Project Summary Voltage-gated ion channels shape electrical signals in excitable cells. There are now high-resolution structures of eukaryotic sodium, potassium and calcium channels, thus providing structural footprint to guide functional hypotheses. In the previous funding period we discovered a dynamic region the pore region of Shaker potassium channels which suggest that state-dependent hydrogen bonding controls the conduction conformation of the channel. These observations have recently been structurally and computationally validated. Further, computational studies indicate that the status of this H-bond network, and thus the conductive state of the channel, are coupled to channel opening at the inner bundle crossing though side-chain to main-chain bonding network along the pore-lining S6 segment. Additionally, voltage gated channels remain high-value pharmacological targets, and the sodium channel Nav1.7 isoform underlies pain sensation. Genetic loss of Nav1.7 abrogates pain sensing in humans, as do adult Nav.17 conditional knock-out animal models. Aryl- and acylsulfonamide compounds target the domain IV (DIV) voltage-sensing domain (VSD) of peripherally expressed sodium channels, such as Nav1.7, with low nanomolar affinity and attenuate inflammatory and neuropathic pain. A structure of the human Nav1.7 DIV VSD in complex with GX-936, a potent arylsulfonamide, suggests that these compounds utilize a unique binding mode whereby the drug simultaneously binds within an aromatic pocket and engages a basic residue (R4) of the activated voltage- sensor. These compounds are useful research tools to advance the understanding of NaV inactivation given the role of the DIV VSD in this process. Further, advancing the chemical details of this drug-bound pose will enable the design of compounds specific activity towards other voltage-sensors. Voltage-gated calcium channels are established drug targets of dihydropyridines (DHP), and recently the binding site was captured in a structure of a bacterial chimeric “CaVAb” – an engineered channel construct with nanomolar DHP binding affinity. However, it is not known if this bacterial chimera faithfully replicated the binding chemistry of the eukaryotic CaV. Not having predictive information on the basis for CaV antagonism severely limits the potential to develop of more critical methods for preclinical screens of therapeutics. The successful execution of these aims will advance the molecular understanding of channel gating and will reveal the binding modes of clinical drugs with high therapeutic value. Further, research tools generated here in will be similarly available to the ion channel research community.