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 α1C subunit gene

Recently we have described a splice variant of the L-type Ca2+ channel (alpha(1C,86)) in which 80 amino acids (1572-1651) of the conventional alpha(1C,77) were substituted by another 81 amino acids due to alternative splicing of exons 40-42, Ba2+ current (I-Ba) through alpha(1C,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" alpha(1C,77) channel with the respective segments of the "fast" alpha(1C,86) gave rise to rapidly inactivating alpha(1C,86)-like channel isoforms, We found that replacement of either motifs (1572)IKTEG(1576) or (1600)LLDQV(1604) of alpha(1C,77) with the respective sequences of alpha(1C,86) caused strong but partial acceleration of I-Ba inactivation. Replacement of both sequences produced an alpha(1C,86)-like fast channel which had no Ca2+-dependent inactivation. These results support the hypothesis that motifs 1572-1576 and 1600-1604 of alpha(1C,77) contribute cooperatively to inactivation kinetics of alpha(1C) and are critical for Ca2+-dependent inactivation of the channel.