Observation Of Structural Changes In Closed K+ Channels By Voltage Clamp Spectroscopy

Ion channels are polymorphic membrane proteins whose states and transitions have been identified by electrophysiology, and whose high-resolution static structures have begun to yield to X-ray and EM techniques. These structures have offered images of individual states, giving us starting points for identifying the complex and transient structural changes that give rise to channel physiology. To understand the structural changes that underlie the gating of voltage-gated K+ channels, we have synthesized fluorescent probes of Kv2 channel activity and used spectral imaging to identify structural changes in the Kv2 complex. We have synthesized chemoselective point mutants of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2 channels, and labeled them with a novel environment-sensitive far-red fluorophore, JP, whose emission is sensitive to the polarity of its surroundings. JP-GxTX fluorescence measured in live cell membranes is dependent on the presence of Kv2 channels, the labeling site on the toxin, and the membrane potential. We collect full emission spectra while varying the membrane potential of patch clamped cells (i.e., voltage clamp spectroscopy, VCS) and have developed curve fitting techniques to identify structural changes of the complex by identifying changes to the environment of the toxin during voltage changes. Emission spectra of the JP27 GxTX mutant comprise 2 major species, polar and non-polar, whose populations change as a function of voltage. These changes occur at far more negative potentials than channel opening, suggesting that they reflect conformational changes of the complex while the channel is closed. VCS of channel-bound fluorophores can bridge the gap between electrophysiology and static structures to offer insight into structural changes of functional channels in live cells.