Voltage-gated potassium (Kv) channels sense voltage and facilitate transmembrane flow of K+ to control the electrical excitability of cells. The Kv2.1 channel subtype is abundant in most brain neurons and its conductance is critical for homeostatic regulation of neuronal excitability. Kv2.1 channel must progress through a series of conformational changes, including consecutive voltage-sensor activation and pore opening, to permit the flux of this potassium conductance. Many forms of regulation modulate Kv2.1 conductance, yet the biophysical mechanisms through which the conductance is modulated are unknown. In my thesis research, I helped develop two molecular tools to track and control Kv2.1 ion channel conformational change to interrogate which conformational changes are modulated to alter Kv2.1 conductance. For the first method, I synthesized GxTX–594, a variant of the peptidyl tarantula toxin guangxitoxin-1E (GxTX-1E), conjugated to a fluorophore (AlexaFluor-594) optimal for two-photon excitation imaging through light-scattering tissue. GxTX–594 targets the voltage sensors of Kv2 proteins and dynamically labels cell surface Kv2 proteins, responding to voltage stimulation and the conformational state of the voltage sensor. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. This tool permitted us to visually determine the conformational state of endogenous Kv2 voltage sensors in live hippocampal tissue. For the second method, we synthesized GxTX Ser13Pra(JP) and GxTX Lys27Pra(JP), different variants of guangxitoxin-1E conjugated to an fluorophore, JP (julolidine phenoxazone), that has an inherent response to the polarity of its immediate surroundings. GxTX–JP variants offer site-specific structural insight into Kv2.1 voltage sensing domain allostery that occurs during membrane depolarization. Using voltage-clamp spectroscopy to collect emission spectra as a function of membrane potential, we found that emission spectra of these tools 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 voltage sensing domain motions at more depolarized potentials. I then deployed these tools to investigate a biophysical mechanism through which Kv2.1 conductance could be modulated by its auxiliary binding partner, AMIGO1. The neuronal adhesion protein AMIGO1 associates with and modulates the voltage-dependence of Kv2.1 channel activation, yet the underlying mechanism for this or any other modulator of Kv2 conformational change was unknown. With voltage clamp recordings and spectroscopy of heterologously expressed Kv2.1 and AMIGO1 in mammalian cell lines, I demonstrated that AMIGO1 modulates Kv2.1 voltage sensor movement to change Kv2.1 conductance. AMIGO1 speeds early voltage sensor movements and shifts the gating charge–voltage relationship to more negative voltages. From the gating charge–voltage relationship I found that AMIGO1 exerts a larger energetic effect on voltage sensor movement than apparent from the conductance–voltage relationship, which is largely dependent on pore opening. I propose that the mechanistic separation between early voltage sensor movements and pore opening makes the magnitude of the AMIGO1 impact dependent on modulation of Kv2 gating. Conductance-voltage measurements made in the presence of GxTX–594 reveal an increased impact of AMIGO1 on Kv2.1 conduction compared to conditions lacking Kv2 gating modulators. Finally, fluorescence measurements from GxTX Lys27Pra(JP) and GxTX Ser13Pra(JP) bound to Kv2.1 indicate that the voltage sensors enter their earliest resting conformation, yet this conformation is less stable upon voltage stimulation. From this work I concluded that AMIGO1 modulates the Kv2.1 conductance activation pathway by destabilizing the earliest resting state of the voltage sensors. Based on a series of thermodynamic calculations, I speculate that removal of AMIGO1 could be functionally equivalent to blocking the majority of Kv2 current in neurons, which would suggest that AMIGO1 plays a supporting role in controlling the electrical excitability of cells. This work has contributed to the greater understanding of the allosteric mechanisms through which ion channel voltage-dependence can be modulated and raises new questions about the molecular interactions that occur during these early resting conformations.