Molecular determinants of L-type Ca2+ channel inactivation. Segment exchange analysis of the carboxyl-terminal cytoplasmic motif encoded by exons 40-42 of the human alpha1C subunit gene.
Recently we have described a splice variant of the L-type Ca2+ channel (alpha1C,86) in which 80 amino acids (1572-1651) of the conventional alpha1C,77 were substituted by another 81 amino acids due to alternative splicing of exons 40-42. Ba2+ current (IBa) through alpha1C,86 exhibited faster inactivation kinetics, was strongly voltage-dependent, and had no Ca2+-dependent inactivation. An oligonucleotide-directed segment substitution and expression of the mutated channels in Xenopus oocytes were used to study the molecular determinants for gating of the channel within the 80-amino acid domain. Replacement of segments 1572-1598 or 1595-1652 of the "slow" alpha1C,77 channel with the respective segments of the "fast" alpha1C,86 gave rise to rapidly inactivating alpha1C,86-like channel isoforms. We found that replacement of either motifs 1572IKTEG1576 or 1600LLDQV1604 of alpha1C,77 with the respective sequences of alpha1C,86 caused strong but partial acceleration of IBa inactivation. Replacement of both sequences produced an alpha1C, 86-like fast channel which had no Ca2+-dependent inactivation. These results support the hypothesis that motifs 1572-1576 and 1600-1604 of alpha1C,77 contribute cooperatively to inactivation kinetics of alpha1C and are critical for Ca2+-dependent inactivation of the channel.
Soldatov
N M
NM
Georgetown University Medical Center, Department of Pharmacology, Washington, D. C. 20007, USA.
Oz
M
M
O'Brien
K A
KA
Abernethy
D R
DR
Morad
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism*; D005091:Exons; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D014982:Xenopus laevis
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D011994:Recombinant Proteins