Voltage dependence and kinetics of activation of CaV1.2 channels are affected by structural changes in pore lining S6 segments of the α1-subunit. Significant effects on channel activation are induced by either proline or threonine substitutions in the lower third of segment IIS6 (‘bundle crossing region’). Here we report that S435P in IS6 results in a large shift of the activation (−26mV) curve and slowed current kinetics. Threonine substitutions in positions Leu429 and Leu434 induced a similar kinetic phenotype with shifted activation curves.Double mutations in segments IS6 and IIS6 induced additive shifts of the activation curves, e.g.: L429T/I781T (-44.0±1.0), L434T/I781T (-50.3±0.8), L429T/L779T (-22.5±0.8) and L434T/L779T (-32.3±0.8). If the gating sensitive residues in the two neighboring segments IS6 and IIS6 do not interact then the change in free energy (ΔGdouble) of the double mutant is equal to the sum of the changes in free energy of the two single mutations (ΔGmut IS6 and ΔGmut IIS6 see scheme, see also Horovitz 1996). Double mutant cycle analysis revealed that the studied IS6 and IIS6 mutations are energetically independent and thus have independent impacts on activation gating.Supported by FWF-Project P19614-B11.View Large Image | View Hi-Res Image | Download PowerPoint Slide