Calpastatin Domain L Is a Partial Agonist of the Calmodulin-binding Site for Channel Activation in Cav1.2 Ca2+ Channels

Abstract

Cav1.2 Ca2؉ channel activity diminishes in inside-out patches (run-down). Previously, we have found that with ATP, calpastatin domain L (CSL) and calmodulin (CaM) recover channel activity from the run-down in guinea pig cardiac myocytes. Because the potency of the CSL repriming effect was smaller than that of CaM, we hypothesized that CSL might act as a partial agonist of CaM in the channel-repriming effect. To examine this hypothesis, we investigated the effect of the competitions between CSL and CaM on channel activity and on binding in the channel. We found that CSL suppressed the channel-activating effect of CaM in a reversible and concentration-dependent manner. The channel-inactivating effect of CaM seen at high concentrations of CaM, however, did not seem to be affected by CSL. In the GST pull-down assay, CSL suppressed binding of CaM to GST fusion peptides derived from C-terminal regions in a competitive manner. The inhibition of CaM binding by CSL was observed with the IQ peptide but not the PreIQ peptide, which is the CaM-binding domain in the C terminus. The results are consistent with the hypothesis that CSL competes with CaM as a partial agonist for the site in the IQ domain in the C-terminal region of the Cav1.2 channel, which may be involved in activation of the channel. Calpastatin (CS)2 is a specific endogenous inhibitor of calpain, a Ca 2ϩ -activated cysteine protease. The calpain and CS complex plays a pivotal role in cell adhesion, shape change, migration, and apoptosis. CS is composed of a unique leader domain (domain L) in the N-terminal and four repetitive homologous domains (domains 1-4), each of which has calpain-inhibiting activity. However, CS domain L (CSL) has no calpain-inhibiting activity (1), and its physiological role remains largely unknown. Previous studies have shown that CS or CSL reprimes activity of Cav1.2 Ca 2ϩ channels, in a concentration-dependent manner, in cardiac myocytes using the inside-out patch clamp technique (2, 3). Subsequent studies have further shown that the effective region of CSL is confined to a region spanning 11 amino acid residues (amino acids 54 -64 in exon 5) and that CSL can bind to a fragment derived from the intracellular C-terminal region of Cav1.2 channels (4, 5). However, it has not been clarified how CSL interacts with the channel and regulates its activity.Calmodulin (CaM) is a small (18 kDa), ubiquitous, Ca 2ϩ -binding protein that mediates Ca 2ϩ -dependent regulation of various target proteins, such as enzymes, ion channels, and transporters (6). The activity of the Cav1.2 Ca 2ϩ channel is controlled by Ca 2ϩ -dependent facilitation (CDF) and inactivation (CDI) (7-11). CaM has been suggested to serve as a Ca 2ϩ sensor and a transducer to produce Ca 2ϩ -dependent conformation changes of the channel protein in both CDF and CDI. In the case of CDF, although another mechanism involving phosphorylation of the channels by Ca 2ϩ /CaM-dependent protein kinase has been suggested (12), the activation of the kinase is also mediated by CaM. In our prev...

The Ca2+‐dependent interaction of calpastatin domain L with the C‐terminal tail of the Cav1.2 channel

“…However, the maximum effect of CS L was only 14% of the CaM effect; the effects of CS L and CaM were not simply additive; and CS L even suppressed the effect of CaM in a concentration-dependent manner [8]. Thus, it is proposed that CS L may compete with CaM for binding with Cav1.2 as a partial agonist [8], and this kind of competitive binding of CaM and CS L was eventually confirmed on the IQ motif [8]. , in IQ motif, significantly affect the CaM binding to the preIQ and IQ peptides, respectively [12].…”

“…The purification of the GST-fusion proteins was as described in the previous reports [8,12,13] As for CS L fusion protein, the GST portion of it was removed by using the PreScission Protease (GE Healthcare). All steps were performed on ice or at 4°C.…”

“…The amount of CS L and GST-fusion peptides was corrected based on the relative optical densities of 0.59 and 0.80 in reference to BSA, respectively [8,13]. …”

“…A study using the pull-down assay method has also shown that CS L integrates with fragments of the proximal region (IQ region) of the C-terminal tail of the Cav1.2 channel [7]. CS L additionally competes with calmodulin (CaM) as a partial agonist for the IQ domain in the C-terminal region of Cav1.2 channels, which may be involved in the Ca 2+ -dependent regulations of the channel [7,8]. However, it has not yet been clarified how CS L interacts with the channel and regulates its activity.…”

Structural Insights into Activation of the Retinal L-type Ca2+ Channel (Cav1.4) by Ca2+-binding Protein 4 (CaBP4)

“…4(1440 -1982)) and the Y1595E mutant containing a GST tag were cloned, expressed, and purified as noted before (22). GST pull-down assays were used to monitor binding of CaBP4 to Cav1.4(1440 -1982)-GST as described previously (23). Protein samples of CaBP4 and Cav1.4(1440 -1982)-GST were dissolved in Buffer A (0.1% Triton X-100, 150 mM NaCl, 10 mM CaCl 2 , 10 mM Tris-HCl, pH 7.4, and protease inhibitors), concentrated to 0.5 ml (10 M each), mixed with 0.5 ml of GST-Sepharose beads for 10 min, and then centrifuged for 5 min at 500 ϫ g. Bead pellets were washed twice with Buffer A for 5 min at 4°C.…”