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Functional expression and FRET analysis of green fluorescent proteins fused to G‐protein subunits in rat sympathetic neurons
Author(s) -
RuizVelasco Victor,
Ikeda Stephen R.
Publication year - 2001
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.2001.00679.x
Subject(s) - yellow fluorescent protein , green fluorescent protein , patch clamp , g protein coupled inwardly rectifying potassium channel , protein subunit , microbiology and biotechnology , fusion protein , förster resonance energy transfer , g alpha subunit , chemistry , homomeric , beta (programming language) , gamma subunit , hek 293 cells , g protein , biophysics , biology , receptor , fluorescence , biochemistry , signal transduction , recombinant dna , physics , quantum mechanics , computer science , gene , programming language
1 cDNA constructs coding for a yellow‐emitting green fluorescent protein (GFP) mutant fused to the N‐terminus of the G‐protein subunit β1 (YFP‐β1) and a cyan‐emitting GFP mutant fused to the N‐terminus of the G‐protein subunit γ2 (CFP‐γ2) were heterologously expressed in rat superior cervical ganglion (SCG) neurons following intranuclear injection of the tagged subunits. The ability of the tagged subunits to modulate effectors, form a heterotrimer and couple to receptors was characterized using the whole‐cell patch‐clamp technique. Fluorescent resonance energy transfer (FRET) was also measured to determine the protein‐protein interaction between the two fusion proteins. 2 Similar to co‐expression of untagged β1/γ2, co‐expression of YFP‐β1/γ2, β1/CFP‐γ2, or YFP‐β1/CFP‐γ2 resulted in a significant increase in basal N‐type Ca 2+ channel facilitation when compared to uninjected neurons. Furthermore, the noradrenaline (NA)‐mediated inhibition of Ca 2+ channels was significantly attenuated. 3 Co‐expression of YFP‐β1/CFP‐γ2 with G‐protein‐gated inwardly rectifying K + channels (GIRK1 and GIRK4) resulted in tonic GIRK currents that were blocked by Ba 2+ . 4 The ability of the tagged subunits to form heterotrimers was tested by co‐injecting either tagged or untagged Gβ1 and Gγ2 with excess Gα oA cDNA. Under these conditions, the NA‐mediated Ca 2+ current inhibition was significantly decreased when compared to uninjected neurons. 5 Coupling to the α2‐adrenergic receptor was reconstituted in neurons expressing pertussis toxin (PTX)‐insensitive Gα oA and either tagged or untagged Gβ1γ2 subunits. Application of NA to PTX‐treated cells resulted in a voltage‐dependent inhibition of N‐type Ca 2+ currents. 6 FRET measurements in the SCG revealed an in vivo interaction between YFP‐β1 and CFP‐γ2. Co‐expression of untagged β1 significantly decreased the interaction between the two fusion proteins. 7 In summary, the attachment of GFP mutants to the N‐terminus of Gβ1 or Gγ2 does not qualitatively impair their ability to form a heterotrimer, modulate effectors (N‐type Ca 2+ and GIRK channels), or couple to receptors.