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

Victor Ruiz-Velasco and Stephen R. Ikeda

1. cDNA constructs coding for a yellow-emitting green fluorescent protein (GFP) mutant fused to the N-terminus of the G-protein subunit 1 (YFP-1) and a cyan-emitting GFP mutant fused to the N-terminus of the G-protein subunit 2 (CFP-2) 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 1/2, co-expression of YFP-1/2, 1/CFP-2, or YFP-1/CFP-2 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-1/CFP-2 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 G1 and G2 with excess G_oA cDNA. Under these conditions, the NA-mediated Ca²⁺ current inhibition was significantly decreased when compared to uninjected neurons.
5. Coupling to the 2-adrenergic receptor was reconstituted in neurons expressing pertussis toxin (PTX)-insensitive G_oA and either tagged or untagged G12 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-1 and CFP-2. Co-expression of untagged 1 significantly decreased the interaction between the two fusion proteins.
7. In summary, the attachment of GFP mutants to the N-terminus of G1 or G2 does not qualitatively impair their ability to form a heterotrimer, modulate effectors (N-type Ca²⁺ and GIRK channels), or couple to receptors.

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