LRET Measurements In The Three Major Conformations Of The Shaker K Channel

Abstract

In Shaker K+ channels, lanthanide binding tags (LBT) were encoded in 4 consecutive positions on the top of the S4 segment and in 4 consecutive positions on the top of the S3 segment. To constrain the LBT position a truncated S3-S4 linker Shaker construct was used.Tagged channels were expressed in Xenopus laevis oocytes and LRET-based distance measurements were conducted between Tb3+ ions bound to the LBT and Bodipy-Fl attached to the pore-blocker Agitoxin-2. Distance measurements for each of the tagged Shaker constructs were repeated with 3 toxins labeled at positions D20C, Q13C and N5C, respectively. Distances were determined in the three main conformational states of the channel: closed, open and open-inactivated.Voltage-dependent K+ channels are comprised of 4 subunits, symmetrically arranged around a central pore. In our measurements each of the subunits carried a LBT. With the toxin bound to the channel pore, energy is transferred from the 4 donors on the channel to the 1 acceptor element on the toxin. Due to this geometry the lifetime of the sensitized emission decay is composed of 4 exponential components corresponding to 4 donor-acceptor distances (Posson, Selvin 2008). We could determine all 4 distances by fitting a geometrical model to the decay and also determine the positions of the bound Tb3+ ion in the LBT in x, y and z. The resultant coordinates are used to refine the models based on the crystal structure of KV1.2 for the closed, open and open-inactivated states. The most important finding of this study is that the position of the voltage sensor changes, not only when going from the closed to the open state, but also when going from the open to the open-inactivated state.Support: AHA07257632(WS), NIHGM30376(FB), NIHGM68044(AMC).