We have previously shown that mutations in pore lining segment IIS6 of Cav1.2 induce leftward shifts of the activation curve reflecting a destabilization of the closed and a simultaneous stabilization of the open channel state (Beyl et al. 2009). Systematic substitutions of a cluster of hydrophobic residues (LAIA motive) by residues of different size and polarity (except proline) revealed a strong correlation between changes in hydrophobicity (ΔH) and the shifts of the activation curve (ΔV0.5). A similar analysis in segment IS6 revealed no correlation between ΔH and ΔV0.5. Here we show that amino acid substitutions in the corresponding sequence stretch of segment IIIS6 (F1191 - I1196) shift the activation curves (ΔV0.5 F1191T=−7.1±1.3mV, V1192T= 8.2± 1.2mV, G1193T=-31.2±1.3mV, F1194T=2.1±1.2mV, V1195T=−10.3±1.1mV, I1196T=−17.9±1.1mV). A reduction in hydrophobicity in positions V1195 (ΔH(V1195T)=−4.9) and I1196 (ΔH(I1196T)=−5.2) apparently destabilized the closed channel state. Other mutations (V1192T, G1193T and F1194T) did not fit this paradigm. Potential interactions between residues in segments IS6, IIS6 and IIIS6 were analysed by means of double mutant cycle analysis. Our data reveal a positional specific interaction between gating determinants in segments IS6 and IIS6 as well as IIS6 and IIIS6. Rate constants of the voltage sensing machinery and pore stability were estimated using a 4 state gating model (Beyl et al. 2009).