Voltage sensing is found in many in biological processes and it is fundamental in excitable tissues. This project aims at understanding the physical bases of voltage sensing and how it acts on channel opening at the molecular level. We propose to find the structural basis of function with the following aims: AIM1. Understanding the voltage sensor domain (VSD)-PORE energy landscape with time-dependent temperature jumps. We have developed a method that allows for fast and homogeneous increase in temperature in the area from which function (gating and ionic currents) and the T are measured. We will explore the energy landscape of the Shaker K channel VSD-pore by applying T jumps at different times and voltages during the development of the gating currents and by modifying the landscape with mutations that stabilize intermediate states, which differentially affects a population of particular states in the energy landscape. The results will be interpreted using a structure- based Brownian motion model of the sensor, which correlates the landscape energetic features with the VSD structure and the physical parameters of the sensor and its medium. AIM 2. The coupling between sensor and pore domains. Our previous findings of non-canonical coupling in Shaker is a guide to explore its relevance and function in other channels. 2a. We will study noncanonical VSD-pore coupling in Nav1.4 sodium channel, guided by the structures and the Shaker results by mutation of residues that may couple the four surfaces to the pore using gating and ionic currents while searching for the origin of interdomain cooperativity. 2b. In BK, a non- domain-swapped channel, we hypothesize that the voltage sensor move only two arginines across a fraction of the field based on our preliminary data. To test the hypothesis we will replace arginines with histidines and look for pores or transporters. The arginine trajectories will be inferred by replaced them by qBBR, a positively charged fluorescent probe that is quenched by tryptophan and tyrosine in their path, as we did with Shaker. We will test with mutations and CryoEM structures a noncanonical coupling of S4 via S5 that may gate the channel either by acting on the selectivity filter or directly on an S6 rotation inducing dewetting. 3. VSD-Pore coupling and Inactivation of the Sodium channel. CryoEM structures give no clear mechanistic understanding of Na channel inactivation. We hypothesize that a docked IFM is not the inactivating particle but triggers inactivation. We will first define the inactivation gate, that we propose is made by bulky hydrophobic residues in S6 segments that rotate into position to obstruct conduction, based on our preliminary data where residues impair inactivation when their volume are decreased, with possible inactivation gate restoration by methylsulfonate conjugation when those residues are replaced by cysteine. The linker connecting the IFM motif receptor to the inactivation is a postulated chain of r...