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

[Image: see text] 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 meta-xylyl (m-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(V)β). We show using circular dichroism spectroscopy, X-ray crystallography, and isothermal titration calorimetry that the m-xylyl staples enhance AID helix formation are structurally compatible with native-like AID:Ca(V)β interactions and reduce the entropic penalty associated with AID binding to Ca(V)β. Importantly, electrophysiological studies reveal that stapled AID peptides act as effective inhibitors of the Ca(V)α(1):Ca(V)β interaction that modulate Ca(V) function in an Ca(V)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(V) modulator design.