Both N- and C-lobes of calmodulin are required for Ca2+-dependent regulations of CaV1.2 Ca2+ channels

Guo, Feng; Minobe, Etsuko; Yazawa, Kazuto; Asmara, Hadhimulya; Xiu-juan, Bai; Han, Dong-Yun; Liang, Hao; Kameyama, Masaki

doi:10.1016/j.bbrc.2009.11.171

Cited by 11 publications

(23 citation statements)

References 22 publications

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“…We hypothesized that CaM is not permanently tethered to the Cav1.2 channel but dynamically interacts with the channel, depending on CaM and Ca 2ϩ concentrations (13,18,19,21). The results of this study further support this hypothesis.…”

Section: Discussionsupporting

confidence: 80%

“…Furthermore, an inhibitory effect of CaM on channel activity has been found at high [CaM], indicating that CaM can manifest both facilitatory and inhibitory effects in the insideout mode. The fact that these CaM effects are Ca 2ϩ -dependent implies that the effects may reflect those seen in CDF and CDI in intact myocytes (13,19,21). In this study, we confirmed the dual CaM effects on channel activity and provided further support for the view that CaM interacts dynamically with the channel, thereby regulating basal activity and mediating CDF and CDI in cardiac myocytes.…”

Section: Discussionsupporting

confidence: 76%

“…Alternatively, both the N terminus and C terminus might collaborate with CaM to trigger CDI. The function of CaM binding to LI-II is largely unknown, although this region was also suggested to be involved in CDI (9,11 dependence at its major component of action (13,21). Ca 2ϩ -dependent binding of CaM to peptides containing PreIQ and/or IQ peptides has been reported in many studies (7, 9 -11, 17, 31, 33, 35) and confirmed in this study.…”

Section: Discussionsupporting

confidence: 70%

“…Our previous studies have revealed that CaM reprimes the channels in a run-down state in inside-out patches (13,19,21). Furthermore, an inhibitory effect of CaM on channel activity has been found at high [CaM], indicating that CaM can manifest both facilitatory and inhibitory effects in the insideout mode.…”

Section: Discussionmentioning

confidence: 99%

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

Minobe

Asmara

Saud

et al. 2011

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 80%

Section: Discussionsupporting

confidence: 76%

Section: Discussionsupporting

confidence: 70%

Section: Discussionmentioning

confidence: 99%

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

Minobe

Asmara

Saud

et al. 2011

Journal of Biological Chemistry

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“…The CaM protein contains two lobes, each with two Ca 2ϩ -binding sites. CaM mutations at Ca 2ϩ -binding sites 1 and 2 in the NH 2 -terminal (N) lobe (CaM 12 ) and mutations at Ca 2ϩ -binding sites 3 and 4 in the COOH-terminal (C) lobe (CaM 34 ) have been used to investigate Ca 2ϩ -dependent regulation of the Ca V 1.2 channel, and both N and C lobes have been shown to be of critical importance in CaM-dependent modulation of Ca 2ϩ channels in our previous studies (13).…”

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confidence: 99%

Low-Mg²⁺ treatment increases sensitivity of voltage-gated Na⁺ channels to Ca²⁺/calmodulin-mediated modulation in cultured hippocampal neurons

Guo

Zhou

Gao

et al. 2015

American Journal of Physiology-Cell Physiology

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Culture of hippocampal neurons in low-Mg(2+) medium (low-Mg(2+) neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg(2+) to hyperexcitability of neurons has not been clarified. Our data, obtained using the patch-clamp technique, show that voltage-gated Na(+) channel (VGSC) activity, which is associated with a persistent, noninactivating Na(+) current (INa,P), was modulated by calmodulin (CaM) in a concentration-dependent manner in normal and low-Mg(2+) neurons, but the channel activity was more sensitive to Ca(2+)/CaM regulation in low-Mg(2+) than normal neurons. The increased sensitivity of VGSCs in low-Mg(2+) neurons was partially retained when CaM12 and CaM34, CaM mutants with disabled binding sites in the N or C lobe, were used but was diminished when CaM1234, a CaM mutant in which all four Ca(2+) sites are disabled, was used, indicating that functional Ca(2+)-binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg(2+) neurons. Furthermore, the number of neurons exhibiting colocalization of CaM with the VGSC subtypes NaV1.1, NaV1.2, and NaV1.3 was significantly higher in low- Mg(2+) than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg(2+) treatment increases sensitivity of VGSCs to Ca(2+)/CaM-mediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na(+), Ca(2+), and Mg(2+), by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg(2+) neurons.

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The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties

Kovalevskaya

Bokhovchuk

Vuister

2012

J Struct Funct Genomics

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Both N- and C-lobes of calmodulin are required for Ca2+-dependent regulations of CaV1.2 Ca2+ channels

Cited by 11 publications

References 22 publications

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

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

Low-Mg²⁺ treatment increases sensitivity of voltage-gated Na⁺ channels to Ca²⁺/calmodulin-mediated modulation in cultured hippocampal neurons

The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties

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Both N- and C-lobes of calmodulin are required for Ca2+-dependent regulations of CaV1.2 Ca2+ channels

Cited by 11 publications

References 22 publications

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

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

Low-Mg2+ treatment increases sensitivity of voltage-gated Na+ channels to Ca2+/calmodulin-mediated modulation in cultured hippocampal neurons

The TRPV5/6 calcium channels contain multiple calmodulin binding sites with differential binding properties

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Low-Mg²⁺ treatment increases sensitivity of voltage-gated Na⁺ channels to Ca²⁺/calmodulin-mediated modulation in cultured hippocampal neurons