Tethered Protein Display Identifies a Novel Kir3.2 (GIRK2) Regulator from Protein Scaffold Libraries

Use of randomized peptide libraries to evolve molecules with new functions provides a means for developing novel regulators of protein activity. Despite the demonstrated power of such approaches for soluble targets, application of this strategy to membrane systems, such as ion channels, remains challenging. Here, we have combined libraries of a tethered protein scaffold with functional selection in yeast to develop a novel activator of the G-protein-coupled mammalian inwardly rectifying potassium channel Kir3.2 (GIRK2). We show that the novel regulator, denoted N5, increases Kir3.2 (GIRK2) basal activity by inhibiting clearance of the channel from the cellular surface rather than affecting the core biophysical properties of the channel. These studies establish the tethered protein display strategy as a means to create new channel modulators and highlight the power of approaches that couple randomized libraries with direct selections for functional effects. Our results further underscore the possibility for the development of modulators that influence channel function by altering cell surface expression densities rather than by direct action on channel biophysical parameters. The use of tethered library selection strategies coupled with functional selection bypasses the need for a purified target and is likely to be applicable to a range of membrane protein systems

A High-Throughput Functional Screen Identifies Small Molecule Regulators of Temperature- and Mechano-Sensitive K_2P Channels

K_2P (KCNK) potassium channels generate “leak” potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K_2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K_2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K_2P channel K_2P2.1 (<i>KCNK2</i>, TREK-1). By combining biophysical, structure–activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K_2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K_2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K_2P activity and facilitate the discovery and development of other K_2P small molecule modulators

Stapled Voltage-Gated Calcium Channel (Ca_V) α‑Interaction Domain (AID) Peptides Act As Selective Protein–Protein Interaction Inhibitors of Ca_V Function

For many voltage-gated ion channels (VGICs), creation of a properly functioning ion channel requires the formation of specific protein–protein interactions between the transmembrane pore-forming subunits and cystoplasmic accessory subunits. Despite the importance of such protein–protein interactions in VGIC function and assembly, their potential as sites for VGIC modulator development has been largely overlooked. Here, we develop <i>meta</i>-xylyl (<i>m</i>-xylyl) stapled peptides that target a prototypic VGIC high affinity protein–protein interaction, the interaction between the voltage-gated calcium channel (Ca_V) pore-forming subunit α-interaction domain (AID) and cytoplasmic β-subunit (Ca_Vβ). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the <i>m</i>-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca_Vβ interactions and reduce the entropic penalty associated with AID binding to Ca_Vβ. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca_Vα₁:Ca_Vβ interaction that modulate Ca_V function in an Ca_Vβ isoform-selective manner. Together, our studies provide a proof-of-concept demonstration of the use of protein–protein interaction inhibitors to control VGIC function and point to strategies for improved AID-based Ca_V modulator design