Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel alpha(1) subunits.
The pore-forming component of voltage-gated calcium channels, alpha(1) 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 I and 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 I and 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, G182Q, 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 VDI 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.
Teng
Jinfeng
J
Department of Biology, Tokyo Gakugei University, 4-1-1 Nukui kita-machi, Koganei-shi, Tokyo 184-8501, Japan.
Iida
Kazuko
K
Ito
Masanori
M
Izumi-Nakaseko
Hiroko
H
Kojima
Itaru
I
Adachi-Akahane
Satomi
S
Iida
Hidetoshi
H
D000595:Amino Acid Sequence; D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D005998:Glycine / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D005973:Glutamine; D018698:Glutamic Acid; D011392:Proline; D005998:Glycine