Genetically targeted chromophore-assisted light inactivation.
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) 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 alpha1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by approximately 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or alpha1C 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.
Tour
Oded
O
Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA.
Meijer
Rene M
RM
Zacharias
David A
DA
Adams
Stephen R
SR
Tsien
Roger Y
RY
D020746:Calcium Channels, L-Type / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005456:Fluorescent Dyes / Q000378:metabolism* / Q000528:radiation effects*; D018390:Gene Targeting / Q000379:methods*; D015640:Ion Channel Gating / Q000502:physiology / Q000528:radiation effects; D008027:Light; D010777:Photochemistry / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000528:radiation effects
D020746:Calcium Channels, L-Type; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins