Calmodulin overexpression does not alter Ca<sub>v</sub>1.2 function or oligomerization state

Findeisen, Felix; Tolia, Alexandra; Arant, Ryan J; Kim, Eun Young; Isacoff, Ehud Y.; Minor, Daniel L.

doi:10.4161/chan.5.4.16821

Cited by 14 publications

(10 citation statements)

References 29 publications

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“…In this work, we demonstrated that CaM binds the N-terminus of Na v 1.5, which resembles CaM binding to the NTD of Ca v 1.2 2,17,33,34 . This interaction seems to be crucial for the dominant-negative effect, in which a Na v 1.5 NTD variant identified in BrS patients negatively regulates wild-type channel function.…”

Section: Discussionmentioning

confidence: 65%

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Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

Wang

Vermij

Sottas

et al. 2020

Preprint

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1The cardiac voltage-gated sodium channel Nav1.5 conducts the rapid inward sodium current crucial for 2 cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias 3 such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain exert 4 a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly 5 understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three 6 mutations in the Nav1.5 N-terminal domain (NTD): Y87C-here newly identified-, R104W and R121W. In 7 addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. 8Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that 9 Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction 10 in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT channel 11 with the ER however did not change in the presence of variants, shown by transfected and stained rat 12 neonatal cardiomyocytes. We next demonstrated that calmodulin binds Nav1.5 N-terminus using in silico 13 modeling, SPOTS, pull-down and proximity ligation assays. This binding is impaired in the R121W variant 14 and in a Nav1.5 construct missing residues 80-105, a predicted calmodulin binding site. 15In conclusion, we present the first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a 16 determinant for the DNE. 17Wang et al.3

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Section: Discussionmentioning

confidence: 65%

“…Firstly, calmodulin binds the NTD of the voltage-gated calcium channel Ca v 1.2 at two different sites 2,17 , and plays a role in C-terminal dimerization of Ca v 1.2 2,34 and Na v 1.5 2 . Secondly, the NTDs of Na v 1.5 and Ca v 1.2 both contain a highly conserved sequence consisting of the residues PIRRA ( Figure 6A,B ), herein the PIRRA motif.…”

Section: Resultsmentioning

confidence: 99%

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

Wang

Vermij

Sottas

et al. 2020

Preprint

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“…Due to endogenous CaM, Ca V 1.2 CDI in oocytes is robust 6; 23; 30; 48 . We had shown previously that even at a 60:1 excess of CaM:Ca V 1.2 mRNA, CaM expression did not further affect CDI as measured by the fraction of current decrease at 300 milliseconds (t i300 ) 49 . In contrast to these results, changes in CaBP1:Ca V 1.2 mRNA ratios caused clear alterations in t i300 and loss of CDI (Fig.…”

Section: Resultsmentioning

confidence: 92%

Apo States of Calmodulin and CaBP1 Control CaV1 Voltage-Gated Calcium Channel Function through Direct Competition for the IQ Domain

Findeisen

Rumpf

Minor

2013

Journal of Molecular Biology

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In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation (CDI) and limits calcium entry, whereas CaBP1 blocks CDI and allows sustained calcium influx. Here, we combine isothermal titration calorimetry (ITC) with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca2+/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium binding properties. The observation that the apo-forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity.

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“…Considering potential roles for CaM at the Na v 1.5 NTD, known effects of CaM binding to the NTD and CTD of Ca v 1.2, and to the CTD of Na v 1.5 may provide valuable insights. At the CTD of Na v 1.5 and Ca v 1.2, CaM is involved in channel dimerization [31][32][33], while CaM binding to the Ca v 1.2 CTD and NTD affects calcium-dependent inactivation [31][32][33]. At the Na v 1.5 C-terminal IQ motif, CaM interaction reduces the late sodium current [40], and affects voltage-dependence of activation in a calcium-dependent manner [33,41,42].…”

Section: Discussionmentioning

confidence: 99%

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

et al. 2020

View full text Add to dashboard Cite

The cardiac voltage-gated sodium channel Na v 1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Na v 1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Na v 1.5 NTD: Y87C-here newly identified-, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Na v 1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Na v 1.5 at the cell surface and in whole-cell lysates. Localization of Na v 1.5 WT channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Na v 1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Na v 1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Na v 1.5 variant Y87C and present first evidence that calmodulin binds to the Na v 1.5 NTD, which seems to be a determinant for the DNE.

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Calmodulin overexpression does not alter Ca_v1.2 function or oligomerization state

Cited by 14 publications

References 29 publications

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

Apo States of Calmodulin and CaBP1 Control CaV1 Voltage-Gated Calcium Channel Function through Direct Competition for the IQ Domain

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

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Calmodulin overexpression does not alter Cav1.2 function or oligomerization state

Cited by 14 publications

References 29 publications

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5

Apo States of Calmodulin and CaBP1 Control CaV1 Voltage-Gated Calcium Channel Function through Direct Competition for the IQ Domain

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5

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Calmodulin overexpression does not alter Ca_v1.2 function or oligomerization state

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5