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Molecular determinant for run-down of L-type Ca2+ channels localized in the carboxyl terminus of the 1C subunit
K J Kepplinger1, G Förstner, H Kahr
1Institute for Biophysics, University of Linz, A-4040 Linz, Austria.
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.
Insights
The C-terminal sequence (1572-1651) of alpha1C subunits significantly impacts L-type calcium channel run-down. This region confers resistance to run-down and may be modulated by calpastatin.
Area of Science:
- Molecular and Cellular Biology
- Cardiovascular Physiology
- Ion Channel Research
Background:
- Calcium (Ca2+) channels are crucial for cellular function, and their activity can be modulated by various factors.
- Channel 'run-down,' a decrease in channel activity over time, affects the reliability of Ca2+ channel studies and function.
- The C-terminal tail of the alpha1C subunit is implicated in the regulation of L-type Ca2+ channel properties.
Purpose of the Study:
- To investigate the role of the specific C-terminal sequence (residues 1572-1651) in the alpha1C subunit of Ca2+ channels.
- To determine how alterations in this sequence affect the phenomenon of channel run-down.
- To explore the potential involvement of calpastatin in modulating Ca2+ channel run-down.
Main Methods:
- Functional characterization of wild-type and mutant alpha1C,77 and alpha1C,86 Ca2+ channel isoforms.
- Co-expression of pore-forming alpha1C subunits with alpha2delta and beta2a subunits in HEK-tsA201 cells.
- Electrophysiological recordings using whole-cell and single-channel patch-clamp techniques.
- Western blot analysis to detect calpastatin expression.
Main Results:
- Replacing amino acids 1572-1651 in alpha1C,77 with those from alpha1C,86 (creating alpha1C,86) significantly altered run-down, slowing it considerably.
- Transferring specific segments (L: 1572-1598 or K: 1595-1652) from alpha1C,86 into alpha1C,77 did not fully replicate the resistance to run-down observed in alpha1C,86.
- The protease inhibitor calpastatin, when added with ATP, could reverse the run-down of alpha1C,77 channels, and calpastatin expression was confirmed in the cells.
Conclusions:
- The C-terminal sequence spanning residues 1572-1651 in the alpha1C subunit plays a critical role in regulating the run-down of L-type Ca2+ channels.
- This specific sequence confers significant resistance to channel run-down.
- Endogenous calpastatin is suggested as a potential modulator of Ca2+ channel run-down, targeting this C-terminal region.