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Auxiliary beta-subunits bound to the cytoplasmic alpha(1)-interaction domain of the pore-forming alpha(1C)-subunit are important modulators of voltage-gated Ca(2+) channels. The underlying mechanisms are not yet well understood. We investigated correlations between differential modulation of inactivation by beta(1a)- and beta(2)- subunits and structural responses of the channel to transition into distinct functional states. The NH(2)-termini of the alpha(1C)- and beta-subunits were fused with cyan or yellow fluorescent proteins, and functionally coexpressed in COS1 cells. Fluorescence resonance energy transfer (FRET) between them or with membrane-trapped probes was measured in live cells under voltage clamp. It was found that in the resting state, the tagged NH(2)-termini of the alpha(1C)- and beta-subunit fluorophores are separated. Voltage-dependent inactivation generates strong FRET between alpha(1C) and beta(1a) suggesting mutual reorientation of the NH(2)-termini, but their distance vis-à-vis the plasma membrane is not appreciably changed. These voltage-gated rearrangements were substantially reduced when the beta(1a)-subunit was replaced by beta(2). Differential beta-subunit modulation of inactivation and of FRET between alpha(1C) and beta were eliminated by inhibition of the slow inactivation. Thus, differential beta-subunit modulation of inactivation correlates with the voltage-gated motion between the NH(2)-termini of alpha(1C)- and beta-subunits and targets the mechanism of slow voltage-dependent inactivation.

The role of the 80-amino acid motif 1572^1651 in the C-terminal tail of K K 1C Ca 2+ channel subunits was studied by comparing properties of the conventional K K 1CY77 channel expressed in HEK-tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 15721 651 in K K 1CY77 with 81 non-identical residues leading to K K 1CY86 impaired membrane targeting and cluster formation of the channel. Similar to K K 1CY86 , substitution of its 1572^1598 (K K 1CY77L ) or 1595^1652 (K K 1CY77K ) segments into the K K 1CY77 channel yielded single-channel Ba 2+ currents with increased inactivation, reduced open probability and unitary conductance, when compared to the K K 1CY77 channel. Thus, the C-terminal sequence 1572^1651 of the K K 1C subunit is important for membrane targeting, permeation and open probability of L-type Ca 2+ channels. ß

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.

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