Sloan-Kettering Institute for Cancer Research

Memorial Sloan Kettering Cancer Center

Gero Miesenböck

Boris Zemelman

James Edward Rothman

Dino De Angelis

Methods and compositions to activate a genetically designated target cell (or population of target cells) artificially, in vivo or in vitro, employ triggering of heterologous stimulus-gated ion channels to activate the cells. The stimulus-gated ion channels are suitably TRPV1, TRPM8 or P2X. A stimulus which leads to opening or 'gating' of the ion channel can be a physical stimulus or a chemical stimulus. Physical stimuli can be provided by heat, or mechanical force, while chemical stimuli can suitably be a ligand, such as capsaicin for TRPV1 or ATP for P2X, or a 'caged ligand,' for example a photolabile ligand derivative, in which case a physical signal in the form of light is used to provide the chemical signal. Selective activation of the cell may be used for various applications including neuronal and neuroendocrine mapping and drug screening.

Heterologous stimulus-gated ion channels and methods of using same

Cells are rendered sensitive to stimulation by introducing into a non-photoreceptor cell nucleic acid sequences encoding at least an opsin gene product, an arrestin gene product, and the alpha subunit of the heterotrimeric G protein of the Gfamily. The introduced sequences are expressed by the cell to yield at least the opsin gene product, the arrestin gene product, and the alpha subunit of the heterotrimeric G protein of the Gfamily. Retinal or a derivative thereof capable of bonding with the opsin gene product to form a rhodopsin is provided to the cell. The cell is then irradiated with light having a wavelength capable of converting the rhodopsin to metarhodopsin. The conversion of rhodopsin to metarhodopsin triggers a cascade of intracellular responses within the cell resulting in an increased intracellular concentration of IPand calcium ions.

Bio-synthetic photostimulators and methods of use

The invention relates to methods and compositions which utilize the emission of light to monitor changes in microenvironments involving cells. The invention is especially useful for monitoring exocytotic activity such as detecting quantal release of synaptic vesicles. Fusion proteins ofand synaptotagmin-I or VAMP/synaptobrevin-2 were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with luciferin present in the extracellular medium. Photon emissions in the presence of a depolarizing stimulus can be observed with these systems. pH-sensitive mutants of green fluorescent protein are also provided, which are useful for visualizing exocytosis and for imaging and measuring the pH of intracellular compartments.

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New York, NY, United States of America

Gero Miesenböck