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

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.

The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

“…At resting membrane potentials, GxTX Ser13Pra(JP) has an emission maximum of 644 nm, consistent with the homology-based prediction that Ser13 of GxTX localizes in an aqueous environment branched away from S4. Conversely, GxTX Lys27Pra(JP) has an emission maximum of 617 nm, consistent with the prediction that Lys27 sits in the polar region of the membrane adjacent to S4 (Fletcher-Taylor et al, 2020). If AMIGO1 were to alter the resting conformation of the Kv2.1 voltage sensor domain, we would expect either of these environmental point detectors, GxTX Ser13Pra(JP) or GxTX Lys27Pra(JP), to exhibit an altered emission maximum.…”

“…Fitting shows emission peaks, 644 nm and 617 nm, respectively, are unchanged with or without AMIGO1-YFP, consistent with the local electrostatic environment surrounding the JP probes positioned on resting Kv2.1 voltage sensors not being altered by AMIGO1 expression. Previous work has shown that GxTX Lys27Pra(JP) emission peak wavelength is sensitive to conformational changes among early resting states of voltage sensors (Fletcher-Taylor et al, 2020). The absence of any AMIGO1-induced change in environment for either of these GxTX sidechains suggests that AMIGO1 does not cause significant changes to the local environment of the GxTX binding site on the S3b segment of Kv2.1, nor the GxTX position in the membrane when bound to the channel.…”

“…To measure the electrostatics of the environment immediately surrounding the Kv2.1 voltage sensor domain complex with and without AMIGO1, we employed far-red polaritysensitive fluorescence (Cohen et al, 2005). The polarity-sensitive fluorophore, JP, was localized to the Kv2.1 voltage sensor by conjugating GxTX to JP at either residue Ser13 or Lys27 (Fletcher-Taylor et al, 2020). When GxTX binds to the S3-S4 extracellular region of the Kv2.1 channel (Gupta et al, 2015), Ser13 and Lys27 occupy positions of distinct polarity.…”

“…GxTX mutants were synthesized by solid phase peptide synthesis as described Fletcher-Taylor et al, 2020;Tilley et al, 2014) with propargylglycine (Pra) substituents at either Ser13 or Lys27. Purified GxTX mutants were conjugated with JP-N3 as reported (Fletcher-Taylor et al, 2020). Stock solutions of 7 and 73 µm, respectively, were dissolved in mM Arginine buffer (1 M Arg-HCl, 50 mM Glu, pH 5.0 with NaOH) and stored at -80 ºC and thawed on ice.…”

“…A conjugate of a cysteine-modified guangxitoxin-1E and the maleimide of fluorophore Alexa594 (GxTX Ser13Cys(Alexa594)) was used to selectively modulate Kv2.1 channel gating and to fluorescently identify surfaceexpressing Kv2.1 channels (45). Conjugates of propargylglycine (Pra)-modified GxTX and the fluorophore JP-N3 (GxTX Ser13Pra(JP) and GxTX Lys27Pra(JP)) were used to monitor the chemical environment surrounding GxTX when localized to the channel (46). All modified GxTX-mutants were synthesized by solid phase peptide synthesis as described (46)(47)(48).…”