Two-pore potassium (K2P) channels are responsible for regulating the resting membrane potential of excitable cells. Though K2P channels are heavily implicated in disease, no approved therapies exist to specifically target K2P channels. Defined pharmacology for these channels has lagged, as K2Ps differ from traditional potassium channels in a number of ways. By harnessing these differences, we aim to discover and characterize new K2P modulators to aid in the study of this unique channel class. Through the structural and functional characterization of small molecule K2P modulators, we will facilitate the understanding of how these channels contribute to physiology and disease.
The introduction of this thesis aims to define and describe the scope of K2P pharmacology, with a specific focus on TREK family modulation. High throughput methods for identifying new classes of TREK family modulators are reviewed, and a synthesis of the selectivity of small-molecule TREK family modulators has been compiled, with special attention to those compounds for which structural information has been elucidated. In chapter two, the action and selectivity of K2P activator BL-1249 is presented and, using a chimeric functional approach, subtle differences between K2P2.1 (TREK-1) and K2P4.1 (TRAAK) channels and their differential response to BL-1249 were pinpointed to specific areas involving M2 and M4 helices. This work highlights how channels within K2P families can differ from one another in their pharmacological profiles and how selective molecules can help to tease apart these important differences. Chapter three presents structural and functional studies of polyruthenium amine inhibitors of K2Ps and creation of a ruthenium red super-binder with low nanomolar efficacy. Through our studies, we define the mechanism of polyruthenium amine inhibitors as an electrostatic block positioned above by coordinating acidic residues. Furthermore, we provide a template for future development of K2P modulators which may target this novel binding site. From these and other recent studies, a multi-site model for K2P pharmacological regulation has emerged and future work will require precise dissection to establish how each of these sites may preferentially affect gating in TREK, and more broadly, all K2Ps.
Finally, chapter four presents the synthetic modulation of a protein-protein interaction (PPI) using stapled peptides in a different context, departing from a focus on K2P channels onto another voltage-gated ion channel (VGIC) family member, voltage-gated calcium channels (CaV). Stapled peptide mimics of the alpha-interacting domain (AID) were found to inhibit the high affinity interaction of the channel domain (CaV-alpha) with its cytosolic subunit (CaV-beta) to affect calcium channel properties. Establishing and improving the modulation of this type of PPI informs the future development of inhibitors of K2P interactions with important cellular partners.