Project Summary Members of the voltage-gated ion channels (VGICs) are critical for electrical and chemical signaling throughout the three kingdoms of life. Dysfunction of ion channels underlie a wide range of pathophysiology and they are one of the primary targets for new drug development. Although they share a common membrane architecture, the channels in this superfamily exhibit surprising diversity of function. Most open in response to a membrane depolarization but some open on hyperpolarization. Many of them are also polymodal- their activity is regulated by second messengers such as cyclic nucleotide or a physical stimulus such as temperature. The main objective of this proposal is to probe the molecular driving forces in order to understand the fundamental mechanisms of voltage-gating and its modulation by temperature and ligand. Current mechanistic approach tends to be structure focused to the extent that protein dynamics is either ignored or treated as secondary. Although the structures of many highly temperature-sensitive ion channels are now available, our understanding of the mechanism of tem- perature-sensitivity remains limited, in large part, due to our inability to directly probe the molecular forces. To address this issue, we are using a multi-pronged approach that combines new and existing tools to systematically characterize the molecular interactions that determine polarity of voltage-gating, exquisite temperature-sensitiv- ity and unusual allostery in VGICs. We are using the HCN channel as a model system to study gating polarity and ligand activation. Using zero model waveguides and newly developed high-throughput analysis algorithms we were able to probe the cooperativity of ligand binding in a model system. We are now poised to extend these studies to full-length channels and receptors. With regards to mechanisms of gating polarity, we have made a surprising discovery that a bipartite switch regulates gating polarity in HCN channels. Microsecond scale simu- lations in Anton supercomputer suggest a gating model which we will be tested further. We will carry out structural studies and combine it with voltage clamp fluorometry in order to annotate these structures. Next, we will also use ancestral protein reconstruction approach, to identify the deep allosteric networks that regulate gating po- larity in these channels. Our studies on temperature-dependent gating is based on two model systems: a) Tem- perature-sensitive Shaker mutant and, b) archaeal MthK channel. In order to determine the essential elements that are responsible for “sensing” temperature, we have to measure the thermodynamic properties such as heat capacity. We propose to develop a new approach involving single molecule force spectroscopy to extract these energetic parameters. Overall, our “molecular forces” focused approach has the potential to provide unparalleled insights into the mechanisms of voltage gating and its regulation by temperature in VGICs.