No abstract

Both tetrodotoxin-sensitive (TTX-S) and TTX-resistant (TTX-R) voltage-dependent Na+ channels are expressed in the human neuroblastoma cell line NB-1, but a gene encoding the TTX-R Na+ channel has not been identified. In this study, we have cloned cDNA encoding the alpha subunit of the TTX-R Na+ channel in NB-1 cells and designated it hNbR1. The longest open reading frame of hNbR1 (accession no. AB158469) encodes 2016 amino acid residues. Sequence analysis has indicated that hNbR1 is highly homologous with human cardiac Nav1.5/SCN5A with > 99% amino acid identity. The presence of a cysteine residue (Cys373) in the pore-loop region of domain I is consistent with the supposition that hNbR1 is resistant to TTX. Analysis of the genomic sequence of SCN5A revealed a new exon encoding S3 and S4 of domain I (exon 6A). In addition, an alternative splicing variant, lacking exon 18, that encodes 54 amino acids in the intracellular loop between domains II and III was found (hNbR1-2; accession no. AB158470). Na+ currents in human embryonic kidney cells (HEK293) transfected with hNbR1 or hNbR1-2 showed electrophysiological properties similar to those for TTX-R I(Na) in NB-1 cells. The IC50 for the TTX block was approximately 8 microM in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.

Abstract. Although calmodulin binding to various sites of the Cav1.2 Ca 2+ channel has been reported, the mechanism of the interaction is not fully understood. In this study we examined calmodulin binding to fragment channel peptides using a semi-quantitative pull-down assay. Calmodulin bound to the peptides with decreasing affinity order: IQ > preIQ > I-II loop > N-terminal peptide. A peptide containing both preIQ and IQ regions (Leu 1599 -Leu 1668 ) bound with approximately 2 mol of calmodulin per peptide. These results support the hypothesis that two molecules of calmodulin can simultaneously bind to the C-terminus of the Cav1.2 channel and modulate its facilitatory and inhibitory activities.

Abstract. The L-type Ca 2+ channel (Ca V 1.2) shows clear Ca 2+

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...