Opto-chemogenetic inhibition of L-type CaV1 channels in neurons through a membrane-assisted molecular linkage

Genetically encoded inhibitors of CaV1 channels that operate via C-terminus-mediated inhibition (CMI) have been actively pursued. Here, we advance the design of CMI peptides by proposing a membrane-anchoring tag that is sufficient to link the inhibitory modules to the target channel as well as chemical and optogenetic modes of system control. We designed and implemented the constitutive and inducible CMI modules with appropriate dynamic ranges for the short and long variants of CaV1.3, both naturally occurring in neurons. Upon optical (near-infrared-responsive nanoparticles) and/or chemical (rapamycin) induction of FRB/FKBP binding, the designed peptides translocated onto the membrane via FRB-Ras, where the physical linkage requirement for CMI could be satisfied. The peptides robustly produced acute, potent, and specific inhibitions on both recombinant and neuronal CaV1 activities, including Ca2+ influx-neuritogenesis coupling. Validated through opto-chemogenetic induction, this prototype demonstrates Ca2+ channel modulation via membrane-assisted molecular linkage, promising broad applicability to diverse membrane proteins. [Display omitted] •Improved designs for genetically encoded peptides inhibiting Cav1 channels•Membrane-assisted molecular linkage enables C-terminus-mediated inhibition mechanism•Opto-chemogenetic control mechanisms by NIR-sensitive nanoparticles and rapamycin•Validated on recombinant CaV1 channels and CaV1 signaling in neurons CaV1 channel inhibitors are of significant interest for both research and therapeutic purposes. Genetically encoded peptides that enact C-terminus-mediated inhibition (CMI) offer potential improvements in controllability, specificity, applicability, and effectiveness compared to small-molecule and other conventional inhibitors. However, several limitations remain in designed CMI systems, including the need to express engineered channels to target certain isoforms and the dynamic range of inhibition. Building on the discovery of a membrane-assisted molecular linkage, we sought to address these limitations and also explore different modes of controlling inhibition, including via optical near-infrared-responsive nanoparticles. Geng et al. present design of opto-chemogenetic CaV1 peptide inhibitors by integrating NIR-sensitive nanoparticles with rapamycin-mediated interactions and membrane translocation by way of a Ras/membrane-assisted molecular linkage. They demonstrate inducible, potent, and specific peptide inhib