Cargando…
Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy
BACKGROUND: Ca(2+)-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca(2+) inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674...
Autores principales: | , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2010
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877719/ https://www.ncbi.nlm.nih.gov/pubmed/20523736 http://dx.doi.org/10.1371/journal.pone.0010873 |
_version_ | 1782181806581219328 |
---|---|
author | Gonçalves, J. Tiago Stühmer, Walter |
author_facet | Gonçalves, J. Tiago Stühmer, Walter |
author_sort | Gonçalves, J. Tiago |
collection | PubMed |
description | BACKGROUND: Ca(2+)-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca(2+) inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674-683, BD-C2: 711-721, BD-N: 151-165) have been identified in a peptide screen and were proposed to mediate binding. The participation of the three sites in CaM binding to the native channel, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we studied the binding of Ca(2+)/CaM to the EAG channel by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by FRET. The results of our cellular approach substantiate that two CaM binding sites are predominantly involved; the high-affinity 1-8-14 based CaM binding domain in the N-terminus and the second C-terminal binding domain BD-C2. Mutations at these sites completely abolished CaM binding to hEAG1. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the BD-N and BD-C2 binding domains are sufficient for CaM binding to the native channel, and, therefore, that BD-C1 is unable to bind CaM independently. |
format | Text |
id | pubmed-2877719 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-28777192010-06-03 Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy Gonçalves, J. Tiago Stühmer, Walter PLoS One Research Article BACKGROUND: Ca(2+)-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca(2+) inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674-683, BD-C2: 711-721, BD-N: 151-165) have been identified in a peptide screen and were proposed to mediate binding. The participation of the three sites in CaM binding to the native channel, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we studied the binding of Ca(2+)/CaM to the EAG channel by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by FRET. The results of our cellular approach substantiate that two CaM binding sites are predominantly involved; the high-affinity 1-8-14 based CaM binding domain in the N-terminus and the second C-terminal binding domain BD-C2. Mutations at these sites completely abolished CaM binding to hEAG1. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the BD-N and BD-C2 binding domains are sufficient for CaM binding to the native channel, and, therefore, that BD-C1 is unable to bind CaM independently. Public Library of Science 2010-05-27 /pmc/articles/PMC2877719/ /pubmed/20523736 http://dx.doi.org/10.1371/journal.pone.0010873 Text en Gonçalves, Stühmer. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Gonçalves, J. Tiago Stühmer, Walter Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy |
title | Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy |
title_full | Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy |
title_fullStr | Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy |
title_full_unstemmed | Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy |
title_short | Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy |
title_sort | calmodulin interaction with heag1 visualized by fret microscopy |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877719/ https://www.ncbi.nlm.nih.gov/pubmed/20523736 http://dx.doi.org/10.1371/journal.pone.0010873 |
work_keys_str_mv | AT goncalvesjtiago calmodulininteractionwithheag1visualizedbyfretmicroscopy AT stuhmerwalter calmodulininteractionwithheag1visualizedbyfretmicroscopy |