Etsuko Minobe

Tetrodotoxin-resistant Na+ channels in human neuroblastoma cells are encoded by new variants of Nav1.5/SCN5A

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 α 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 INa in NB‐1 cells. The IC50 for the TTX block was ≈ 8 µm in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.

Calpastatin domain L is a partial agonist of the calmodulin-binding site for channel activation in Cav1.2 Ca2+ channels

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.

CaMKII phosphorylates a threonine residue in the C-terminal tail of Cav1.2 Ca(2+) channel and modulates the interaction of the channel with calmodulin.

We have previously found that both CaMKII-mediated phosphorylation and calmodulin (CaM) binding to the channels are required for maintaining basal activity of the Cav1.2 Ca2+ channels. In this study, we investigated the hypothetical CaMKII phosphorylation site on Cav1.2 that contributes to the channel regulation. We found that CaMKII phosphorylates the Thr1603 residue (Thr1604 in rabbit) within the preIQ region in the C-terminal tail of the guinea-pig Cav1.2 channel. Mutation of Thr1603 to Asp (T1603D) slowed the run-down of the channel in inside-out patch mode and abolished the time-dependency of the CaM’s effects to reverse run-down. We also found that CaMKII-mediated phosphorylation of the proximal C-terminal fragment (CT1) increased, while dephosphorylation of CT1 decreased its binding with CaM. These findings suggest that CaMKII regulates the CaM binding to the channel, and thereby maintains basal activity of the Cav1.2 Ca2+ channel.

A new phosphorylation site in cardiac L-type Ca2+ channels (Cav1.2) responsible for its cAMP-mediated modulation.

Cardiac L-type Ca(2+) channels are modulated by phosphorylation by protein kinase A (PKA). To explore the PKA-targeted phosphorylation site(s), five potential phosphorylation sites in the carboxyl (COOH) terminal region of the α1C-subunit of the guinea pig Cav1.2 Ca(2+) channel were mutated by replacing serine (S) or threonine (T) residues with alanine (A): S1574A (C1 site), S1626A (C2), S1699A (C3), T1908A, (C4), S1927A (C5), and their various combinations. The wild-type Ca(2+) channel activity was enhanced three- to fourfold by the adenylyl cyclase activator forskolin (Fsk, 5 μM), and that of mutants at C3, C4, C5, and combination of these sites was also significantly increased by Fsk. However, Fsk did not modulate the activity of the C1 and C2 mutants and mutants of combined sites involving the C1 site. Three peptides of the COOH-terminal tail of α1C, termed CT1 [corresponding to amino acids (aa) 1509-1789, containing sites C1-3], CT2 (aa 1778-2003, containing sites C4 and C5), and CT3 (aa 1942-2169), were constructed, and their phosphorylation by PKA was examined. CT1 and CT2, but not CT3, were phosphorylated in vitro by PKA. Three CT1 mutants at two sites of C1-C3 were also phosphorylated by PKA, but the mutant at all three sites was not. The CT2 mutant at the C4 site was phosphorylated by PKA, but the mutant at C5 sites was not. These results suggest that Ser(1574) (C1 site) may be a potential site for the channel modulation mediated by PKA.

Adenosine triphosphate regulates the activity of guinea pig Cav1.2 channel by direct binding to the channel in a dose-dependent manner

The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca(2+) channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATP-biotin maintains Ca(2+) channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca(2+) was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6-140) and proximal COOH-terminal region (amino acids 1,509-1,789) of the main subunit (α1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca(2+) dependent.

The individual N- and C-lobes of calmodulin tether to the Cav1.2 channel and rescue the channel activity from run-down in ventricular myocytes of guinea-pig heart

The present study examined the binding of the individual N‐ and C‐lobes of calmodulin (CaM) to Cav1.2 at different Ca2+ concentration ([Ca2+]) from ≈ free to 2 mM, and found that they may bind to Cav1.2 Ca2+‐dependently. In particular, using the patch‐clamp technique, we confirmed that the N‐ or C‐lobes can rescue the basal activity of Cav1.2 from run‐down, demonstrating the functional relevance of the individual lobes. The data imply that at resting [Ca2+], CaM may tether to the channel with its single lobe, leading to multiple CaM molecule binding to increase the grade of Ca2+‐dependent regulation of Cav1.2.

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

To demonstrate the interaction of calpastatin (CS) domain L (CSL) with Cav1.2 channel, we investigated the binding of CSL with various C‐terminus‐derived peptides at ≈ free, 100 nM, 10 μM, and 1 mM Ca2+ by using the GST pull‐down assay method. Besides binding with the IQ motif, CSL was also found to bind with the PreIQ motif. With increasing [Ca2+], the affinity of the CSL–IQ interaction gradually decreased, and the affinity of the CSL–PreIQ binding gradually increased. The results suggest that CSL may bind with both the IQ and PreIQ motifs of the Cav1.2 channel in different Ca2+‐dependent manners.

Role of protein phosphatases in the run down of guinea pig cardiac Cav1.2 Ca2+ channels.

This study aimed to investigate protein phosphatases involved in the run down of Cav1.2 Ca(2+) channels. Single ventricular myocytes obtained from adult guinea pig hearts were used to record Ca(2+) channel currents with the patch-clamp technique. Calmodulin (CaM) and ATP were used to restore channel activity in inside-out patches. Inhibitors of protein phosphatases were applied to investigate the role of phosphatases. The specific protein phosphatase type 1 (PP1) inhibitor (PP1 inhibitor-2) and protein phosphatase type 2A (PP2A) inhibitor (fostriecin) abolished the slow run down of Cav1.2 Ca(2+) channels, which was evident as the time-dependent attenuation of the reversing effect of CaM/ATP on the run down. However, protein phosphatase type 2B (PP2B, calcineurin) inhibitor cyclosporine A together with cyclophilin A had no effect on the channel run down. Furthermore, PP1 inhibitor-2 mainly prolonged the open time constants of the channel, specifically, the slow open time. Fostriecin primarily shortened the slow close time constants. Our data suggest that PP1 and PP2A were involved in the slow phase of Cav1.2 Ca(2+) channel run down. In addition, they exerted different effects on the open-close kinetics of the channel. All above support the view that PP1 and PP2A may dephosphorylate distinct phosphorylation sites on the Cav1.2 Ca(2+) channel.

PKA and phosphatases attached to the CaV1.2 channel regulate channel activity in cell-free patches

Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca(2+) channels (Ca(V)1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the Ca(V)1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of Ca(V)1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. Ca(V)1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the Ca(V)1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced Ca(V)1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the Ca(V)1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.

Lobe-related concentration- and Ca2+-dependent interactions of calmodulin with C- and N-terminal tails of the CaV1.2 channel

This study examined the bindings of calmodulin (CaM) and its mutants with the C- and N-terminal tails of the voltage-gated Ca2+ channel CaV1.2 at different CaM and Ca2+ concentrations ([Ca2+]) by using the pull-down assay method to obtain basic information on the binding mode, including its concentration- and Ca2+-dependencies. Our data show that more than one CaM molecule could bind to the CaV1.2 C-terminal tail at high [Ca2+]. Additionally, the C-lobe of CaM is highly critical in sensing the change of [Ca2+] in its binding to the C-terminal tail of CaV1.2, and the binding between CaM and the N-terminal tail of CaV1.2 requires high [Ca2+]. Our data provide new details on the interactions between CaM and the CaV1.2 channel.