The voltage-dependent inhibition of single N-type Ca(2+) channels by noradrenaline (NA) and the delta-opioid agonist D-Pen(2)-D-Pen (5)-enkephalin (DPDPE) was investigated in cell-attached patches of human neuroblastoma IMR32 cells with 100 mM Ba(2+) and 5 microM nifedipine to block L-type channels. In 70% of patches, addition of 20 microM NA + 1 microM DPDPE delayed markedly the first channel openings, causing a four- to fivefold increase of the first latency at +20 mV. The two agonists or NA alone decreased also by 35% the open probability (P(o)), prolonged partially the mean closed time, and increased the number of null sweeps. In contrast, NA + DPDPE had little action on the single-channel conductance (19 versus 19.2 pS) and minor effects on the mean open time. Similarly to macroscopic Ba(2+) currents, the ensemble currents were fast activating at control but slowly activating and depressed with the two agonists. Inhibition of single N-type channels was effectively removed (facilitated) by short and large depolarizations. Facilitatory pre-pulses increased P(o) significantly and decreased fourfold the first latency. Ensemble currents were small and slowly activating before pre-pulses and became threefold larger and fast decaying after facilitation. Our data suggest that slowdown of Ca(2+) channel activation by transmitters is mostly due to delayed transitions from a modified to a normal (facilitated) gating mode. This single-channel gating modulation could be well simulated by a Monte Carlo method using previously proposed kinetic models predicting marked prolongation of first channel openings.

Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin-3-gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H2O2 release in cell cultures, and exogenous catalase (CAT) abrogated EGCG-induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3-loaded, EGCG-exposed MMe cells showed significant [Ca2+]i rise, prevented by CAT, dithiothreitol or the T-type Ca2+ channel blockers mibefradil and NiCl2. Cell loading with dihydrorhodamine 123 revealed EGCG-induced ROS production, prevented by CAT, mibefradil or the Ca2+ chelator BAPTA-AM. Direct exposure of cells to H2O2 produced similar effects on Ca2+ and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Cav3.2 T-type Ca2+ channels in these cells, compared to normal mesothelium. Also, Cav3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Cav3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T-type Ca2+ channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target.

The high-voltage-activated (HVA) Ba2+ currents of rat insulinoma RINm5F cells insensitive to dihydropyridines (DHP) and omega-conotoxin GVIA (omega-CTx-GVIA) have been studied for their sensitivity to omega-agatoxin-IVA (omega-Aga-IVA) and omega-CTx-MVIIC. Blockade of HVA currents by omega-Aga-IVA was partial (mean 24%), reversible and saturated around 350 nM (half block approximately 60 nM). Blockade by omega-CTx-MVIIC was more potent (mean 45%), partly irreversible and saturated above 3 microM. The effects of both toxins were additive with that of nifedipine (5 microM) and were more pronounced at positive potentials. omega-Aga-IVA action was additive with that of omega-CTx-GVIA (3 microM) but was largely prevented by cell pre-treatment with omega-CTx-MVIIC (3 microM). In contrast, omega-CTx-MVIIC block was attenuated by omega-CTx-GVIA treatment (approximately 15%), suggesting that omega-CTx-MVIIC blocks the N-type (approximately 15%) and the non-L-, non-N-type channel sensitive to omega-Aga-IVA (approximately 30%). Consistent with this, cells deprived of most non-L-type channels by pre-incubation with omega-CTx-GVIA and omega-CTx-MVIIC exhibited predominant L-type currents that activated at more negative potentials than in normal cells (-30 mV in 5 mM Ba2+) and were effectively depressed by nifedipine (maximal block of 95% from -30 mV to +40 mV). Our results suggest that, besides L- and N-type channels, insulin-secreting RINm5F cells possess also a non-L-, non-N-type channel that contributes significantly to the total current (approximately 30%). Although the pharmacology of this channel is similar to Q-type and alpha 1 class A channels, its range of activation (> -20 mV) and its slow inactivation time course resemble more that of N- and P-type channels. The channel is therefore referred to as "Q-like".

Helothermine, a recently isolated toxin from the venom of the Mexican beaded lizard Heloderma horridum horridum was tested on K+ currents of newborn rat cerebellar granule cells. In whole-cell voltage-clamp experiments, cerebellar granule neurons exhibited at least two different K+ current components: a first transient component which is similar to an IA-type current, is characterized by fast activating and inactivating kinetics and blocked by 4-aminopyridine; a second component which is characterized by noninactivating kinetics, is blocked by tetraetylammonium ions and resembles the classical delayed-rectifier current. When added to the standard external solution at concentrations ranging between 0.1 and 2 microM, helothermine reduced the pharmacologically isolated IA-type current component in a voltage- and dose-dependent way, with a half-maximal inhibitory concentration (IC50) of 0.52 microM. A comparison between control and helothermine-modified peak transient currents shows a slowdown of activation and inactivation kinetics. The delayed-rectifier component inhibition was concentration dependent (IC50 = 0.86 microM) but not voltage dependent. No frequency- or use-dependent block was observed on both K+ current types. Perfusing the cells with control solution resulted in quite a complete current recovery. We conclude that helothermine acts with different affinities on two types of K+ current present in central nervous system neurons.

Ca2+ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5-20 mM Ba2+, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed approximately 20% L-, approximately 45% N- and approximately 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20-200 nM omega-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nM in 5 mM Ba2+), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca2+ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 microM omega-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the alphaE channel. The current activates around -20 mV in 20 mM Ba2+; inactivates slowly and independently of Ca2+; is blocked by low [Cd2+] and high [Ni2+]; and is larger with Ba2+ than Ca2+. The uncovered R-type calcium current can account for part of the presynaptic Ca2+ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP-and omega-CTx-GVIA, and displays anomalous sensitivity to omega-Aga-IVA and omega-CTx-MVIIC.