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

Victor Ruiz-Velasco, Stephen R. Ikeda

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

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 GαoA 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 GαoA 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.

Original languageEnglish (US)
Pages (from-to)679-692
Number of pages14
JournalJournal of Physiology
Volume537
Issue number3
DOIs
StatePublished - Dec 15 2001

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Energy Transfer
Protein Subunits
Green Fluorescent Proteins
GTP-Binding Proteins
Neurons
Superior Cervical Ganglion
Norepinephrine
Pertussis Toxin
Proteins
Complementary DNA
Inwardly Rectifying Potassium Channel
Patch-Clamp Techniques
Adrenergic Receptors
Injections

All Science Journal Classification (ASJC) codes

  • Physiology

Cite this

@article{2d892a317c214591bf7477071ce75ca2,
title = "Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons",
abstract = "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 GαoA 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 GαoA 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.",
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Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons. / Ruiz-Velasco, Victor; Ikeda, Stephen R.

In: Journal of Physiology, Vol. 537, No. 3, 15.12.2001, p. 679-692.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons

AU - Ruiz-Velasco, Victor

AU - Ikeda, Stephen R.

PY - 2001/12/15

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N2 - 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 GαoA 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 GαoA 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.

AB - 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 GαoA 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 GαoA 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.

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