Determination of the stoichiometry between alpha- and gamma 1 subunits of the BK channel using LRET
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Two 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.
National 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-F
Artículo de publicación ISI
Quote ItemBiophysical Journal Volumen: 114 Número: 11 Páginas: 2493-2497