Chemical biology of voltage-gated cation channels

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Voltage-gated ion channels shape electrical signaling in the excitable cells of nerve and muscle. Sodium (NaV) and calcium channels (CaV) drive membrane depolarization and activate second messenger pathways via gated cellular entry of their namesake ions. In skeletal and cardiac cells, CaV channels trigger muscle contraction. Voltage-gated potassium channels (KV) allow the release of potassium ions from within the cell to drive membrane repolarization. In concert, these channels provide the molecular foundation for thought, perception, and contraction. High-resolution protein structures of human voltage-gated channels are now providing the first glimpses of the types of poses they may adopt in cellular environments. However, understanding the ultimate link between how these proteins look and how they support physiological mechanisms is a major challenge that will require innovative approaches. For one, transmembrane voltage is absent in a structural experiment thus depicting voltage-gated channels in an essentially non-physiological environment. We are therefore developing photochemical `stapling' approaches to covalently trap high-value protein conformations in live cell membranes prior to purification for structural determination. Further, we have begun to identify mechanisms of channel function by introducing modified chemistries at the peptide backbone in the transmembrane segments that form voltage-sensors and channel gates. In cellular settings, ion channels are also critical amplifiers of transduction pathways. During the fight-or-fight response, for instance, the near instantaneous phosphorylation of CaV1.2 channels results in faster and sustained channel opening, leading to a more forceful and rapid heart rate. Yet the absolute speed and complexity of the process is a challenge to experimentally parse individual molecular events that result in channel gating modifications. We describe newly validated methods that enable light controlled, site-specific phosphorylation, for the careful deconstruction and identification of key steps and players is this process. Lastly, CaV channels can be therapeutically inhibited to manage pain, epilepsy, arrythmia, high blood pressure, and alternatively, activated to treat heart failure. Surprisingly, both of these effects (channel activation and inactivation) can be elicited by medicines binding a common extracellular binding site on the channel. Conversely, unintended blockade of cardiac hERG potassium channels by otherwise useful therapeutics cause 90% of drug induced long-QT syndrome, a potentially lethal cardiac arrhythmia. All of these chemical binding events rely on aromatic rich binding sites formed by the side-chains of phenylalanine and tyrosine residues in CaV and hERG channels. To better understand these chemical interactions, we have developed a high-resolution method that allows for energetic and nuanced dissection of these aromatics within the CaV and KV drug binding aromatic boxes in the environment of mammalian cells. The successful execution of this research program will provide cutting edge training opportunities, advance the molecular understanding of channel gating, and will reveal the binding modes of clinical drugs with high therapeutic value.