Opto-chemogenetic inhibition of L-type Ca_V1 channels in neurons through a membrane-assisted molecular linkage

Genetically encoded inhibitors of Ca_V1 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 Ca_V1.3, 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 Ca_V1 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.