efficacy of PHT or CBZ. Inhibition of T-type ICa by PHT may enhance the efficacy of its anticonvulsant action. CBZ had little effect on ICa. The anticonvulsant activity may be related to the blockade of T-type ICa in the case of VPA and ZNS.

Reduced calcium currents in subendocardial Purkinje myocytes that survive in the 24- and 48-hour infarcted heart.

The abnormal transmembrane action potentials of subendocardial Purkinje fibers that survive 24 to 48 hours after coronary artery occlusion can be a source of the multiform ventricular tachycardias that occur during this time. A change in the density or function of either or both the T-type and L-type cardiac Ca2+ channels may contribute to the altered electrical activity of these Purkinje myocytes. The purpose of this study was to determine the function of the T- and L-type Ca2+ currents (iCat and iCaL, respectively) in Purkinje myocytes dispersed from the subendocardium of the left ventricle 24 and 48 hours after coronary artery occlusion (IZPC24 and IZPC48, respectively). To do this we compared whole-cell Ca2+ currents from Purkinje myocytes enzymatically dispersed from free-running fiber bundles (SPCs), from the subendocardium of the noninfarcted canine heart (NZPCs), and from IZPC24 and IZPC48. ICaL and iCat were recorded with Cs(+)- and EGTA-rich pipettes and in Na(+)-K(+)-free external solutions to eliminate overlapping currents. ICaL density was significantly reduced in IZPC48 compared with NZPC or IZPC24. This was not accompanied by a shift in the current-voltage relation or by a change in the time course of decay of iCaL. Replacement of Ca2+ with equimolar Ba2+ increased iCaL density in all cell types, but peak iBaL of IZPC48 remained reduced compared with control iBaL values. T-type Ca2+ currents were recorded in all SPCs and NZPCs. In IZPC24 and IZPC48 there was a reduction in peak iCat amplitudes and densities. This was not accompanied by a shift in the current-voltage relation or by a change in the time course of decay of peak iCat. However, there was a hyperpolarizing shift in the steady-state availability relations in both IZPC24 and IZPC48. In addition, the maximally available iCat in IZPC24 was not different from control, whereas it was significantly reduced in IZPC48. The L-type ICa density in subendocardial Purkinje myocytes that survive in the infarcted heart is significantly decreased by 48 hours after the time of coronary artery occlusion. The peak T-type ICa density is decreased in subendocardial Purkinje myocytes that survive in the infarcted heart at 24 hours, but further reduction occurs in these myocytes by 48 hours. This loss in Ca2+ channel function could contribute to the abnormal transmembrane potentials of these myocytes surviving in the infarcted heart.

Voltage-dependent ionic currents in dissociated paratracheal ganglion cells of the rat.

1. Conventional whole-cell voltage-clamp technique was used to study the electrophysiological and pharmacological properties of voltage-dependent Na+, K+ and Ca2+ channels in parasympathetic neurones enzymatically dissociated from the paratracheal ganglia of rat trachea. The voltage-dependent Na+, K+ and Ca2+ currents (INa, IK and ICa) were separated by the use of ion subtraction and pharmacological treatments. 2. INa was activated by a step depolarization more positive than -50 mV and fully activated at positive potentials more than +10 mV. The inactivation phase of INa consisted of fast and slow exponential components (tau if and tau is, respectively). The tau if and tau is were voltage dependent and decreased with a more positive step pulse. 3. The time course for recovery of INa from the complete inactivation exhibited two exponential processes. 4. The reversal potential of INa was equal to the Na+ equilibrium potential (ENa) and resembled a simple Na+ electrode depending only on external Na+ concentration. 5. Tetrodotoxin (TTX) reduced INa without affecting the current kinetics in a concentration-dependent manner, and the concentration of half-maximal inhibition (IC50) was 6 x 10(-9) M. There was no TTX-resistant component of INa in any of the cells tested. 6. Scorpion toxin increased the peak amplitude of INa and prolonged the inactivation phase in a time- and concentration-dependent manner. In the presence of toxin, both tau is and the fractional contribution of the slow current component to total INa increased concentration dependently. 7. High-threshold (L-type) ICa was activated by a step depolarization more positive than -30 mV and reached a peak at near 0 mV in the external solution with 2.5 mM Ca2+. The current was inactivated to only a small extent (< 10%) during 100 ms of depolarizing step pulse. There was no low-threshold (T-type) ICa in this preparation. 8. The maximum ICa in individual current-voltage (I-V) relationships was saturated by an increase in extracellular Ca2+ concentration ([Ca2+]o). The I-V relationships were also shifted along the voltage axis to the more positive potential with increasing [Ca2+]o. 9. The inactivation process of the L-type ICa was dependent on Ca2+ influxes (ICa-dependent inactivation). 10. Relative maximum peak currents of divalent cations passing through the L-type Ca2+ channels were in the order of IBa > ICa > ISr. 11. Organic and inorganic Ca2+ antagonists blocked the ICa in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Apamin, a highly potent fetal L-type Ca2+ current blocker in single heart cells.

