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Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca2+/calmodulin (CaM) binds to VGSC type II (NaV1.2) isoleucine and glutamine (IQ) motif. An autism-associated mutation in NaV1.2 IQ motif, Arg1902Cys (R1902C), has been reported to affect the combination between CaM and the IQ motif compared to that of the wild type IQ motif. However, the detailed properties for the Ca2+-regulated binding of CaM to NaV1.2 IQ (1901Lys-1927Lys, IQwt) and mutant IQ motif (IQR1902C) remains unclear. Here, the binding ability of CaM and CaM's constituent proteins including N- and C lobe to the IQ motif of NaV1.2 and its mutant was investigated by protein pull-down experiments. We discovered that the combination between CaM and the IQ motif was U-shaped with the highest at [Ca2+] ≈ free and the lowest at 100 nM [Ca2+]. In the IQR1902C mutant, Ca2+-dependence of CaM binding was nearly lost. Consequently, the binding of CaM to IQR1902C at 100 and 500 nM [Ca2+] was increased compared to that of IQwt. Both N- and C lobe of CaM could bind with NaV1.2 IQ motif and IQR1902C mutant, with the major effect of C lobe. Furthermore, CaMKII had no impact on the binding between CaM and NaV1.2 IQ motif. This research offers novel insight to the regulation of NaV1.2 IQwt and IQR1902C motif, an autism-associated mutation, by CaM.

Cav1.2 Ca2+ channels, a type of voltage-gated L-type Ca2+ channel, are ubiquitously expressed, and the predominant Ca2+ channel type, in working cardiac myocytes. Cav1.2 channels are regulated by the direct interactions with calmodulin (CaM), a Ca2+-binding protein that causes Ca2+-dependent facilitation (CDF) and inactivation (CDI). Ca2+-free CaM (apoCaM) also contributes to the regulation of Cav1.2 channels. Furthermore, CaM indirectly affects channel activity by activating CaM-dependent enzymes, such as CaM-dependent protein kinase II and calcineurin (a CaM-dependent protein phosphatase). In this article, we review the recent progress in identifying the role of apoCaM in the channel ‘rundown’ phenomena and related repriming of channels, and CDF, as well as the role of Ca2+/CaM in CDI. In addition, the role of CaM in channel clustering is reviewed.

Parkinson's disease (PD), a common neurodegenerative disease characterized by motor dysfunction, results from the death of dopaminergic neurons in the substantia nigra pars compacta (SNc). Although the precise causes of PD are still unknown, several risk factors for PD have been determined, including aging, genetic mutations, environmental factors, and gender. Currently, the molecular mechanisms underlying risk factor-related neurodegeneration in PD remain elusive. Endoplasmic reticulum stress, excessive reactive oxygen species production, and impaired autophagy have been implicated in neuronal death in the SNc in PD. Considering that these pathological processes are tightly associated with intracellular Ca

Voltage-gated sodium channels (VGSCs) are fundamental to the initiation and propagation of action potentials in excitable cells. Ca

Activity of cardiac Cav1.2 channels is enhanced by cyclic AMP-PKA signaling. In this study, we studied the effects of PKA phosphorylation on the binding of calmodulin to the fragment peptide of the proximal C-terminal tail of α1C subunit (CT1, a.a. 1509–1789 of guinea-pig). In the pull-down assay, in vitro PKA phosphorylation significantly decreased calmodulin binding to CT1 (61%) at high [Ca2+]. The phosphoresistant (CT1SA) and phosphomimetic (CT1SD) CT1 mutants, in which three PKA sites (Ser1574, 1626, 1699) were mutated to Ala and Asp, respectively, bound with calmodulin with 99% and 65% amount, respectively, compared to that of wild-type CT1. In contrast, at low [Ca2+], calmodulin-binding to CT1SD was higher (33–35%) than that to CT1SA. The distal C-terminal region of α1C (CT3, a.a. 1942–2169) is known to interact with CT1 and inhibit channel activity. CT3 bound to CT1SD was also significantly less than that to CT1SA. In inside-out patch, PKA catalytic subunit (PKAc) facilitated Ca2+ channel activity at both high and low Ca2+ condition. Altogether, these results support the hypothesis that PKA phosphorylation may enhance channel activity and attenuate the Ca2+-dependent inactivation, at least partially, by modulating calmodulin-CT1 interaction both directly and indirectly via CT3-CT1 interaction. Keywords: Ca2+ channels, Cav1.2, Calmodulin, PKA, Phosphorylation

