Aims: 

Positively charged S4 segments in domains I to IV form a putative voltage sensor of voltage-dependent calcium channels. We have investigated their participation in deactivation of the Ca_V3.1 calcium channel.

Methods: 

Uppermost basic amino acids of the S4 segments from each domain were replaced by neutral cysteines using a PCR-based method. Four single mutants (R180C, R834C, R1379C, and R1717C) and four double mutants combining mutations in two neighbouring domains were constructed and transfected into HEK 293 cells. Whole-cell patch-clamp was used.

Results: 

Replacement of an arginine in domain II accelerated, while other single mutations slowed down channel deactivation. Acceleration of deactivation by the mutation in domain II was further enhanced when mutation in the adjacent domain I or III was introduced. Voltage dependencies of deactivation time constants (t_deact) were shifted along the voltage axis. Mutations, which increased t_deacts shifted their voltage dependence to more negative potentials (IIIS4, IVS4 and III+IVS4). Decrease of t_deact was accompanied by the shift towards more depolarised membrane potentials (I+IIS4). Voltage sensitivity of channel deactivation reflected in slope factor of voltage dependence of t_deact was not altered by introduced mutations except for the single mutation in the domain IV.

Conclusion: 

The kinetics of channel deactivation reflects the stability of the open channel state. Removal of a basic amino acid in domains IV and III and, to a lesser extent, in the domain I stabilised an open channel state while neutralisation of the basic amino acid in domain II destabilised it.

Supported by VEGA 2/7001 and APVV-51-027404