Apamin, a bee venom polypeptide, was reported to block the naturally occurring Ca2+ slow action potentials (APs) in cultured cell reaggregates from old chick hearts [Bkaily, G. et al. Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H961-H965, 1985] as well as the tetrodotoxin (TTX)- and Mn(2+)-insensitive slow Na+ current in young embryonic chick heart cells (Bkaily, G. In Vitro Toxicology. Academic, In press; Bkaily et al. J. Mol. Cell. Cardiol. 23: 25-39, 1991). With the use of the whole cell voltage-clamp technique in single ventricular cells from 10-day-old chick embryos and 17- to 20-wk-old human fetuses, two types of Ca2+ currents (ICa), T and L, were found. These two types of slow inward current in both heart preparations were nearly similar in their voltage, kinetics, and pharmacology. Apamin, a slow Ca2+ action potential blocker in old embryonic chick heart, was found to block the L-type ICa (IL) in a dose-dependent manner without affecting the T-type ICa in both heart cell preparations. The blockade of the IL by apamin was completely reversible upon washout with apamin-free solution. Therefore, when compared with nifedipine or to PN 200-110, apamin seems to be a highly potent L-type Ca2+ channel blocker in heart cells.

Actions of Ca2+ antagonists on two types of Ca2+ channels in rat aorta smooth muscle cells in primary culture.

Mechanisms of blockade of two types of Ca2+ channels by the organic Ca2+ antagonists, nicardipine, diltiazem, verapamil, and flunarizine, were examined in rat aorta smooth muscle cells in primary culture by using the whole-cell voltage-clamp method. T-type Ca2+ current (T-type ICa) was isolated by an internal perfusion of 5 mM F-, which irreversibly suppressed the L-type ICa, without affecting T-type ICa. L-type ICa was isolated by setting a holding potential at -60 mV, at which most of the T-type Ca2+ channels were inactivated. L-type ICa is halved by 0.1 microM nicardipine, 3.0 microM diltiazem, 0.6 microM verapamil, and 0.1 microM flunarizine, whereas T-type ICa is halved by the same drugs at 0.6, 30, 30, and 0.1 microM, respectively. Diltiazem and verapamil accelerated the decay of L-type ICa and cumulatively blocked L-type ICa during repetitive step depolarizations elicited every 30 seconds ("use-dependent block"). Diltiazem and verapamil neither changed the decay of T-type ICa nor showed a use-dependent block of T-type ICa. Nicardipine and flunarizine blocked both L- and T-type ICa from the first depolarization step after drug treatment ("tonic block") and shifted their steady-state inactivation curves to the left. The estimated binding constants of nicardipine and flunarizine for the inactivated state of T-type Ca2+ channels (48 and 19 nM, respectively) were smaller than those for the resting state of L-type Ca2+ channels (160 and 90 nM, respectively). A low concentration (0.1 microM) of nicardipine initially potentiated T-type ICa and then reduced it. We conclude from these results that 1) nicardipine and flunarizine block not only the resting state but, more preferentially, the inactivated state of both the L- and T-type Ca2+ channels; 2) verapamil and diltiazem preferentially act on the open state of the L-type Ca2+ channel and on the resting and inactivated state of the T-type Ca2+ channel; and 3) the T-type Ca2+ channel of the rat aorta smooth muscle cells appears to be more sensitive to nicardipine and flunarizine than does the L-type Ca2+ channel at around the resting membrane potential.