3 research outputs found
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<sub>2P</sub> Channels
K<sub>2P</sub> (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<sub>2P</sub>s lack specific pharmacology.
Addressing this issue has been complicated by the challenges that
the leak nature of K<sub>2P</sub> 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<sub>2P</sub> channel K<sub>2P</sub>2.1 (<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<sub>2P</sub> 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<sub>2P</sub> modulators presented here,
together with the yeast-based assay, should enable both mechanistic
and physiological studies of K<sub>2P</sub> activity and facilitate
the discovery and development of other K<sub>2P</sub> small molecule
modulators
Stapled Voltage-Gated Calcium Channel (Ca<sub>V</sub>) α‑Interaction Domain (AID) Peptides Act As Selective Protein–Protein Interaction Inhibitors of Ca<sub>V</sub> 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<sub>V</sub>) pore-forming subunit α-interaction domain (AID) and
cytoplasmic β-subunit (Ca<sub>V</sub>β). 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<sub>V</sub>β interactions and reduce the entropic penalty associated
with AID binding to Ca<sub>V</sub>β. Importantly, electrophysiological
studies reveal that stapled AID peptides act as effective inhibitors
of the Ca<sub>V</sub>α<sub>1</sub>:Ca<sub>V</sub>β interaction
that modulate Ca<sub>V</sub> function in an Ca<sub>V</sub>β
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<sub>V</sub> modulator design