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

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2018
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Carrasquel Ursulaez, Willy
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Determination of the stoichiometry between alpha- and gamma 1 subunits of the BK channel using LRET
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  • Carrasquel Ursulaez, Willy;
  • Alvarez, Osvaldo;
  • Bezanilla, Francisco;
  • Latorre, Ramón;
Abstract
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.
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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
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URI: https://repositorio.uchile.cl/handle/2250/152334
DOI: 10.1016/j.bpj.2018.04.008
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Biophysical Journal 114, 2493–2497, June 5, 2018
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