In Vivo Measurements Of a Ca2+- And Voltage-Activated K+ Channel Intramolecular Distances Using Genetically Encoded Reporters

Abstract

The large-conductance Ca2+ and voltage-activated K+ (BKCa) channel possesses an Alfa pore-forming subunit and 4 different tissue specific regulatory Beta subunits. The Alfa subunit of BKCa channels contains the S4-based voltage sensor of Kv channels, but is an exception inside the S4 superfamily of ion channels because it is made up of seven (instead of 6) transmembrane segments (S0-S6) with the N terminus facing the extracellular side. Our knowledge is limited of how this extra transmembrane segment affects the architecture of BKCa channels. Equally mysterious is the actual structure of the Alfa-Beta subunit complex. Here we use the genetically encoded Lanthanide Binding Tag (LBT) that binds Tb3+ as LRET donor and the Charibdotoxin (CTX) labeled with Tetramethylrhodamine (TMR) for in vivo spectroscopic studies of intramolecular distances and interactions between Alfa and Beta1 subunit of BKCa channel. We have measured the distance between the extracellular end of S0, S1, S2, and S3 in the Alfa subunit to the TMR in the CTX blocking the pore with and without Beta1 subunit. We have also measured the distance from TM1, TM2 and loop of Beta1 subunit to the TMR in the toxin. We found that segment S0 is further away than the other segments with respect to the center of the pore in the absence of Beta1 subunit, locating it in the periphery of the molecule. However, it becomes closer to the center when Alfa is co expressed with Beta1 subunit. Beta1 co-expression also changes the position of S2. We found that some transmembrane domains of Alfa are further away than Beta1’s transmembranes domains, indicating that the Beta subunit is embedded in the protein. Support: NIHGM30376 and FONDECYT 1070049.