Both recombinant glutathione-S-transferase (GST)-fused and GST-cleaved fragments of an L-type voltage-gated Ca2+ channel (Cav1.2) are used frequently in GST pull-down assays to investigate the interactions between regulatory proteins and the Cav1.2 channel. However, GST-fused and GST-cleaved proximal C-terminal fragments of the guinea-pig cardiac Cav1.2 channel (CT1, amino acids 1509-1791) heterologously expressed in Escherichia coli (E. coli) are difficult to be recovered in a bioactive form because they are only poorly soluble. In this study, we developed a new method to solubilize and purify CT1. GST-CT1 expressed in E. coli was extracted and treated with an inclusion body solubilization and renaturation kit. Then, after adsorption to glutathione Sepharose beads, GST-CT1 was treated with protease to release CT1. However, the cleaved CT1 was insoluble and remained attached to the beads. Therefore, CT1 was treated again with the inclusion body solubilization and renaturation kit. Using this method, GST-CT1 and CT1 were purified with a high yield. GST pull-down experiments showed a dose-dependent interaction between GST-CT1 and calmodulin (CaM), and between GST-CaM and CT1, suggesting recovered bioactivity of GST-CT1 and CT1. This protocol may also be applied to purify other insoluble GST-fused proteins.

Calmodulin (CaM) is well known as an activator of calcium/calmodulin-dependent protein kinase II (CaMKII). Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are crucial molecular targets for nervous system agents. However, the way in which Ca2+/CaM/CaMKII cascade modulates NaV1.1 IQ (isoleucine and glutamine) domain of VGSCs remains obscure. In this study, the binding of CaM, its mutants at calcium binding sites (CaM12, CaM34, and CaM1234), and truncated proteins (N-lobe and C-lobe) to NaV1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca2+/CaM to the NaV1.1 IQ was concentration-dependent. ApoCaM (Ca2+-free form of calmodulin) bound to NaV1.1 IQ domain preferentially more than Ca2+/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to NaV1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca2+/CaM to NaV1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain. This study provides novel mechanisms for the modulation of NaV1.1 by the Ca2+/CaM/CaMKII axis. For the first time, we uncover the effect of Ca2+, lobe-specificity and CaMKII on CaM binding to NaV1.1.

Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca2+channels (CaV1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the CaV1.2 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 CaV1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. CaV1.2 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 CaV1.2 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 CaV1.2 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 CaV1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.

The activity of Cav1.2 Ca(2+) channels is maintained in the presence of calmodulin and ATP, even in cell-free patches, and thus a channel ATP-binding site has been suggested. In this study, we examined whether other nucleotides, such as GTP, UTP, CTP, ADP and AMP, could be substituted for ATP in guinea-pig ventricular myocytes. We found that all the nucleotides tested could re-prime the Ca(2+) channels in the presence of 1 μM calmodulin in the inside-out mode. The order of efficacy was ATP > GTP > UTP > ADP > CTP ≈ AMP. Thus, the presumed nucleotide-binding site in the channel seemed to favor a purine rather than pyrimidine base and a triphosphate rather than a di- or mono-phosphate group. Furthermore, a high concentration (10 mM) of GTP, UTP, CTP, ADP and AMP had inhibitory effects on the channel activity. These results provide information on the putative nucleotide-binding site(s) in Cav1.2 Ca(2+) channels.

