The pore-forming component of voltage-gated calcium channels, a., subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains land II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2-S3 linkers of domains land II of Ca(v)1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Ca(v)1.2s; G182E, d182Q, G579E, G579Q and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current-voltage relationships of the four mutants were the same as those of wild-type Ca(v)1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly(182) in domain I is involved in the membrane trafficking or surface expression of alpha(1) subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Ca(v)1.2, although G579Q showed a normal VDI, implying that Gly(579) in domain II is involved in the regulation of Vol and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for alpha(1) subunit to become functional. (C) 2010 Elsevier B.V. All rights reserved.