Molecular determinant for run‐down of L‐type Ca²⁺ channels localized in the carboxyl terminus of the α_1C subunit

Abstract

The role of the sequence 1572‐1651 in the C‐terminal tail of the α1C subunit in run‐down of Ca2+ channels was studied by comparing functional properties of the conventional α1C,77 channel with those of three isoforms carrying alterations in this motif. The pore‐forming α1C subunits were co‐expressed with α2δ and β2a 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. Replacement of amino acids 1572‐1651 in α1C,77 with 81 different amino acids leading to α1C,86 significantly altered run‐down behaviour. Run‐down of Ba2+ currents was rapid with α1C,77 channels, but was slow with α1C,86. Transfer of the α1C,86 segments L (amino acids 1572‐1598) or K (amino acids 1595‐1652) into the α1C,77 channel yielded α1C,77L and α1C,77K channels, respectively, the run‐down of which resembled more that of α1C,77. These results demonstrate that a large stretch of sequence between residues 1572 and 1652 of α1C,86 renders Ca2+ channels markedly resistant to run‐down. The protease inhibitor calpastatin added together with ATP was able to reverse the run‐down of α1C,77 channels. Calpastatin expression was demonstrated in the HEK‐tsA cells by Western blot analysis. These results indicate a significant role of the C‐terminal sequence 1572‐1651 of the α1C subunit in run‐down of L‐type Ca2+ channels and suggest this sequence as a target site for a modulatory effect by endogenous calpastatin.

Inhibition of Recombinant L-Type Voltage-Gated Calcium Channels by Positive Allosteric Modulators of GABAA Receptors

“…In control cells transfected with Ca v 1.3 LVGCCs (n ϭ 4) in which no drug was applied, the average percentage current remaining 2, 5, and 10 min from the start of recording was 71 Ϯ 11, 57 Ϯ 13, and 42 Ϯ 9%, respectively. This extent of rundown was similar to that of Ca v 1.2 LVGCCs in this study and as reported previously (Kepplinger et al, 2000). There was no apparent correlation between the extent of rundown in different cells and drug concentrationresponse, suggesting that the mechanisms causing the rundown phenomenon were not a significant factor in modifying L-VGCC inhibition by GABA A R modulators.…”

Structure-Functional Diversity of Human L-Type Ca2+Channel: Perspectives for New Pharmacological Targets: Figure 1

“…Replacements in the cytoplasmic 80-amino acid segment leading to ␣ 1C,86 , ␣ 1C,77L , and ␣ 1C,77K variants affected voltage sensors for activation and inactivation (Soldatov et al, 1997(Soldatov et al, , 1998a, and reduced open probability and single channel conductance (Kepplinger et al, 2000b) thus suggesting that both Ca 2ϩ -sensing domains interact with a pore jpet.aspetjournals.org region and affect the voltage-gating mechanism of the channel. Combined disruption of motifs L and K in ␣ 1C,86 completely eliminated the characteristic run-down property of the channel (Kepplinger et al, 2000a). This region may involve determinants for the rescue effect of calpastatin (Romanin et al, 1991).…”

β3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

“…In these studies, the drug induced a small, approximately 20%, maximal reduction of basal I Ca,L , and the effect was increased to approximately 30% when the myocytes were isolated from failing hearts (29,53). Surprisingly, the effects of BRL37344 persisted after washout of the drug, which is a concern when considering that I Ca,L is subjected to substantial spontaneous rundown in mammalian cardiomyocytes (55,56). However, the inhibitory effects of BRL37344 were attenuated by preincubation of the cells with the NOS inhibitor N G -nitrol-arginine methyl ester (L-NAME) (50,53) or after treatment of the myocytes with Bordetella pertussis toxin (53), which is consistent with earlier findings that a β 3 -AR signaling cascade involved G i/o protein and NOS3 activation (8,16,17).…”