Project Summary: Broad Impact: Ion channels such as the big conductance potassium channels (BK) are responsible for the excitable nature of excitable cells, such as neurons, skeletal muscle cells, and cardiac muscle cells. For BK channels specifically, their dual sensitivity to both membrane voltage and intracellular calcium changes make them a perfect tool to combine both stimuli to modulate membrane voltage. Recent and past work show that BK channels work alongside voltage-sensitive, -modulated ion channels that pass calcium for intracellular signaling. Recent work also shows that patients with mutations in BK channels can have developmental, neurological, and other health issues. As such, understanding BK channel gating is of utmost importance to understanding how intracellular calcium signaling is modulated. Recently, there are have work that debates the formerly accepted idea of independence between the voltage-sensor and the calcium- sensors. Here, this project will aim to satisfy this debate by utilizing gate-disrupted mutants to isolate each interaction between the gate, the voltage-sensor, and the calcium-sensor. Aim 1: To reveal the unmodulated voltage-sensor due to only voltage and the interactions between the sensors without contributions from an open pore, constitutively-closed channels will be used. Aim 2: Constitutively-open channels will be used to reveal the fully modulated voltage-sensor due to an open channel and the modulated interactions between the sensors with full contributions from an open pore. Training: This project aims to teach the applicant how to record and analyze gating currents. Additional training will to teach the applicant to think about input-output modelling and input interactions that might influence output of a model. This proposal will also include opportunities to interact with experts in ion channel biophysics as well as to branch into other techniques used in ion channel biophysics such as imaging techniques and molecular dynamic simulations. Alongside a thorough approach to professional development, this proposal will both develop skills needed for the applicant to be a contributing career scientist as well as increase our collective knowledge on gating mechanisms of ion channels.