Blue light-activated ion channel molecules and uses thereof
a technology molecules, applied in the field of altering cell activity and function and the use of light-activated ion channels, can solve problems such as limiting their usefulness
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example 1
Introduction
[0122]The present invention describes the use of light-gated channels to modify the transmembrane potential (and / or ionic composition) of cells (and / or their sub-cellular regions, and their local environment). In particular, the use of inwardly rectifying cationic channels will depolarize cells by moving positively charged ions from the extracellular environment to the cytoplasm. Under certain conditions, their use can decrease the intracellular pH (and / or increase the intracellular cation concentration) or increase the extracellular pH (and / or decrease the extracellular cation concentration). Compared to the currently reported natural gene sequences used to depolarize neurons in the prior art [see for example, Zhang, F. et al. Nature 446, 633-639, (2007) and Han, X & Boyden, E. S. PloS one 2, e299, (2007), the content of each of which is incorporated herein by reference] (this disclosure notwithstanding), ChR64 and ChR86 have demonstrably improved photocurrent generatio...
example 2
[0137]Studies were performed to prepare sequences and to express light-activated ion channels in cells, tissues, and subjects. Non-limiting exemplary methods are set forth Example 1. General methods also applicable to light-activated channel molecules and methods for their use are disclosed in publications such as US Published Application No. 2010 / 0234273, US Published Application No. 20110165681, Chow B Y, et. al. Methods Enzymol. 2011; 497:425-43; Chow, B Y, et al. Nature 2010 Jan. 7; 463(7277):98-102, the content of each of which is incorporated by reference herein.
[0138]Studies were performed to prepare sequences and to express light-activated ion channels in cells, tissues, and subjects. Non-limiting exemplary methods are set forth below.
Plasmid Construction and Site Directed Mutagenesis.
[0139]Opsins were mammalian codon-optimized, and synthesized by Genscript (Genscript Corp., NJ). Opsins were fused in frame, without stop codons, ahead of GFP (using BamHI and AgeI) in a lentiv...
example 3
[0149]ChR64 (SEQ ID NO:2); ChR86 (SEQ ID NO:4); ChR64 with E154A substitution (SEQ ID NO:7); ChR86 with D124A substitution (SEQ ID NO:8) and ChR2 including a ChR2 H134R substitution mutant were expressed in HEK293 cells using methods described in Examples. Normalized action spectrum were recorded in the cells under physiological conditions with the voltage clamped to −65 mV. Equal photon flux was used at each wavelength.
[0150]FIG. 1 shows action spectra recorded in HEK293 cells.
[0151]FIG. 2A-D shows blue light photocurrent and kinetic comparisons in cultured hippocampal neurons.
[0152]FIG. 3A-D shows improvements in trafficking leading from ChR64 to CheRiff FIG. 3A shows photomicrographic image of a cultured neuron expressing wild-type SdChR.
[0153]SdChR typically aggregated and formed puncta in the soma. FIG. 3B shows photomicrographic image of a neuron expressing SdChR with an additional trafficking sequence from Kir2.1 between the C-terminus of SdChR and the N-terminus of eGFP. Thi...
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Abstract
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