Significance Electrically excitable cells, such as neurons, exhibit tremendous variation in their patterns of electrical signals. These variations arise from the collection of ion channels present in any specific cell, but understanding which ion channels are at the root of particular electrical signals remains a significant challenge. Here, we describe novel probes, derived from a tarantula venom peptide, that are able to report the activity of voltage-gated ion channels in living cells. This technology uses state-selective binding to optically monitor the activation of ion channels during cellular electrical signaling. Activity-reporting probes based on these prototypes could potentially identify when endogenous ion channels contribute to electrical signaling, thus facilitating the identification of ion channel targets for therapeutic drug intervention.

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.

1A primary goal of molecular physiology is to understand how conformational changes of 2 proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging 3 method for measuring the conformational changes of a voltage-sensing protein within tissue. We 4 synthesized a fluorescent molecular probe, compatible with two-photon microscopy, that targets 5 a resting conformation of Kv2-type voltage gated K + channel proteins. Voltage-response 6 characteristics were used to calibrate a statistical thermodynamic model relating probe labeling 7 intensity to the conformations adopted by unlabeled Kv2 proteins. Two-photon imaging of rat 8 brain slices labeled with the probe revealed fluorescence consistent with conformation-selective 9 labeling of endogenous neuronal Kv2 proteins. In principle, this method of quantifying 10 endogenous protein conformational change from fluorescence images is generalizable to other 11 proteins labeled with conformation-selective probes. 12