Figure 2. Replacing Rem distal C-terminus for novel spatio-temporal control of Ca_V channel inhibition.

A. Rem structure consists of a guanine nucleotide binding domain (G-domain) flanked by N- and C-termini. The Rem distal C-terminus (DCT), comprised of the last 32 residues of the protein, is a polybasic peptide that mediates binding to the plasma membrane and is necessary for Ca_V channel inhibition. B. Replacing Rem DCT with C1 domain from protein kinase C γ (C1_PKCγ) enables acute recruitment of the engineered Rem to the plasma membrane with a small molecule phorbol ester, PdBu. Co-expressed Ca_V1/Ca_V2 channels are inhibited concomitantly with Rem₂₆₅-C1_PKCγ association with the plasma membrane. This chemo-genetic configuration provides acute temporal control over Ca_V channel inhibition that is slowly reversible. C. Optogenetic control of Rem inhibition of Ca_V channels was achieved using the photodimerizer pair, iLID (LOV2-ssrA) and sspB. The Rem DCT was replaced with sspB via varying linkers (creating optoRGK) while iLID was constitutively anchored to the plasma membrane. Exposure of cells to blue light (470 nm) enabled acute recruitment of optoRGK to the plasma membrane and inhibition of Ca_V1.2 channels. Both plasma membrane association of optoRGK and Ca_V1.2 channel inhibition were reversed in the dark. (C) Replacing Rem DCT with a caveolae-targeting peptide enabled selective inhibition of caveolae-targeted Ca_V1.2 channels in cardiomyocytes while sparing dyadic Ca_V1.2 channels that mediate cardiac excitation-contraction coupling.