The Timothy syndrome is a multisystem disorder associated with the mutation of a Gly residue (G402 or G406) in the Ca(v)1.2 Ca(2+) channel. G406 is localized at the end of the IS6 segment and just before the intracellular I-II loop, which is important for the regulation of channel inactivation and the binding of the Ca(v)beta subunit. This Gly residue is conserved in all Ca(v)1 and Ca(v)2 channels, and the G to R exchange produces a strong decrease of inactivation not only in Ca(v)1.2 but also in Ca(v)2.3. Here, we show that the mutation into Arg or Glu of the homologous Gly residue in Ca(v)2.1 (G363) produces also a slowing of inactivation. However, the G-to-A exchange that decreases the inactivation rate in Ca(v)1.2 and Ca(v)2.3 increases inactivation in Ca(v)2.1. Each mutation affects specifically the gating properties of Ca(v)2.1 that remain nevertheless modulated by the co-expressed beta subunit as with wild-type channel. The strong decrease of inactivation produced by the G363R or G363E mutations was reminiscent to that previously described for a specific splice variant of Ca(v)2.1 that contains a single Val residue inserted in the I-II loop (V421). We unexpectedly found that the V421 insertion does not affect the inactivation rate of Ca(v)2.1 and that the effects previously attributed to this insertion, including those on G-protein regulation, can be reproduced by the G363E mutation. Altogether, our results highlight the role of G363 in gating properties, inactivation kinetics, and G-protein regulation of Ca(v)2.1 and the lack of effect of V421 insertion on inactivation.