Distinguishing Potassium Channel Resting State Conformations in Live Cells with Environment-Sensitive Fluorescence

Sebastian Fletcher-Taylor, Parashar Thapa, Rebecka J. Sepela, Rayan Kaakati, Vladimir Yarov-Yarovoy, Jon T. Sack, Bruce E. Cohen

Research output: Contribution to journalArticlepeer-review

Abstract

Ion channels are polymorphic membrane proteins whose high-resolution structures offer images of individual conformations, giving us starting points for identifying the complex and transient allosteric changes that give rise to channel physiology. Here, we report live-cell imaging of voltage-dependent structural changes of voltage-gated Kv2.1 channels using peptidyl tarantula toxins labeled with an environment-sensitive fluorophore, whose spectral shifts enable identification of voltage-dependent conformation changes in the resting voltage sensing domain (VSD) of the channel. We synthesize a new environment-sensitive, far-red fluorophore, julolidine phenoxazone (JP) azide, and conjugate it to tarantula toxin GxTX to characterize Kv2.1 VSD allostery during membrane depolarization. JP has an inherent response to the polarity of its immediate surroundings, offering site-specific structural insight into each channel conformation. Using voltage-clamp spectroscopy to collect emission spectra as a function of membrane potential, we find that they vary with toxin labeling site, the presence of Kv2 channels, and changes in membrane potential. With a high-affinity conjugate in which the fluorophore itself interacts closely with the channel, the emission shift midpoint is 50 mV more negative than the Kv2.1 gating current midpoint. This suggests that substantial conformational changes at the toxin-channel interface are associated with early gating charge transitions and these are not concerted with VSD motions at more depolarized potentials. These fluorescent probes enable study of conformational changes that can be correlated with electrophysiology, putting channel structures and models into a context of live-cell membranes and physiological states.

Original languageEnglish (US)
Pages (from-to)2316-2326
Number of pages11
JournalACS Chemical Neuroscience
Volume11
Issue number15
DOIs
StatePublished - Aug 5 2020

Keywords

  • allostery
  • fluorophore
  • Potassium channel
  • spectroscopy
  • structure
  • toxin

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Cognitive Neuroscience
  • Cell Biology

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