Selective fluorophore-assisted light inactivation of voltage-gated calcium channels.
Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Ca(v)1.2 (L-type) and Ca(v)3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Ca(v)1.2 calcium channels containing EYFP-labeled α(1C)subunits but does not affect the EYFP-α(1G) Ca(v)3.1 calcium channels or Ca(v)1.2 channels containing EYFP-labeled β subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.
National Institute on Aging, National Institutes of Health, Baltimore, MD, USA.
Evgeny
E
Kobrinsky
Jung-Ha
JH
Lee
Nikolai M
NM
Soldatov
D000818:Animals; D019556:COS Cells; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D020747:Calcium Channels, T-Type / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D061885:Chromophore-Assisted Light Inactivation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D008856:Microscopy, Fluorescence / Q000379:methods; D038761:Photobleaching; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
D020746:Calcium Channels, L-Type; D020747:Calcium Channels, T-Type; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins