Asako Kameyama

Tissue extract recovers cardiac calcium channels from ‘run-down’

Effects of cardiac-tissue extract on the activity of L-type Ca2+ channels were investigated in guinea-pig ventricular myocytes with the patch-clamp method. In most patches, Ca2+-channel current recorded with a pipette solution containing 50 mM Ba2+ and 3 μM Bay K 8644 ran down within 5 min after excision of the patches into a solution containing EGTA. This run-down of Ca2+ channels was prevented when pathches were excised into a solution containing a supernatant fraction of homogenate of guinea-pig or bovine heart. Furthermore, this tissue extract was able to restore channel activity after run-down. This channel-activating effect of the extract was abolished by heat treatment or trypsin digestion. Fractionation of the extract by gel filtration suggested that the channel-activating factor(s) had an apparent molecular weight of 2–3×105. These results suggest that some cytoplasmic protein(s) maintains the activity of the cardiac L-type Ca2+ channel.

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.

ATP regulates cardiac Ca2+ channel activity via a mechanism independent of protein phosphorylation

Abstract The role of adenosine triphosphate (ATP) in the regulation of L-type Ca2+ channel activity was investigated in inside-out patches from guinea-pig ventricular cells, in which the Ca2+ channel activity had been reprimed by application of cytoplasm from bovine heart. Passing the cytoplasm through a diethylaminoethyl (DEAE)-sepharose column or heating at 60°C for 20 min attenuated the induction Ca2+ channel activity to 6–13% of that in the preceding cell-attached patch. Addition of 10 mM MgATP to the cytoplasm greatly improved the potency of cytoplasm in restoring Ca2+ channel activity (to 83 ± 22%, mean ± SE). This effect of MgATP was also produced, although with lower potency, by K2ATP (61 ± 20%) or 5′-adenylylimidodiphosphate (AMP-PNP, 39 ± 7%), a non-hydrolyzable ATP analogue, suggesting that hydrolysis of ATP is not required for the stimulatory effect on channel activity. A non-specific protein kinase inhibitor H8 (50–100 μM) did not inhibit the effect of cytoplasm + MgATP on channel activity, suggesting the involvement of a pathway independent of phosphorylation. We conclude that ATP regulates Ca2+ channel activity in dual pathways: one with, and the other without, protein phosphorylation.

Run-down of the cardiac Ca2+ channel: characterization and restoration of channel activity by cytoplasmic factors

Abstract Possible mechanisms for run-down in the Ca2+ channel, such as proteolysis or dephosphorylation of the channel, were examined in guinea-pig ventricular myocytes. The Ca2+ channel current, recorded in inside-out patches using a pipette solution containing 50 mM Ba2+ and 3 μM Bay K 8644, ran down with a mean survival time of 2.35 min. The survival time was not significantly affected by adenosine triphosphate (ATP) (3 mM), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) (2 mM), isoprenaline (l–5 μM), phosphate (l20 mM) and leupeptin (l0 μM). Stimulation of guanosine triphosphate (GTP)-binding proteins was also ineffective. The catalytic subunit of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase (PKA, 0.5–2 μM) slightly and transiently increased channel activity, but had minimal effects on the channel when applied after complete run-down. On the other hand, cytoplasm from the heart, skeletal muscle, brain and liver, but not kidney, induced channel activity. There was a positive correlation between NPo (the product of the number of channels N and the open probability Po) value before run-down and that after the application of cytoplasm, suggesting that the activity of once-active channels was restored ba the exogenous cytoplasm. The potency of cytoplasm in tissues in inducing channel activity was not related to PKA activity nor to the number of dihydropyridine binding sites. These results suggest that the run-down of the cardiac Ca2+ channel is not mediated by dephosphorylation or proteolysis of the channel, but involves other factor(s), possibly interaction of the channel protein with a cytoplasmic regulatory protein.

A cytoplasmic factor, calpastatin and ATP together reverse run‐down of Ca2+ channel activity in guinea‐pig heart

1 The cytoplasmic extract of bovine heart was separated into four fractions by gel filtration: H (molecular mass > 300 kDa), P (250‐300 kDa), L1 (180‐250 kDa) and L2 (< 180 kDa). The effects of these fractions on the run‐down of L‐type Ca2+ channel activity were investigated in guinea‐pig ventricular myocytes. 2 After run‐down induced by inside‐out patch formation, Ca2+ channel activity was restored by P or H (+ 3 mM ATP) to 7·5 and 5·8 % of that in the cell‐attached mode, respectively, but to as high as 86 % by P + H + ATP. 3 The reversal of run‐down brought about by the P fraction was mimicked by calpastatin. 4 The restorative effect of calpastatin + ATP showed a biphasic time course: 38 % in the early transient phase and 11 % in the late phase. However, calpastatin + H + ATP showed a sustained effect: 66 % in the early transient phase, and 87 % in the late phase. 5 The effective component of the H fraction showed a protein‐like nature: heat and trypsin sensitivity. 6 The activities of cAMP‐dependent protein kinase, casein kinase I, casein kinase II, protein tyrosine kinase, protein serine/threonine or tyrosine phosphatases were measured. However, these kinases and phosphatases were not confirmed as the effective component of cytoplasm or the H fraction. 7 Run‐down was not prevented by 2 μM phalloidin or 2 μM paclitexal, suggesting that neither actin filaments nor microtubules are directly involved in the run‐down. 8 Our results support the view that the basal activity of the Ca2+ channel is maintained by at least three factors: a protein‐like factor in the H fraction, calpastatin, and ATP.

