期刊
NATURE BIOTECHNOLOGY
卷 21, 期 12, 页码 1505-1508出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt914
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资金
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [P01DK054441] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE [R37NS027177, R01NS027177] Funding Source: NIH RePORTER
- NIDDK NIH HHS [P01 DK54441] Funding Source: Medline
- NINDS NIH HHS [NS27177] Funding Source: Medline
Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore- assisted light inactivation (CALI)(1) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye- labeled nonfunction- blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and alpha(1C) L- type calcium channels, each tagged with one or two small tetracysteine (TC) motifs(2) that specifically bind the membrane- permeant, red biarsenical dye, ReAsH3,4. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by similar to 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or α(1C) tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH- mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.
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