G protein β2 subunit-derived peptides for inhibition and induction of G protein pathways -: Examination of voltage-gated Ca2+ AND G Protein inwardly rectifying K+ channels

Voltage-gated Ca2+ channels of the N-, P/Q-, and R-type and G protein inwardly rectifying K+ channels (GIRK) are modulated via direct binding of G proteins. The modulation is mediated by G protein beta gamma subunits. By using electrophysiological recordings and fluorescence resonance energy transfer, we characterized the modulatory domains of the G protein beta subunit on the recombinant P/Q- type channel and GIRK channel expressed in HEK293 cells and on native non-L-type Ca2+ currents of cultured hippocampal neurons. We found that G beta(2) subunit-derived deletion constructs and synthesized peptides can either induce or inhibit G protein modulation of the examined ion channels. In particular, the 25-amino acid peptide derived from the G beta(2) N terminus inhibits G protein modulation, whereas a 35-amino acid peptide derived from the G beta(2) C terminus induced modulation of voltage-gated Ca2+ channels and GIRK channels. Fluorescence resonance energy transfer ( FRET) analysis of the live action of these peptides revealed that the 25-amino acid peptide diminished the FRET signal between G protein beta(2)gamma(3) subunits, indicating a reorientation between G protein beta(2)gamma(3) subunits in the presence of the peptide. In contrast, the 35-amino acid peptide increased the FRET signal between GIRK1,2 channel subunits, similarly to the G beta gamma-mediated FRET increase observed for this GIRK subunit combination. Circular dichroism spectra of the synthesized peptides suggest that the 25-amino acid peptide is structured. These results indicate that individual G protein beta subunit domains can act as independent, separate modulatory domains to either induce or inhibit G protein modulation for several effector proteins.