Molecular determinant for run-down of L-type Ca2+ channels localized in the carboxyl terminus of the alpha(1C) subunit

1. The role of the sequence 1572-1651 in the C-terminal tail of the alpha ( 1C) subunit in run-down of Ca2+ channels was studied by comparing functiona l properties of the conventional alpha (1C,77) channel with those of three isoforms carrying alterations in this motif. 2. The pore-forming alpha (1C) subunits were co-expressed with alpha (2)del ta and beta (2a) subunits in HEK-tsA201 cells, a subclone of the human embr yonic kidney cell line, and studied by whole-cell and single-channel patch- clamp techniques. 3. Replacement of amino acids 1572-1651 in alpha (1C,77) with 81 different amino acids leading to alpha (1C,86) significantly altered run-down behavio ur. Run-down of Ba2+ currents was rapid with alpha (1C,77) channels, but wa s slow with alpha (1C,86). 4. Transfer of the alpha (1C,86) segments L (amino acids 1572-1598) or K (a mino acids 1595-1652) into the alpha (1C,77) channel yielded alpha (1C,77L) and alpha (1C,77K) channels, respectively, the run-down of which resembled more that of alpha (1C,77). These results demonstrate that a large stretch of sequence between residues 1572 and 1652 of alpha (1C,86) renders Ca2+ c hannels markedly resistant to run-down. 5. The protease inhibitor calpastatin added together with ATP was able to r everse the run-down of alpha (1C,77) channels. Calpastatin expression was d emonstrated in the HEK-tsA cells by Western blot analysis. 6. These results indicate a significant role of the C-terminal sequence 157 2-1651 of the alpha (1C) subunit in run-down of L-type Ca2+ channels and su ggest this sequence as a target site for a modulatory effect by endogenous calpastatin.