The present study examined the binding of the individual N- and C-lobes of calmodulin (CaM) to Cav1.2 at different Ca(2+) concentration ([Ca(2+)]) from ≈ free to 2mM, and found that they may bind to Cav1.2 Ca(2+)-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 [Ca(2+)], CaM may tether to the channel with its single lobe, leading to multiple CaM molecule binding to increase the grade of Ca(2+)-dependent regulation of Cav1.2.

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

Cardiac Cav1.2 channels, coupling membrane stimulation to intracellular Ca2+ signaling, are regulated by multiple cytoplasmic factors, such as calmodulin (CaM), phosphorylation, Ca2+, ATP and intramolecular fragments of the channel. The interaction between distal and proximal C-terminal regulatory domains (DCRD and PCRD) of Cav1.2 channel is suggested to inhibit the channel activity, while PKA-mediated phosphorylation facilitates Cav1.2 channel by releasing such an interaction. Here, we report that the interaction between the distal C-terminus (CT3) and the proximal C-terminus (CT1) are inhibited by CaM in a Ca2+-dependent manner. Furthermore, CT3D (a short CT3 with DCRD truncated) interacts with CT1B (a short CT1 with EF-hand and PCRD truncated), revealing a new interaction between distal and proximal C-terminus. Ca2+/CaM inhibited the binding of CT3D to CT1B more strongly than the binding between CT3 and CT1, implying that the interaction of DCRD/PCRD (in CT3/CT1) might cooperate with the binding of CT3D to CT1B. We name the new CT1B-binding region of CT3D as CaM-competitive domain (CCD). The electrophysiological experiments show that CT3D inhibits while CT1B facilitates Cav1.2 channel activity in inside-out patches in guinea-pig ventricular myocytes. These results suggest that distal C-terminus inhibits Cav1.2 channel through modulation of the CaM-binding property of the channels.

Cav1.2 Ca(2+) 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.

In this study, we investigated the roles of calmodulin kinase II (CaMKII) and calmodulin (CaM) in the reversal of run-down of L-type Ca2+ channels. Single Ca2+-channel activities in guinea-pig ventricular myocytes were recorded using the patch-clamp technique, and run-down of the channel activities was induced by inside-out patch formation in the basic internal solution. At 1 min after patch excision, 1 – 30 μM CaMKII mutant T286D (CaMKIIT286D), a constitutively active type of CaMKII, induced the Ca2+-channel activities to only 2% – 10% of that recorded in the cell-attached mode. However, in the presence of CaMKIIT286D, the time-dependent attenuation of CaM’s effects in the reversal of run-down was abolished. A GST-fusion protein containing amino acids 1509 – 1789 of the C-terminal region of guinea-pig Cav1.2 (CT1) was prepared. In pull-down assays, CT1 treated with CaMKIIT286D showed a higher affinity for CaM compared with CT1 treated with phosphatase. We propose a model in which CaMKII-mediated phosphorylation of the channels regulates the binding of CaM to the channels in the reversal of run-down of L-type Ca2+ channels. Keywords:: calmodulin, calmodulin-dependent protein kinase II, cardiac myocyte, L-type Ca2+ channel

The roles of calmodulin (CaM)-dependent protein kinase II (CaMKII) in the maintenance of basal activity and the reversion of run-down of L-type Ca2+ channels were studied in guinea-pig ventricular myocytes by the patch-clamp technique. In the cell-attached configuration, the Ca2+-channel activity was inhibited to 82% – 26% by 1 – 10 μM KN-93 and to 92% – 66% by 0.1 – 1 μM autocamtide-2–related inhibitory peptide (AIP) myristoylated. In the inside-out configuration, the bovine cardiac cytoplasm recovered Ca2+-channel activity to 87% of that recorded in the cell-attached configuration, while the CaMKII inhibitor 281-301 at 10 μM reduced the recovery effect to 19%. CaM + ATP recovered the channel activity to 93% and 28% of that recorded in the cell-attached configuration when applied at 1 and 5 min after run-down, respectively, showing a time-dependent attenuation. However, in the presence of 0. 33 μM CaMKII, this attenuation was abolished, showing 85% and 75% recovery when applied at 1 and 5 min after run-down, respectively. This recovery effect was suppressed by 10 μM AIP, applied at 5 min, but not at 1 min after run-down. We concluded that CaMKII activation is required in the maintenance of basal activity of L-type Ca2+ channels. Keywords:: calmodulin-dependent protein kinase II (CaMKII), calmodulin, cardiac myocyte, L-type Ca2+ channel

