title: Genetic targeting of a voltage-sensitive dye by enzymatic activation of phosphonooxymethyl-ammonium derivative.
abstract: Optical recording of action potentials in individual neurons requires cell-selective targeting with a fluorescent, voltage-sensitive probe. We report on a new labeling system that takes advantage of recent developments in prodrug-based chemistry and allows for the targeting of a lipophilic dye into the plasma membrane of genetically specified cells. With the introduction of two phosphonooxymethylammonium zwitterions into the hydrocarbon chains of an amphiphilic, voltage-sensitive hemicyanine dye, a precursor dye was formed that is water-soluble to an extent that it can no longer bind into cell membranes and hence prevents unspecific staining. Glycosylphosphatidylinositol anchored placental alkaline phosphatase expressed in HEK293 cells converted the precursor dye to a homologue of the widely used dye di-4-ANEPPS and gave rise to excellent levels of plasma membrane localized staining. The voltage sensitivity of the enzymatically activated dye was tested and shown to be similar to sensitivity reported for di-4-ANEPPS.
authors.affiliation: Department of Membrane and Neurophysics, Max Planck Institute for Biochemistry, D-82152 Martinsried-München, Germany.
authors.forename: David N
authors.initials: DN
authors.lastname: Ng
authors.forename: Peter
authors.initials: P
authors.lastname: Fromherz
mesh_terms: D000200:Action Potentials / Q000187:drug effects*; D000469:Alkaline Phosphatase / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D011355:Prodrugs / Q000737:chemistry; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D058851:GPI-Linked Proteins; D007527:Isoenzymes; D011355:Prodrugs; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C050019:1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental