A Molecular Model For The Bkca Channel And The Location Of B1 In The B1/A Subunit Complex

We describe two approaches for the construction of a 3D molecular model for BK. First, we used a LRET technique in which an encoded Lanthanide Binding Tag (LBT) that binds Tb3+ plays the role of the donors and SulphoRodhamine Methanothiosulfonate (TMSR) attached to Charibdotoxin (ChTX) ) plays the role of the acceptor. The data obtained allowed us to determine the distances from the center of symmetry of the channel to the external aspect of S0, S1, S2, S3-S4 linker in the α subunit. The distances from the channel center of symmetry to TM1, TM2 and three positions in the β1 loop were obtained using the same methodology, by inserting LBT's in the β1 subunit. Second, molecular simulations of ChTX-TMSR complex bound to the BK pore were used to obtain the conformational space of TMSR bound to ChTX. This simulation was used to evaluate the population of the different conformations that TMSR can adopt. The conformational sampling of TMSR was used to recalculate all the distances and estimate the position of the donors in 3D coordinates. The new distance values were used as distance restrains to build a final model of BK. The initial homology model of BK was built using as reference structure the Kv chimeric crystal of Kv1.2-Kv2.1 mammalian channel. Our model refined with LRET experiments shows a structure of α subunit alone with the extra-transmembrane segment S0 located in a pocket in the voltage sensor domain, and a concave shape for BK extracellular face. The β1 external loop, on the other hand, lies very near to the BK pore and forms a structure similar to an alpha greek letter. Supported by FONDECYT Nº1070049, AnilloCientifico ACT/24 and NIHGM030376