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 C 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_V2.2 and Ca_V1.2 channels were inhibited concomitantly with C1_PKC-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.

1. Calcium Signals Laboratory, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 720 Rutland Avenue, 726 Traylor Building, Baltimore, Maryland 21205, USA.
2. Present address: Department of Physiology and Cellular Biophysics and Department of Pharmacology, Columbia University, College of Physicians and Surgeons, 630 West 168^th Street, P&S 7-422, New York, New York 10032, USA.

Correspondence to: Henry M Colecraft^1,2 Email: hcolecr1@jhmi.edu

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