Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket
<p dir="ltr">Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcop...
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| مؤلفون آخرون: | , , , , , , , , , |
| منشور في: |
2023
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| _version_ | 1864513530402701312 |
|---|---|
| author | Angelos Thanassoulas (2822861) |
| author2 | Vyronia Vassilakopoulou (17807600) Brian L. Calver (17807603) Luke Buntwal (17807606) Adrian Smith (2385022) Christopher Lai (144808) Iris Kontogianni (17807609) Evangelia Livaniou (2528806) George Nounesis (2202139) F. Anthony Lai (17714499) Michail Nomikos (17563188) |
| author2_role | author author author author author author author author author author |
| author_facet | Angelos Thanassoulas (2822861) Vyronia Vassilakopoulou (17807600) Brian L. Calver (17807603) Luke Buntwal (17807606) Adrian Smith (2385022) Christopher Lai (144808) Iris Kontogianni (17807609) Evangelia Livaniou (2528806) George Nounesis (2202139) F. Anthony Lai (17714499) Michail Nomikos (17563188) |
| author_role | author |
| dc.creator.none.fl_str_mv | Angelos Thanassoulas (2822861) Vyronia Vassilakopoulou (17807600) Brian L. Calver (17807603) Luke Buntwal (17807606) Adrian Smith (2385022) Christopher Lai (144808) Iris Kontogianni (17807609) Evangelia Livaniou (2528806) George Nounesis (2202139) F. Anthony Lai (17714499) Michail Nomikos (17563188) |
| dc.date.none.fl_str_mv | 2023-04-01T00:00:00Z |
| dc.identifier.none.fl_str_mv | 10.1016/j.bbagen.2023.130313 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/Life-threatening_arrhythmogenic_CaM_mutations_disrupt_CaM_binding_to_a_distinct_RyR2_CaM-binding_pocket/25018253 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biological sciences Biochemistry and cell biology Biomedical and clinical sciences Cardiovascular medicine and haematology Calmodulin Ryanodine receptor RyR2 Arrhythmias Cardiac disease |
| dc.title.none.fl_str_mv | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| dc.type.none.fl_str_mv | Text Journal contribution info:eu-repo/semantics/publishedVersion text contribution to journal |
| description | <p dir="ltr">Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2<sup>+</sup>) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaM<sup>N98I</sup>, CaM<sup>D132E</sup>, CaM<sup>D134H</sup> and CaM<sup>Q136P</sup>) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [<sup>3</sup>H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca<sup>2+</sup>, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association.</p><h2>Other Information</h2><p dir="ltr">Published in: Biochimica et Biophysica Acta (BBA) - General Subjects<br>License: <a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1016/j.bbagen.2023.130313" target="_blank">https://dx.doi.org/10.1016/j.bbagen.2023.130313</a></p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_efb36bd906cb670f68bb73fa5ce05696 |
| identifier_str_mv | 10.1016/j.bbagen.2023.130313 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/25018253 |
| publishDate | 2023 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocketAngelos Thanassoulas (2822861)Vyronia Vassilakopoulou (17807600)Brian L. Calver (17807603)Luke Buntwal (17807606)Adrian Smith (2385022)Christopher Lai (144808)Iris Kontogianni (17807609)Evangelia Livaniou (2528806)George Nounesis (2202139)F. Anthony Lai (17714499)Michail Nomikos (17563188)Biological sciencesBiochemistry and cell biologyBiomedical and clinical sciencesCardiovascular medicine and haematologyCalmodulinRyanodine receptorRyR2ArrhythmiasCardiac disease<p dir="ltr">Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2<sup>+</sup>) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaM<sup>N98I</sup>, CaM<sup>D132E</sup>, CaM<sup>D134H</sup> and CaM<sup>Q136P</sup>) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [<sup>3</sup>H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca<sup>2+</sup>, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association.</p><h2>Other Information</h2><p dir="ltr">Published in: Biochimica et Biophysica Acta (BBA) - General Subjects<br>License: <a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1016/j.bbagen.2023.130313" target="_blank">https://dx.doi.org/10.1016/j.bbagen.2023.130313</a></p>2023-04-01T00:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.1016/j.bbagen.2023.130313https://figshare.com/articles/journal_contribution/Life-threatening_arrhythmogenic_CaM_mutations_disrupt_CaM_binding_to_a_distinct_RyR2_CaM-binding_pocket/25018253CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/250182532023-04-01T00:00:00Z |
| spellingShingle | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket Angelos Thanassoulas (2822861) Biological sciences Biochemistry and cell biology Biomedical and clinical sciences Cardiovascular medicine and haematology Calmodulin Ryanodine receptor RyR2 Arrhythmias Cardiac disease |
| status_str | publishedVersion |
| title | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_full | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_fullStr | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_full_unstemmed | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_short | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| title_sort | Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket |
| topic | Biological sciences Biochemistry and cell biology Biomedical and clinical sciences Cardiovascular medicine and haematology Calmodulin Ryanodine receptor RyR2 Arrhythmias Cardiac disease |