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 beta 1 (YFP-beta 1) and a cyan-emitting GFP mutant fused to the N-terminus of the G-protein subunit gamma 2 (CFP-gamma 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 beta 1/gamma 2, co-expression of YFP-beta 1/gamma 2, beta 1/CFP-gamma 2, or YFP-beta 1/CFP-gamma 2 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-beta 1/CFP-gamma 2 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 G beta 1 and G gamma 2 with excess G alpha(oA) cDNA. Under these conditions, the NA-mediated Ca(2+) current inhibition was significantly decreased when compared to uninjected neurons. 5. Coupling to the alpha 2-adrenergic receptor was reconstituted in neurons expressing pertussis toxin (PTX)-insensitive G alpha(oA) and either tagged or untagged G beta 1 gamma 2 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-beta 1 and CFP-gamma 2. Co-expression of untagged beta 1 significantly decreased the interaction between the two fusion proteins. 7. In summary, the attachment of GFP mutants to the N-terminus of G beta 1 or G gamma 2 does not qualitatively impair their ability to form a heterotrimer, modulate effectors (N-type Ca(2+) and GIRK channels), or couple to receptors.
Ruiz-Velasco
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Laboratory of Molecular Physiology, Guthrie Research Institute, 1 Guthrie Square, Sayre, PA 18840, USA. vruizv@inet.guthrie.org
Ikeda
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SR
D000818:Animals; D020864:Calcium Channels, N-Type / Q000378:metabolism; D004351:Drug Resistance; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000502:physiology; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics*; D008297:Male; D009474:Neurons / Q000378:metabolism*; D037342:Pertussis Toxin; D015221:Potassium Channels / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000378:metabolism; D013564:Sympathetic Nervous System / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D020864:Calcium Channels, N-Type; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D015221:Potassium Channels; D020033:Protein Isoforms; D011942:Receptors, Adrenergic, alpha; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D049452:Green Fluorescent Proteins; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins