title: Molecular determinant for run-down of L-type Ca2+ channels localized in the carboxyl terminus of the 1C subunit.
abstract: 1. The role of the sequence 1572-1651 in the C-terminal tail of the alpha1C subunit in run-down of Ca2+ channels was studied by comparing functional properties of the conventional alpha1C,77 channel with those of three isoforms carrying alterations in this motif. 2. The pore-forming alpha1C subunits were co-expressed with alpha2delta and beta2a subunits in HEK-tsA201 cells, a subclone of the human embryonic kidney cell line, and studied by whole-cell and single-channel patch-clamp techniques. 3. Replacement of amino acids 1572-1651 in alpha1C,77 with 81 different amino acids leading to alpha1C,86 significantly altered run-down behaviour. Run-down of Ba2+ currents was rapid with alpha1C,77 channels, but was slow with alpha1C,86. 4. Transfer of the alpha1C,86 segments L (amino acids 1572-1598) or K (amino acids 1595-1652) into the alpha1C,77 channel yielded alpha1C,77L and alpha1C,77K channels, respectively, the run-down of which resembled more that of alpha1C,77. These results demonstrate that a large stretch of sequence between residues 1572 and 1652 of alpha1C,86 renders Ca2+ channels markedly resistant to run-down. 5. The protease inhibitor calpastatin added together with ATP was able to reverse the run-down of alpha1C,77 channels. Calpastatin expression was demonstrated in the HEK-tsA cells by Western blot analysis. 6. These results indicate a significant role of the C-terminal sequence 1572-1651 of the alpha1C subunit in run-down of L-type Ca2+ channels and suggest this sequence as a target site for a modulatory effect by endogenous calpastatin.
authors.affiliation: Institute for Biophysics, University of Linz, A-4040 Linz, Austria.
authors.forename: K J
authors.initials: KJ
authors.lastname: Kepplinger
authors.forename: G
authors.initials: G
authors.lastname: Förstner
authors.forename: H
authors.initials: H
authors.lastname: Kahr
authors.forename: K
authors.initials: K
authors.lastname: Leitner
authors.forename: P
authors.initials: P
authors.lastname: Pammer
authors.forename: K
authors.initials: K
authors.lastname: Groschner
authors.forename: N M
authors.initials: NM
authors.lastname: Soldatov
authors.forename: C
authors.initials: C
authors.lastname: Romanin
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology; D001464:Barium / Q000378:metabolism; D015153:Blotting, Western; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000494:pharmacology / Q000502:physiology; D002154:Calpain / Q000037:antagonists & inhibitors; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004594:Electrophysiology; D049452:Green Fluorescent Proteins; D006801:Humans; D007536:Isomerism; D007668:Kidney / Q000378:metabolism; D008164:Luminescent Proteins; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics
chemical_list: D020746:Calcium Channels, L-Type; D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001464:Barium; C033668:calpastatin; D002154:Calpain