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Role of L-type and T-type Ca2+ channels in neuronal excitability

Principal Investigator: Prof. Franz Hofmann
International Collaborator: Ľubica Lacinová

Duration: January 2002 – February 2004
Coordinating Organization: Institute of Pharmacology and Toxicology, Technical University of Munich, Munich, Germany


Structure, function and regulation of the T-type calcium channel and the role of voltage-gated calcium channels in neuronal excitability were investigated.

Properties of the charge movement measured from the Cav3.1 channel expressed in HEK 293 cells were characterized. Threshold of its activation was by 10 mV more negative than the threshold for current activation. Slope of voltage dependence of charge movement was extremely shallow compared to voltage dependence of current activation. Prolonged depolarisation did not immobilise the charge movement. Coexpression of a2d-2a subunit or the g5 subunit improved charge movement – channel opening coupling probably by facilitation of transitions between individual closed states and the transition between last closed state and an open state. Gating of the Cav3.1 channel is modulated by Ca2+ ions, which facilitate the transition of the channel from conducting, i.e. open channel state into non-conducting, i.e., closed and inactivated states and backwards transition from non-conducting states into conducting state. Channel is not regulated by phoshorylation through the protein tyrosine kinase (PTK) dependent pathway. Nonspecific PTK inhibitor genistein effectively inhibits the channel by PTK-independent mechanism involving specific interaction with the voltage sensor of the channel together with the channel pore occlusion. Acute inhibition of the Cav3.1 channel both organic (methylmercury, MeHg) and inorganic (Hg2+) mercury as well as its potentiation by chronic exposure to nanomolar concentrations of MeHg may contribute to pathology of acute and chronic mercury poisoning. When uppermost arginines in S4 segments of domains I to IV were replaced by neutral cysteines all aspects of channel gating were altered. With the exception of mutation in domain IV all other mutations significantly shifted activation towards more negative membrane voltages and increased slope factor of channel activation. Similarly, inactivation was shifted towards more negative potentials and its slope factor was increased. When mutations in two neighboring domains were combined, effects on channel activation were only slightly enhanced, while effects on channel inactivation were additive. Recovery from the inactivation was slowed down by mutations in IS4 or IIIS4 segments. S4 segments in individual domains contributed differently to channel activation and inactivation with S4 segment in the domain III playing the most important role and S4 segment in the domain IV having the smallest impact.

Contribution of Cav1.2 voltage-gated calcium channel to the excitability of the hippocampal CA1 region was investigated in Hippocampus a1C Knock-Out (HCKO) mice. Approximately 90% decrease in the amount of Cav1.2 protein was demonstrated by Western blots in both hippocampus and neocortex of young mice between 8 and 15 weeks old. 10 mM of (+/-)isradipine inhibited 22.3±2.8% of the current amplitude in hippocampal slices from control, but only 4.4±1.8% of the current amplitude in slices from HCKO mice. The resting membrane potential was not altered by inactivation of the Cav1.2 gene (‑64.0±1.0 mV in control vs. ‑65.3±0.8 mV in mutant mice. The input resistance of CA1 pyramidal neurons measured at a membrane potential of –70 mV was slightly, but not significantly, increased (79.4±2.2 MW in control vs. 86.6±3.1 MW in mutant mice). As expected, the knock-out had no effect on the shape of single action potentials (AP). Maximal slope of the ascending and the descending phase, half-maximal width as well as threshold and amplitude single AP induced by brief 5 ms current pulse were not significantly different between the two genotypes. The threshold for generating a serie of APs from the resting membrane potential of –70 mV was significantly enhanced and the AP frequency within an AP serie was lowered in CA1 neurons from HCKO mice compared with the control. The Cav1.2 channels facilitate initiation of burst firing but are less important for steady state activity of hippocampal neurons. They do not participate in settling of resting potential.