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.

Figure 1. Whole-cell characteristics of the α_1C,77, α_1C,86, α_1C,77K and α_1C,77L channels

Representative whole-cell current traces with 15 mm Ba²⁺ as charge carrier obtained with α_1C,77 (A), α_1C,86 (B), α_1C,77K (E) and α_1C,77L (F) channels by repetitive (0.2 Hz) depolarizations from a holding potential of -80 mV to test potentials between -5 and 55 mV with an incremental increase of 10 mV. C and G, corresponding averaged current-voltage relationships derived from the indicated number of experiments, each normalized to maximum current. D and H, whole-cell traces recorded from α_1C,77 and α_1C,86 (D) as well as α_1C,77K and α_1C,77L channels (H) at a test potential of 25 mV were scaled with respect to the peak current size. Current decays were fitted by mono-exponential (α_1C,77) and double-exponential (α_1C,86, α_1C,77K and α_1C,77L) functions yielding the indicated time constants. Dotted lines in A and B, and D–H denote zero current level.

Figure 2. Time dependence of whole-cell Ba²⁺ currents through α_1C,77 and α_1C,86 channels

A, peak currents generated by repetitive depolarizations to 25 mV (α_1C,77) and 35 mV (α_1C,86). Comparison of currents at 4 min indicated a significant (P < 0.01) difference in current size between the α_1C,77 and α_1C,86 channels. B, representative current traces recorded for α_1C,77 and α_1C,86 channels at 1 and 4 min as indicated in A. Dotted lines denote zero current level. C, the initial current (0 min) measured in each individual experiment for the respective channel is plotted against the normalized current reached after 4 min.

Figure 3. Time dependence of single-channel currents of α_1C,77 and α_1C,86 channels in cell-attached and inside-out patch configurations

Average channel activity (NP_o) of α_1C,77 (A) and α_1C,86 channels (B) recorded in the cell-attached (c.a.) patch followed by inside-out (i.o.) patch configuration. Inset in A shows mean channel activity reached 1.5-2.5 min after patch excision normalized to the previous activity in the cell-attached patch. Consecutive single-channel traces corresponding to the experiments in A and B are depicted in C and D both in the cell-attached and inside-out patch. Dotted lines indicate zero current level.

Figure 4. Time dependence of whole-cell currents of α_1C,77K and α_1C,77L channels

A, peak currents normalized to initial currents are shown as dependent on time for α_1C,77K and α_1C,77L channels recorded at test potentials of 35 mV. Comparison of currents at 4 min indicated no significant (P > 0.05) difference in current size between the α_1C,77K and α_1C,77L channels. B, representative current traces recorded for α_1C,77K and α_1C,77L channels at 1 and 4 min as indicated in A. Dotted lines denote zero current level.

Figure 5. Time dependence of single-channel Ba²⁺ currents through α_1C,77K and α_1C,77L channels in cell-attached and inside-out patch configurations

Average channel activity (NP_o) of α_1C,77K (A) and α_1C,77L (B) channels recorded in the cell-attached (c.a.) patch followed by inside-out (i.o.) patch configuration. Inset in A shows mean channel activity reached 1.5-2.5 min after patch excision normalized to the preceding activity in the cell-attached patch. Consecutive single-channel traces corresponding to the experiments in A and B are depicted in C and D both in the cell-attached and inside-out patch. Single channel openings are visible as downward deflections.

Figure 6. Effect of calpastatin + ATP on the activity of single α_1C,77 and α_1C,86 channels in the inside-out patch

Time course of average channel activity (NP_o) of α_1C,77 (A) and α_1C,86 (B) in the cell-attached (c.a.) and inside-out (i.o.) patch before and after addition of calpastatin (2 U ml⁻¹) + Na₂ATP (1 mm). Single-channel activity of α_1C,77 almost completely recovered from run-down in the inside-out (i.o.) patch, whereas almost no effect was observed on channel activity of α_1C,86, which was resistant to run-down in the i.o. patch. C and D depict single-channel traces for the corresponding experiments in A and B, recorded under the indicated conditions. Single channel openings are visible as downward deflections. E, mean channel activity of α_1C,77 and α_1C,86 in the presence of calpastatin + ATP, normalized to the preceding channel activity in the cell-attached configuration. F, representative Western blot analysis (n = 4) of Sigma calpastatin and HEK-tsA201 cell 12000 g supernatant using an anti-calpastatin antibody.