Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells
Ca2+ influx through voltage-gated Ca2+ channels (CaVs) at the plasma membrane is the major pathway responsible for the elevation of the intracellular Ca2+ concentration ([Ca2+]i), which activates various physiological activities. Calmodulin (CaM) is known to be involved in the Ca2+-dependent inactiv...
Main Authors: | , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Elsevier
2017-06-01
|
Series: | IBRO Reports |
Subjects: | |
Online Access: | http://www.sciencedirect.com/science/article/pii/S2451830116300255 |
id |
doaj-8e40fc5794ec4643a60502f05acc2d43 |
---|---|
record_format |
Article |
spelling |
doaj-8e40fc5794ec4643a60502f05acc2d432020-11-24T21:03:17ZengElsevierIBRO Reports2451-83012017-06-012C637110.1016/j.ibror.2017.03.002Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cellsChih-Hung Chi0Chih-Yung Tang1Chien-Yuan Pan2Department of Life Science, National Taiwan University, Taipei, TaiwanDepartment of Physiology, College of Medicine, National Taiwan University, Taipei, TaiwanDepartment of Life Science, National Taiwan University, Taipei, TaiwanCa2+ influx through voltage-gated Ca2+ channels (CaVs) at the plasma membrane is the major pathway responsible for the elevation of the intracellular Ca2+ concentration ([Ca2+]i), which activates various physiological activities. Calmodulin (CaM) is known to be involved in the Ca2+-dependent inactivation (CDI) of several types of CaVs; however, little is known about how CaM modulates CaV2.2. Here, we expressed CaV2.2 with CaM or CaM mutants with a Ca2+-binding deficiency in HEK293T cells and measured the currents to characterize the CDI. The results showed that CaV2.2 displayed a fast inactivation with Ca2+ but not Ba2+ as the charge carrier; when CaV2.2 was co-expressed with CaM mutants with a Ca2+-binding deficiency, the level of inactivation decreased. Using glutathione S-transferase-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of CaV2.2 in the presence or absence of Ca2+. However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. CaV2.2 with mutations in the CaM-binding site showed a greatly reduced current that could be rescued by CaM12 (Ca2+-binding deficiency at the N-lobe) overexpression; in addition, CaM12 enhanced the total expression level of CaV2.2, but the ratio of CaV2.2 present in the membrane to the total fraction remained unchanged. Together, our data suggest that CaM, with different Ca2+-binding abilities, modulates not only the inactivation of CaV2.2 but also its expression to regulate Ca2+-related physiological activities.http://www.sciencedirect.com/science/article/pii/S2451830116300255BiotinylationCa2+-dependent inactivationCalmodulinCaV2.2Voltage-gated Ca2+ channels |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Chih-Hung Chi Chih-Yung Tang Chien-Yuan Pan |
spellingShingle |
Chih-Hung Chi Chih-Yung Tang Chien-Yuan Pan Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells IBRO Reports Biotinylation Ca2+-dependent inactivation Calmodulin CaV2.2 Voltage-gated Ca2+ channels |
author_facet |
Chih-Hung Chi Chih-Yung Tang Chien-Yuan Pan |
author_sort |
Chih-Hung Chi |
title |
Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells |
title_short |
Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells |
title_full |
Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells |
title_fullStr |
Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells |
title_full_unstemmed |
Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells |
title_sort |
calmodulin modulates the ca2+-dependent inactivation and expression level of bovine cav2.2 expressed in hek293t cells |
publisher |
Elsevier |
series |
IBRO Reports |
issn |
2451-8301 |
publishDate |
2017-06-01 |
description |
Ca2+ influx through voltage-gated Ca2+ channels (CaVs) at the plasma membrane is the major pathway responsible for the elevation of the intracellular Ca2+ concentration ([Ca2+]i), which activates various physiological activities. Calmodulin (CaM) is known to be involved in the Ca2+-dependent inactivation (CDI) of several types of CaVs; however, little is known about how CaM modulates CaV2.2. Here, we expressed CaV2.2 with CaM or CaM mutants with a Ca2+-binding deficiency in HEK293T cells and measured the currents to characterize the CDI. The results showed that CaV2.2 displayed a fast inactivation with Ca2+ but not Ba2+ as the charge carrier; when CaV2.2 was co-expressed with CaM mutants with a Ca2+-binding deficiency, the level of inactivation decreased. Using glutathione S-transferase-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of CaV2.2 in the presence or absence of Ca2+. However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. CaV2.2 with mutations in the CaM-binding site showed a greatly reduced current that could be rescued by CaM12 (Ca2+-binding deficiency at the N-lobe) overexpression; in addition, CaM12 enhanced the total expression level of CaV2.2, but the ratio of CaV2.2 present in the membrane to the total fraction remained unchanged. Together, our data suggest that CaM, with different Ca2+-binding abilities, modulates not only the inactivation of CaV2.2 but also its expression to regulate Ca2+-related physiological activities. |
topic |
Biotinylation Ca2+-dependent inactivation Calmodulin CaV2.2 Voltage-gated Ca2+ channels |
url |
http://www.sciencedirect.com/science/article/pii/S2451830116300255 |
work_keys_str_mv |
AT chihhungchi calmodulinmodulatestheca2dependentinactivationandexpressionlevelofbovinecav22expressedinhek293tcells AT chihyungtang calmodulinmodulatestheca2dependentinactivationandexpressionlevelofbovinecav22expressedinhek293tcells AT chienyuanpan calmodulinmodulatestheca2dependentinactivationandexpressionlevelofbovinecav22expressedinhek293tcells |
_version_ |
1716773569292140544 |