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Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca(2+)-Dependent Inactivation

[Image: see text] Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are inhibited by millimollar Ca(2+), but the affinity of RyR2 for inhibitory Ca(2+) is ∼10 times lower than that of RyR1. Previous studies demonstrated that the C-terminal quarter of RyR has c...

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Autores principales: Gomez, Angela C., Yamaguchi, Naohiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985739/
https://www.ncbi.nlm.nih.gov/pubmed/24521037
http://dx.doi.org/10.1021/bi401586h
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author Gomez, Angela C.
Yamaguchi, Naohiro
author_facet Gomez, Angela C.
Yamaguchi, Naohiro
author_sort Gomez, Angela C.
collection PubMed
description [Image: see text] Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are inhibited by millimollar Ca(2+), but the affinity of RyR2 for inhibitory Ca(2+) is ∼10 times lower than that of RyR1. Previous studies demonstrated that the C-terminal quarter of RyR has critical domain(s) for Ca(2+) inactivation. To obtain further insights into the molecular basis of regulation of RyRs by Ca(2+), we constructed and expressed 18 RyR1–RyR2 chimeras in HEK293 cells and determined the Ca(2+) activation and inactivation affinities of these channels using the [(3)H]ryanodine binding assay. Replacing two distinct regions of RyR1 with corresponding RyR2 sequences reduced the affinity for Ca(2+) inactivation. The first region (RyR2 amino acids 4020–4250) contains two EF-hand Ca(2+) binding motifs (EF1, amino acids 4036–4047; EF2, amino acids 4071–4082), and the second region includes the putative second transmembrane segment (S2). A RyR1–backbone chimera containing only EF2 from RyR2 had a modest (not significant) change in Ca(2+) inactivation, whereas another chimera channel carrying only EF1 from RyR2 had a significantly reduced level of Ca(2+) inactivation. The results suggest that EF1 is a more critical determinant for RyR inactivation by Ca(2+). In addition, activities of the chimera carrying RyR2 EF-hands were suppressed at 10–100 μM Ca(2+), and the suppression was relieved by 1 mM Mg(2+). The same effects have been observed with wild-type RyR2. A mutant RyR1 carrying both regions replaced with RyR2 sequences (amino acids 4020–4250 and 4560–4618) showed a Ca(2+) inactivation affinity comparable to that of RyR2, indicating that these regions are sufficient to confer RyR2-type Ca(2+)-dependent inactivation on RyR1.
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spelling pubmed-39857392015-02-12 Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca(2+)-Dependent Inactivation Gomez, Angela C. Yamaguchi, Naohiro Biochemistry [Image: see text] Skeletal (RyR1) and cardiac muscle (RyR2) isoforms of ryanodine receptor calcium channels are inhibited by millimollar Ca(2+), but the affinity of RyR2 for inhibitory Ca(2+) is ∼10 times lower than that of RyR1. Previous studies demonstrated that the C-terminal quarter of RyR has critical domain(s) for Ca(2+) inactivation. To obtain further insights into the molecular basis of regulation of RyRs by Ca(2+), we constructed and expressed 18 RyR1–RyR2 chimeras in HEK293 cells and determined the Ca(2+) activation and inactivation affinities of these channels using the [(3)H]ryanodine binding assay. Replacing two distinct regions of RyR1 with corresponding RyR2 sequences reduced the affinity for Ca(2+) inactivation. The first region (RyR2 amino acids 4020–4250) contains two EF-hand Ca(2+) binding motifs (EF1, amino acids 4036–4047; EF2, amino acids 4071–4082), and the second region includes the putative second transmembrane segment (S2). A RyR1–backbone chimera containing only EF2 from RyR2 had a modest (not significant) change in Ca(2+) inactivation, whereas another chimera channel carrying only EF1 from RyR2 had a significantly reduced level of Ca(2+) inactivation. The results suggest that EF1 is a more critical determinant for RyR inactivation by Ca(2+). In addition, activities of the chimera carrying RyR2 EF-hands were suppressed at 10–100 μM Ca(2+), and the suppression was relieved by 1 mM Mg(2+). The same effects have been observed with wild-type RyR2. A mutant RyR1 carrying both regions replaced with RyR2 sequences (amino acids 4020–4250 and 4560–4618) showed a Ca(2+) inactivation affinity comparable to that of RyR2, indicating that these regions are sufficient to confer RyR2-type Ca(2+)-dependent inactivation on RyR1. American Chemical Society 2014-02-12 2014-03-04 /pmc/articles/PMC3985739/ /pubmed/24521037 http://dx.doi.org/10.1021/bi401586h Text en Copyright © 2014 American Chemical Society
spellingShingle Gomez, Angela C.
Yamaguchi, Naohiro
Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca(2+)-Dependent Inactivation
title Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca(2+)-Dependent Inactivation
title_full Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca(2+)-Dependent Inactivation
title_fullStr Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca(2+)-Dependent Inactivation
title_full_unstemmed Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca(2+)-Dependent Inactivation
title_short Two Regions of the Ryanodine Receptor Calcium Channel Are Involved in Ca(2+)-Dependent Inactivation
title_sort two regions of the ryanodine receptor calcium channel are involved in ca(2+)-dependent inactivation
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985739/
https://www.ncbi.nlm.nih.gov/pubmed/24521037
http://dx.doi.org/10.1021/bi401586h
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