This study aimed to investigate protein phosphatases involved in the run down of Cav1.2 Ca2+channels. Single ventricular myocytes obtained from adult guinea pig hearts were used to record Ca2+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 Ca2+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 Ca2+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 Ca2+channel.

Calpastatin, an endogenous inhibitor of calpain, is composed of domain L and four repetitive homologous domains 1–4. Domains 1–4 inhibit calpain, whereas domain L partially reprimes L-type Ca 2+ channels for voltage-gated activation. In the present study, the effects on Ca 2+ channel activity of four isoforms and a series of fragments of calpastatin domain L were investigated in guinea-pig ventricular myocytes with the patch–clamp method. With one exception, all the isoforms and fragment peptides that contained amino acid residues 54–64 of domain L reprimed the Ca 2+ channels to comparable levels (9–15% of control activity) to those observed previously with a full-length form of calpastatin. These results suggest that the region containing amino acid residues 54–64 (EGKPKEHTEPK) is responsible for the Ca 2+ channel repriming function of calpastatin domain L.

Cardiac L-type Ca2+ 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 Ca2+ 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 Ca2+ 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 Ser1574 (C1 site) may be a potential site for the channel modulation mediated by PKA.

hsp105alpha is a stress protein characteristically highly expressed in the brain compared with other tissues in mammals. Here, to examine whether hsp105alpha plays a pivotal role in the nervous system, we tested the capability of hsp105alpha to protect against apoptosis in rat neuronal PC12 cells. Various stress treatments such as serum deprivation, heat shock, hydrogen peroxide, etoposide, and actinomycin D induced apoptosis in PC12 cells with characteristic shrinking of nuclei and chromatin. However, PC12 cells that constitutively overexpressed mouse hsp105alpha exhibited a strong protective effect against apoptosis induced by these stress treatments. Cleavage of poly(ADP-ribose) polymerase induced in PC12 cells by these treatments was inhibited in the constitutively overexpressed hsp105alpha cells. Furthermore, c-Jun N-terminal kinase (JNK) was activated in the cells treated with heat shock but not other treatments, and the heat-induced JNK activation was inhibited by the constitutive expression of hsp105alpha.Thus, hsp105alpha prevents not only heat-induced apoptosis by inhibiting JNK activation, but also prevents the apoptosis induced by other stressors through different pathways, and may play important roles in neuronal protection.

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. Ca2+ 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 Ca2+ 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, Ca2+ 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 Ca2+ dependent.

Although Cav1.2 Ca(2+) channels are modulated by reactive oxygen species (ROS), the underlying mechanisms are not fully understood. In this study, we investigated effects of hydrogen peroxide (H2O2) on the Ca(2+) channel using a patch-clamp technique in guinea pig ventricular myocytes. Externally applied H2O2 (1 mM) increased Ca(2+) channel activity in the cell-attached mode. A specific inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) KN-93 (10 μM) partially attenuated the H2O2-mediated facilitation of the channel, suggesting both CaMKII-dependent and -independent pathways. However, in the inside-out mode, 1 mM H2O2 increased channel activity in a KN-93-resistant manner. Since H2O2-pretreated calmodulin did not reproduce the H2O2 effect, the target of H2O2 was presumably assigned to the Ca(2+) channel itself. A thiol-specific oxidizing agent mimicked and occluded the H2O2 effect. These results suggest that H2O2 facilitates the Ca(2+) channel through oxidation of cysteine residue(s) in the channel as well as the CaMKII-dependent pathway.

