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

[Image: see text] 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.