External Architecture of the Large-Conductance Ca2+ and Voltage-Activated K+ (BKCa) Revealed by a Spectroscopic Ruler

Abstract

BKCa channels are involved in a large variety of physiological processes and regulatory β subunits are one of the mechanisms responsible of creating BKCa channel diversity fundamental to the adequate function of many tissues. Regardless the proven importance of this channel little is known about its detailed structure. Here we disclose the external architectural intimacies of BKCa channels using Lanthanide based Resonance Energy Transfer (LRET) as a molecular ruler to measure intra and intermolecular distances. We introduced a genetically encoded lanthanide binding tag (LBT that binds Tb3+ with high affinity) at several positions of the external loops of the α and β1 subunits, and constructed a fluorescent molecule of BODIPY-FL linked to a scorpion toxin, iberiotoxin (Bodipy FL-IbTX), that was used as an acceptor for the LRET interaction with Tb3+. These functional LBT-BKCa constructs were expressed in Xenopus laevis oocytes that were voltage clamped with two microelectrodes to obtain simultaneously electrical and LRET recordings under physiological ionic conditions. Sensitized emission (SE) recordings from different LBT-BKCa positions had different kinetics indicating different relative positions for each construct. We analyzed SE records with a novel method developed by our group that determines the position of LBT-tagged sites of BKCa to obtain an external structural map, including the β1 subunit. Interestingly, when the BKCa α subunit was co-expressed with the regulatory β1 subunit, SE becomes slower, indicating a large conformational change of the BKCa channel structure. The methodology presented here gives us the first glimpses to the BKCa channel external surface structure in its different functional states with and without the β1 subunit. Supported by Fondecyt grant 1110430 and NIH grants U54GM087519 and GM030376. CINV is a Scientific Millennium Institute.