We investigated the concentration- and Ca(2+)-dependent effects of CaM mutants, CaM(12) and CaM(34), in which Ca(2+)-binding to its N- and C-lobes was eliminated, respectively, on the Ca(V)1.2 Ca(2+) channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM(12) and CaM(34) (0.7-10muM) applied with 3mM ATP produced channel activity after "rundown". Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca(2+)], suggesting that both functional lobes of CaM were necessary for the Ca(2+)-dependent shift. However, channel activity induced by the CaM mutants showed Ca(2+)-dependent decrease, implying a Ca(2+) sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca(2+)-dependent regulations of Ca(V)1.2 Ca(2+) channels.

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 Ca(2+) 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 Ca(2+) channel.

Calpastatin is an endogenous inhibitor of calpain and composed of domain L (CS(L)), which interacts with the Cav1.2 channels, and four repetitive calpain inhibitory domains. We have previously found that CS(L) reprimes activity of the Cav1.2 channels in cell-free patches of cardiac myocytes [L.Y. Hao, A. Kameyama, S. Kuroki, J. Takano, E. Takano, M. Maki, M. Kameyama, Calpastatin domain L is involved in the regulation L-type of Ca2+ channels in guinea pig cardiac myocytes, Biochem. Biophys. Res. Commun. 279 (2000) 756-761; E. Minobe, L.Y. Hao, Z.A. Saud, J.J. Xu, A. Kameyama, M. Maki, K.K. Jewell, T. Parr, R.G. Bardsley, M. Kameyama, A region of calpastatin domain L that reprimes cardiac L-type Ca2+ channels, Biochem. Biophys. Res. Commun. 348 (2006) 288-294]. In this study, we explored the CS(L) interaction site in the Ca2+ channel by the pull-down method, using glutathione-S-transferase-fused fragment peptides of the Cav1.2 channel. CS(L) bound directly to a proximal region of the C-terminal tail of the channel, but not with the N-terminal tail, a distal region of the C-terminal tail or cytoplasmic loops between repeats I-II, II-III or III-IV. Furthermore IQ domain, but not EF-hand-like region or CB domain, in the C-terminal tail was found to bind with CS(L) in a partially Ca2+-dependent manner and in a probably competitive manner with calmodulin. These results suggest that CS(L) modulates Ca2+-channel activity through interacting with the calmodulin-binding site on the C-terminal tail of the Cav1.2 channel.

L-type Ca(2+) channels have two opposing forms of autoregulatory feedback, Ca(2+) -dependent facilitation (CDF) and Ca(2+) -dependent inactivation (CDI), in response to increases in intracellular Ca(2+) concentration. Calmodulin (CaM) has been reported to mediate the two feedbacks. Although both the direct binding of CaM and the phosphorylation mediated by Ca(2+)/CaM -dependent protein kinase II (CaMKII) have been suggested as underlying mechanisms, the detailed features remain to be clarified. In this study, we investigated the effects of CaM and CaMKII inhibitors on CDF and CDI with patch clamp cell-attached recordings in guinea-pig ventricular myocytes. We confirmed that a high-K(+) and high-Ca(2)(+) could induce an increase of the intracellular Ca(2+) concentration and subsequent CDF and CDI. We then found that CDF and CDI were both depressed and were finally abolished by treatment with a CaM inhibitor chlorpromazine (1-100 microM) in a concentration-dependent manner. Another CaM antagonist calmidazolium (1 microM) showed a similar effect. In contrast, CaMKII inhibitors, KN-62 (0.1-3 microM) and autocamtide 2 -related inhibitory peptide (1 microM), delayed the development of CDF and CDI significantly, but they did not depress either CDF or CDI. These results imply that CaM is necessary and possibly sufficient for the two mechanisms. We propose a hypothesis that CaM is a key molecule to bifurcate the Ca(2+) signal to CDF and CDI and that CaMKII plays a modulatory role in them both.

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 1mM Ca(2+) 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 [Ca(2+)], 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 Ca(2+)-dependent manners.