Opto-chemogenetic inhibition of L-type Ca _V 1 channels in neurons through a membrane-assisted molecular linkage

Acute, specific, and robust inhibition of L-type Ca ²⁺ (Ca _V 1) channels has been sought after for both research and therapeutic applications. Compared to other available Ca _V 1 antagonists, genetically-encoded modulators, such as CMI (C-terminus mediated inhibition) peptides encoded by Ca _V 1 DCT (distal C-terminus), hold great potentials due to its affirmative mechanisms of action on both gating and signaling. Here, we find that membrane-anchoring with a Ras tag could essentially help form a type of intramolecular-equivalent linkage, by which the tag anchored-peptide appears to dimerize with another protein or peptide on the membrane, supported by the evidence from patch-clamp electrophysiology and FRET imaging. We then design and implement the constitutive and inducible CMI modules, with appropriate dynamic ranges targeting the short and long variants of CaV1.3, both naturally occurring in neurons. Upon optical (infrared-responsive nanoparticles) and/or chemical (rapamycin) induction of FRB/FKBP binding, DCT peptides with no CMI in the cytosol acutely translocate onto the membrane via FRB-Ras, where the physical linkage requirement could be fulfilled. The peptides robustly produce acute and potent inhibitions on both recombinant CaV1.3 channels and neuronal CaV1 activities, and thus the Ca ²⁺ influx-neuritogenesis coupling. Validated through opto-chemogenetic induction, this prototype demonstrates channel modulation via membrane-assisted molecular linkage, promising broad applicability to diverse membrane proteins.