FacePalm: Probing dynamic S-palmitoylation of CaV channel complexes using engineered depalmitoylases.

Abstract

S-palmitoylation is a powerful post-translational modification of ion channels that tunes its function and localization. Unlike other lipid modifications, palmitoylation is a reversible process orchestrated by zDHHC family of protein palmitoyl-transferases and acyl-protein thioesterases that attach and excise palmitic groups, respectively. Even so, dissecting spatial and temporal dynamics of palmitoylation of large channel complexes remains challenging, owing to limitations in molecular approaches to acutely manipulate palmitoylation of specific targets. Here, we develop a new approach to rapidly and inducible depalmitoylate target proteins by engineering the ABHD17 (α/β-hydrolase domain containing protein 17) family of protein depalmitoylases with an FKBP/FRBP chemically inducible dimerization system. This approach is termed FKBP/FRB-activated chemogenetic excision of Palmitoylation or FacePalm. Application of FacePalm revealed multifaced regulation of CaV1/2 channel complexes by palmitoylation. First, time-lapse confocal imaging revealed dynamic redistribution of the CaVβ2A subunit from the plasma membrane to the cytoplasm. Second, whole-cell and single-channel electrophysiology show differences in both inactivation and activation of CaV2 channels depending on palmitoylation of distinct sites on either the β2A or the pore-forming α-subunit. Third, targeting FacePalm to distinct subcellular compartments revealed intrinsic differences in the rate of depalmitoylation of CaVβ2A subunit in different organelles. To further demonstrate generality, we applied FacePalm to various cellular proteins and found substrate-specific differences in palmitoylation dynamics. In all, these studies provide critical insights into the exquisite molecular and cellular mechanisms regulating CaV channel function. Furthermore, FacePalm opens new frontiers to dissect dynamic palmitoylation of ion channel complexes in distinct physiological settings.