Analysis of four novel variants of Nav1.5/SCN5A cloned from the brain.

Na+ currents with tetrodotoxin resistance (TTX-R) have been observed in neurons, but the full-length cDNAs encoding the TTX-R Nav1.5 channels in human and rat brains have not been identified. In this study, four full-length cDNAs encoding the alpha-subunits of the Nav1.5 channels in human and rat cerebral cortexes were cloned and designated hB1, hB2, rN1 and rN2 (accession number: EF629346, EF629347, EF618549, EF618550). The longest open reading frame of hB1 or rN1 encodes 2016 amino acid residues. Sequence analysis has indicated that hB1 is highly homologus with human cardiac Nav1.5/SCN5A (hH1) with >98% amino acid identity. Genomic sequence analysis of Nav1.5/SCN5A revealed that it is exon6A rather than exon6 splice variant of Nav1.5 which is expressed in human and rat brains. Alternative splicing variants hB2 and rN2, which lack exon24 and encode proteins of 1998 amino acids, were also identified. Furthermore, the total Nav1.5 mRNA and Navbeta1 mRNA were detected in 16 different tissue types of developing Wistar rats by reverse polymerase chain reaction (RT-PCR), and their expression patterns varied among different tissue types with age development. These results suggest that Nav1.5 channels in human and rat brains are encoded by new variants of Nav1.5/SCN5A and Nav1.5 is more widely distributed and expressed than previously thought.

New Variants of Nav1.5/SCN5A Encode Na+ Channels in the Brain

Exon6A of Nav1.5/SCN5A was first found in the cloning of Nav1.5 from the human neuroblastoma cell line NB-1 (Ou et al., 16), but its expression in brain and non-brain tissue had not been identified. In this study, we have further investigated this new exon and compared it with exon6 of Nav1.5/SCN5A. Reverse transcription-polymerase chain reaction (RT-PCR) and sequence analysis both confirmed that it is exon6A that encodes Nav1.5 in brain tissue, and it is exon6 that encodes Nav1.5 in non-brain tissue. The expression of exon6A in different parts of the brain is different, with expression levels in the order of hippocampus > cerebral cortex > brain stem > cerebellum. Different expression levels of exon6 in different tissues of Wistar rats were also found. These results suggest that exon6A is unique in encoding the Nav1.5 channels in the central nervous system. In addition, novel alternative splicing of Nav1.5/SCN5A, lacking exon24, was first found in our study. This alternative splicing was also found in other tissues, such as heart, lung and testis. However, the ratio of the two variants changed differently in different types of tissues in developing rats. These results suggest that Nav1.5/SCN5A has a newly identified alternative splicing, and the Nav1.5 channels in the brain are encoded by new variants of Nav1.5/SCN5A.

Effects of calciseptine on unitary barium channel currents in guinea-pig portal vein

Effects of synthesized calciseptine (CaS), found naturally in the venom of the black mamba, on voltage-dependent Ca2+ channels in smooth muscle cells of the guinea-pig portal vein were investigated. In the whole-cell voltage-clamp configuration, extracellular application of CaS (≥ 10 nM) inhibited the inward current in a concentration- and voltage-dependent manner at a holding potential of −90 mV. The Ca2+ current recorded at a high holding potential (−50 mV) was approximately 8 times more sensitive to CaS than that at a more negative holding potential (−90 mV). CaS (50 nM) shifted to the left the steady-state inactivation curve obtained by using single 8-s conditioning pulses of various amplitudes. When CaS (≥ 200 nM) was present in the pipette, the Ca2+ current remained for the duration of the experiments (more than 60 min) in the whole-cell configuration. Two different Ca2+ channel conductances are present in this tissue (25-pS and 12-pS channels). Both channels are blocked by dihydropyridine (DHP) derivatives, but have different sensitivities. In the cell-attached condition, CaS hardly changed the activity of either unitary Ca2+ channel current. To prevent the “run down” of the Ca2+ channels in cell-free conditions, we added cardiac cytosol, a supernatant from homogenized cardiac cells and an endogenous Ca2+ channel activating factor, in the pipette. The unitary Ca2+ channel currents were then recorded using the outside-out membrane patch configuration. Application of CaS (1 μM) in the bath completely blocked the open events of the 25-pS Ca2+ channel. CaS (10 nM) in the bath reduced the mean open time and channel availability, resulting in a decrease in the open probability of the 25-pS channel currents without affecting the amplitude of the single-channel conductance. CaS also reduced the open probability (though less potently) and channel availability of the 12-pS Ca2+ channel without a change in its amplitude. From these results, we conclude that CaS has inhibitory effects on the voltage-dependent Ca2+ current that are similar to those of DHP derivatives and that it acts from the outside of the membrane.

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.