• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.1-6
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• 1994

We have studied the relation between Ca$^{2+}$-ATPase and trigger peptidase(TPase) which are membeane protein well known as their significant role for signal transduction of mating pheromone in heterobasidiomycetous yeast. Rhodosporidium toruloides. We found out that there were Ca $^{2+}$-ATPase and TPase together in isolated calmodulim binding protein(CBP), usion calmodulin affinity column chromatography after solubilization of mation type a cell membrane protein, and that the dependence of enzyme activity of both the enzymes on Ca$^{2+}$, phospholipid and nonionic detergent are similar. However, Ca$^{2+}$-ATPase hed quite absolute dependence on calmodulin and, on the other hand, TPase didn't have any dependence. Judging from the fact that there are both enzymes in CBP which the dependence of calmodulin are quite different, we found out that both enzymes were made to their compound and existed in mating type a cell membrane.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1177-1181
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• 2007

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety CaM-binding proteins (CaMBPs). Because eukaryotes have multiple CaMBPs, it is important to isolate and characterize them in different tissues and conditions. So far a number of CaMBPs have been identified through classical screening methods. Many classes of proteins have been predicted to bind CaMs based on their structural homology with already known targets. In an effort to develop a method for large-scale analysis of CaMBPs in Arabidopsis, we have generated a transgenic plants overexpressing AtCaM2-GFP. We performed protein pull-down assay to test whether exogenously expressed AtCaM2-GFP proteins can interact with CaMBPs. The exogenously expressed AtCaM2-GFP could strongly interact with a CaMBP, AS1 protein. This result suggests that AtCaM2-GFP in transgenic plants may interact with many CaMBPs in plant cell. Therefore, we will be able to isolate kinds of CaMBPs by using these transgenic plants in many different tissue and environments.

• Journal of Plant Biotechnology
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• v.38 no.1
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• pp.62-68
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• 2011

Calmodulin (CaM), a $Ca^{2+}$ binding protein in eukaryotes, mediates cellular $Ca^{2+}$ signals in response to a variety of biotic and abiotic external stimuli. The $Ca^{2+}$-bound CaM transduces signals by modulating the activities of numerous CaM-binding proteins. As a CaM binding protein, AtCBP63 ($\b{A}$rabidopsis thaliana $\b{C}$aM-binding protein $\underline{63}$ kD) has been known to be positively involved in plant defense signaling pathway. To investigate the pathogen resistance function of AtCBP63 in potato, we constructed transgenic potato (Solanum tuberosum L.) plants constitutively overexpressing AtCBP63 under the control of cauliflower mosaic virus (CaMV) 35S promoter. The overexpression of the AtCBP63 in potato plants resulted in the high level induction of pathogenesis-related (PR) genes such as PR-2, PR-3 and PR-5. In addition, the AtCBP63 transgenic potato showed significantly enhanced resistance against a pathogen causing bacterial soft rot, Erwinia carotovora ssp. Carotovora (ECC). These results suggest that a CaM binding protein from Arabidopsis, AtCBP63, plays a positive role in pathogen resistance in potato.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.207-207
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• 2005

• Journal of Ginseng Research
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• v.33 no.1
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• pp.59-64
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• 2009

$Ca^{2+}$ and calmodulin (CaM), a key $Ca^{2+}$ sensor in all eukaryotes, have been implicated for defense responses of plants. Eukaryotic CaM contains four structurally and functionally similar $Ca^{2+}$ domains named I, II, III and IV. Each $Ca^{2+}$ binding loop consists of 12 amino acid residues with ligands arranged spatially to satisfy the octahedral symmetry of $Ca^{2+}$ binding. To investigate the altered gene expression and the role of CaM in ginseng plant defense system, cDNA clone containing a CaM gene, designated PgCaM was isolated and sequenced from Panax ginseng. PgCaM, which has open reading frame of 450 nucleotides predicted to encode a precursor protein of 150 amino acid residues. Its sequence shows high homologies with a number of other CaMs, with more similarity to CaM of Daucus carota (AAQ63461). The expression of PgCaM in different P. ginseng organs was analyzed using real time PCR. The results showed that PgCaM expressed at different levels in young leaves, shoots, and roots of 3-week-old P. ginseng. In addition, the expressions of PgCaM under different abiotic stresses were analyzed at different time intervals.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.3
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• pp.219-227
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• 2019

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when co-expressed with CaM and $CaM{\triangle}N$. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ${\triangle}EF$-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

