Article abstract
Nature Chemical Biology 3, 795 - 804 (2007)
Published online: 21 October 2007 | doi:10.1038/nchembio.2007.42
Genetically encoded molecules for inducibly inactivating CaV channels
Tingting Yang1, Yasir Suhail1, Stanislava Dalton1, Timothy Kernan1 & Henry M Colecraft1,2
Abstract
Voltage-gated Ca2+ (CaV) 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 CaV channels (GEMIICCs). GEMIICCs are derivatives of Rem, a Ras-like GTPase that constitutively inhibits Ca2+ currents (ICa). C terminus–truncated Rem1–265 lost the ability to inhibit ICa owing to loss of membrane targeting. Fusing the C1 domain of protein kinase C
to yellow fluorescent protein (YFP)-Rem1–265 generated a molecule that rapidly translocated from cytosol to plasma membrane with phorbol-12,13-dibutyrate in human embryonic kidney cells. Recombinant CaV2.2 and CaV1.2 channels were inhibited concomitantly with C1PKC
-YFP-Rem1–265 membrane translocation. The generality of the approach was confirmed by creating a GEMIICC using rapamycin-dependent heterodimerization of YFP-FKBP-Rem1–265 and a constitutively membrane-targeted rapamycin-binding domain. GEMIICCs reduced ICa 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.
- Calcium Signals Laboratory, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 720 Rutland Avenue, 726 Traylor Building, Baltimore, Maryland 21205, USA.
- Present address: Department of Physiology and Cellular Biophysics and Department of Pharmacology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, P&S 7-422, New York, New York 10032, USA.
Correspondence to: Henry M Colecraft1,2 e-mail: hcolecr1@jhmi.edu
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