Determination of the Stoichiometry between alpha- and gamma 1 Subunits of the BK Channel Using LRET

作者：Carrasquel Ursulaez Willy; Alvarez Osvaldo; Bezanilla Francisco; Latorre Ramon^*

来源：Biophysical Journal, 2018, 114(11): 2493-2497.

DOI：10.1016/j.bpj.2018.04.008

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.

• 出版日期2018-6-5