Genetically encoded molecules for inducibly inactivating CaV channels

Abstract

Voltage-gated Ca2+ (Ca(V)) channels are central to the biology of excitable cells, and therefore regulating their activity has widespread applications. We describe genetically encoded molecules for inducibly inhibiting Ca(V) channels (GEMIICCs). GEMIICCs are derivatives of Rem, a Ras-like GTPase that constitutively inhibits Ca2+ currents (I(Ca)). C terminus-truncated Rem(1-265) lost the ability to inhibit I(Ca) owing to loss of membrane targeting. Fusing the C1 domain of protein kinase Cgamma to yellow fluorescent protein (YFP)-Rem(1-265) generated a molecule that rapidly translocated from cytosol to plasma membrane with phorbol-12,13-dibutyrate in human embryonic kidney cells. Recombinant Ca(V)2.2 and Ca(V)1.2 channels were inhibited concomitantly with C1(PKCgamma)-YFP-Rem(1-265) membrane translocation. The generality of the approach was confirmed by creating a GEMIICC using rapamycin-dependent heterodimerization of YFP-FKBP-Rem(1-265) and a constitutively membrane-targeted rapamycin-binding domain. GEMIICCs reduced I(Ca) without diminishing gating charge, thereby ruling out decreased number of surface channels and voltage-sensor immobilization as mechanisms for inhibition. We introduce small-molecule-regulated GEMIICCs as potent tools for rapidly manipulating Ca2+ signals in excitable cells.