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 α(1C) subunit gene

Recently we have described a splice variant of the L-type Ca²⁺ channel (α(1C,86)) in which 80 amino acids (1572-1651) of the conventional α(1C,77) were substituted by another 81 amino acids due to alternative splicing of exons 40-42. Ba²⁺ current (I(Ba)) through α(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' α(1C,77) channel with the respective segments of the 'fast' α(1C,86) gave rise to rapidly inactivating α(1C,86) like channel isoforms. We found that replacement of either motifs ¹⁵⁷²IKTEG¹⁵⁷⁶ or ¹⁶⁰⁰LLDQV¹⁶⁰⁴ of α(1C,77) with the respective sequences of α(1C,86) caused strong but partial acceleration of I(Ba) inactivation. Replacement of both sequences produced an α(1C,86)-like fast channel which had no Ca²⁺ -dependent inactivation. These results support the hypothesis that motifs 1572-1576 and 1600-1604 of α(1C,77) contribute cooperatively to inactivation kinetics of α(1C) and are critical for Ca²⁺ -dependent inactivation of the channel.