NGF-induced neurite outgrowth in PC12 cells is independent of calcium entry through L-type calcium channels.

Neuronal growth factor (NGF) induces neurodifferentiation of PC12 cells into cholinergic neurons-like cells. It was shown that intracellular Ca2+ ions participate in regulation of the differentiation of PC12 cells. We tested whether L-type calcium channels contribute to Ca2+ entry which supports neurite outgrowth accompanying NGF-activated differentiation process. Development of morphological changes did correlate with increase of functional expression of L-type calcium channels. However, inhibition of L-type calcium channels by 1 µM of isradipine did not affect significantly an NGF-activated neurite outgrowth.

T-type calcium channel blockers - new and notable.

Since cloning of the T-type or Ca(V)3.n calcium channel family in 1998-1999 much progress was made in investigation of their regulation. Most effective metal Ca(V)3 channel blockers are trivalent cations from lanthanide group together with transition metals La(3+) and Y(3+). Divalent cations Zn(2+), Cu(2+) and Ni(2+) inhibit Ca(V)3.2 channels more efficiently than Ca(V)3.1 and Ca(V)3.3 channels via second high-affinity binding site including histidine H191 specific for the Ca(V)3.2 channel. Dihydropyridines and phenylalkylamines in addition to block of L-type calcium channel can inhibit Ca(V)3 channels in clinically relevant concentration.

Haloperidol moderately inhibits cardiovascular L-type calcium current.

Effects of haloperidol on L-type CaV1.2 channel were studied. Calcium current was measured in whole cell patch-clamp using calcium as a charge carrier. Inhibition by haloperidol was investigated in CaV1.2 channel natively expressed in rat cardiac myocytes and recombinant cardiac (CaV1.2a) and vascular (CaV1.2b) splice variants of the channel expressed in HEK 293 cells. Haloperidol inhibited L-type calcium current in a concentration-dependent manner with a threshold of 1 nmol/l. 1 micromol/l haloperidol inhibited 20.6 +/- 3.6% of calcium current amplitude in cardiomyocytes, 25.4 +/- 2.6% of current amplitude through the CaV1.2b channel and 28.0 +/- 2.7% of current through the CaV1.2a channel. Inhibition was not accompanied by alteration of current waveform or by shift of current-voltage relation. In a current clamp haloperidol suppressed action potential generation. 1 micromol/l of the drug shortened the action potential duration in part of the cells and suppressed fully action potential in other cells. Moderate inhibition of the L-type calcium channels by haloperidol might cause shortening of action potential. Complete abolishment of action potential must have been mediated by inhibition of another, likely sodium channel.

The effect of the outermost basic residues in the S4 segments of the Ca(V)3.1 T-type calcium channel on channel gating.

The contribution of voltage-sensing S4 segments in domains I to IV of the T-type Ca(V)3.1 calcium channel to channel gating was investigated by the replacement of the uppermost charged arginine residues by neutral cysteines. In each construct, either a single (R180C, R834C, R1379C or R1717C) or a double (two adjacent domains) mutation was introduced. We found that the neutralisation of the uppermost arginines in the IS4, IIS4 and IIIS4 segments shifted the voltage dependence of channel activation in a hyperpolarising direction, with the most prominent effect in the IS4 mutant. In contrast, the voltage dependence of channel inactivation was shifted towards more negative membrane potentials in all four single mutant channels, and these effects were more pronounced than the effects on channel activation. Recovery from inactivation was affected by the IS4 and IIIS4 mutations. In double mutants, the effects on channel inactivation and recovery from inactivation, but not on channel activation, were additive. Exposure of mutant channels to the reducing agent dithiothreitol did not alter channel properties. In summary, our data indicate that the S4 segments in all four domains of the Ca(V)3.1 calcium channels contribute to voltage sensing during channel inactivation, while only the S4 segments in domains I, II and III play such role in channel activation. Furthermore, the removal of the outermost basic amino acids from the IVS4 and IIIS4 and, to a lesser extent, from IS4 segments stabilised the open state of the channel, whereas neutralization from that of IIS4 destabilised it.

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel.

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.

Inorganic mercury and methylmercury inhibit the Cav3.1 channel expressed in human embryonic kidney 293 cells by different mechanisms.

Part of the neurotoxic effects of inorganic mercury (Hg(2+)) and methylmercury (MeHg) was attributed to their interaction with voltage-activated calcium channels. Effects of mercury on T-type calcium channels are controversial. Therefore, we investigated effects of Hg(2+) and MeHg on neuronal Ca(v)3.1 (T-type) calcium channel stably expressed in the human embryonic kidney (HEK) 293 cell line. Hg(2+) acutely inhibited current through the Ca(v)3.1 calcium channel in concentrations 10 nM and higher with an IC(50) of 0.63 +/- 0.11 microM and a Hill coefficient of 0.73 +/- 0.08. Inhibition was accompanied by strong deceleration of current activation, inactivation, and deactivation. The current-voltage relation was broadened, and its peak was shifted to a more depolarized membrane potentials by 1 microM Hg(2+). MeHg in concentrations between 10 nM and 100 microM inhibited the current through the Ca(v)3.1 calcium channel with an IC(50) of 13.0 +/- 5.0 microM and a Hill coefficient of 0.47 +/- 0.09. Low concentration of MeHg (10 pM to 1 nM) had both positive and negative effects on the current amplitude. Micromolar concentrations of MeHg reduced the speed of current activation and accelerated current inactivation and deactivation. The current-voltage relation was not affected. Up to 72 h of exposure to 10 nM MeHg had no significant effect on current amplitude, whereas 72-h-long exposure to 1 nM MeHg increased significantly current density. Acute treatment with Hg(2+) or MeHg did not affect HEK 293 cell viability. In conclusion, interaction with the Ca(v)3.1 calcium channel may significantly contribute to neuronal symptoms of mercury poisoning during both acute poisoning and long-term environmental exposure.