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Determination of the stoichiometry between alpha- and gamma 1 subunits of the BK channel using LRET

Authordc.contributor.authorCarrasquel Ursulaez, Willy 
Authordc.contributor.authorAlvarez, Osvaldo 
Authordc.contributor.authorBezanilla, Francisco 
Authordc.contributor.authorLatorre, Ramón 
Cita de ítemdc.identifier.citationBiophysical Journal Volumen: 114 Número: 11 Páginas: 2493-2497es_ES
Abstractdc.description.abstractTwo families of accessory proteins, beta and gamma, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca2+, the gamma 1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how alpha- and gamma 1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled alpha/gamma 1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the alpha- or the gamma 1 subunits to bind Tb+3 as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that gamma 1 associates with the alpha-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.es_ES
Patrocinadordc.description.sponsorshipNational Fund for Scientific and Technological Development (FONDECYT) 1150273 US Air Force Office of Scientific Research (AFOSR) FA9550-16-1-0384 Anillo Grant ACT-1107 National Institutes of Health GM030376 U54GM087519 Millennium Scientific Initiative of the Chilean Ministry of Economy, Development, and Tourism P029-022-Fes_ES
Publisherdc.publisherCell Presses_ES
Sourcedc.sourceBiophysical Journales_ES
Keywordsdc.subjectCa2+-activated k+ channeles_ES
Keywordsdc.subjectIuminescence energy-transferes_ES
Títulodc.titleDetermination of the stoichiometry between alpha- and gamma 1 subunits of the BK channel using LRETes_ES
Document typedc.typeArtículo de revistaes_ES
Indexationuchile.indexArtículo de publicación ISIes_ES
Access notedct.AccessRightsSin acceso a texto completoes_ES

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