Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons

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 Ca²⁺ channel facilitation when compared to uninjected neurons. Furthermore, the noradrenaline (NA)-mediated inhibition of Ca²⁺ 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 Ba²⁺. 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ and GIRK channels), or couple to receptors.