• Molecules and Cells
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• v.37 no.9
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• pp.656-663
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• 2014

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits $[Ca^{2+}]_i$ transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier $K^+$ ($I_{Ks}$) channel is a cardiac $K^+$ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating $I_{Ks}$ channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human $I_{Ks}$ channel activity by expressing human $I_{Ks}$ channels in Xenopus oocytes. We found that gintonin enhances $I_{Ks}$ channel currents in concentration- and voltage-dependent manners. The $EC_{50}$ for the $I_{Ks}$ channel was $0.05{\pm}0.01{\mu}g/ml$. Gintonin-mediated activation 1 of the $I_{Ks}$ channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an $IP_3$ receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the $I_{Ks}$ channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 $[Ca^{2+}]_i$/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on $I_{Ks}$ channel. However, gintonin had no effect on hERG $K^+$ channel activity. These results show that gintonin-mediated enhancement of $I_{Ks}$ channel currents is achieved through binding of the $[Ca^{2+}]_i$/CaM complex to the C terminus of KCNQ1 subunit.

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.128.1-128.1
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• 2003

VR1, a capsaicin receptor, is now known to playa major role in mediating inflammatory thermal nociception. Although the physiological role or biophysical properties of VR1 are known, its activation mechanisms by ligands are poorly understood. Here, we show that VR1 requires phosphorylation by $Ca^{2+}$-calmodulin-dependent kinase II (CaMKII) for its activation by capsaicin. In contrast, dephosphorylation by calcineurin, leads to desensitization of the receptor. Point mutation of VR1 at two putative consensus sites for CaMKII fails to elicit capsaicin-sensitive currents with concomitant reduction in phosphorylation of VR1 in vivo. (omitted)

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.2
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• pp.74-82
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• 2015

Ryanodine receptor (RyR) is one of the two major $Ca^{2+}$ channels in membranes of intracellular $Ca^{2+}$ stores and is found in sarcoplasmic reticulum (SR), endoplasmic reticulum (ER). RyR1 is also the major calmodulin-binding protein of sarcoplasmic reticulum membranes. Residues 4064-4210 in the RyR1 polypeptide chain has similar primary sequence with calmodulin (CaM) and was designated as CaM-like domain (CaMLD). When expressed as a recombinant peptide, CaMLD showed several CaM-like properties in previous studies. Still, previous studies of CaMLD were focused on protein-protein interactions rather than its own properties. Here, we studied the expression of CaMLD and its sub-domains corresponding to each lobe of CaM in Escherichia coli. CaMLD could be obtained only as inclusion body, and it was refolded using urea solubilization followed by dialysis. Using spectroscopic approaches, such as NMR, circular dichroism, and gel filtration experiment, we found that the refolded CaMLD exists as nonspecific aggregate, even though it has alpha helical secondary structure. In comparison, the first half of CaMLD (R4061-4141) could be obtained as natively soluble protein with thioredoxin fusion. After the removal of the fusion tag, it exhibited folded and helical properties as shown by NMR and circular dichroism experiments. Its oligomeric status was different from CaMLD, existing as dimeric form in solution. However, the second half of the protein could not be obtained as soluble protein regardless of fusion tag. Based on these results, we believe that CaMLD, although similar to CaM in sequence, has quite different physicochemical properties and that the second half of the protein renders it the aggregative properties.

• Journal of Life Science
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• v.21 no.5
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• pp.671-677
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• 2011

We previously reported the isolation and characterization of a gene, SCA1 (for soybean $Ca^{2+}$-ATPase 1), encoding a calmodulin-regulated $Ca^{2+}$-ATPase that is located in the plasma membrane in soybean. Here, a $Ca^{2+}$-ATPase designated as SCA2 was isolated from soybean. The two $Ca^{2+}$-ATPases, SCA1 and SCA2, share a remarkably high degree of similarity (78%). Ten transmemebrane domains were predicted by hydropathy analysis. Using gel overlay assays, CaM was found to bind to SCA2 in a $Ca^{2+}$-dependent manner. Southern blot analysis revealed the presence of two copies of the $Ca^{2+}$-ATPase gene in the soybean genome. An N-terminal truncation mutant that deletes sequence through the putative calmodulin binding site was able to complement a yeast mutant (K616) that was deficient in two endogenous $Ca^{2+}$ pumps. Our results indicate that SCA2 is structurally highly conserved with type IIB $Ca^{2+}$ pumps in plants.