title: G protein beta2 subunit-derived peptides for inhibition and induction of G protein pathways. Examination of voltage-gated Ca2+ and G protein inwardly rectifying K+ channels.
abstract: 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 betagamma 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 Gbeta2 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 Gbeta2 N terminus inhibits G protein modulation, whereas a 35-amino acid peptide derived from the Gbeta2 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 beta2gamma3 subunits, indicating a reorientation between G protein beta2gamma3 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 Gbetagamma-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.
authors.affiliation: Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.forename: Alexander
authors.initials: A
authors.lastname: Hümmer
authors.forename: Jing
authors.initials: J
authors.lastname: Han
authors.forename: Mian
authors.initials: M
authors.lastname: Xie
authors.forename: Katya
authors.initials: K
authors.lastname: Melnik-Martinez
authors.forename: Rosa L
authors.initials: RL
authors.lastname: Moreno
authors.forename: Matthias
authors.initials: M
authors.lastname: Buck
authors.forename: Melanie D
authors.initials: MD
authors.lastname: Mark
authors.forename: Stefan
authors.initials: S
authors.lastname: Herlitze
mesh_terms: D000595:Amino Acid Sequence; D015220:Calcium Channels / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D015220:Calcium Channels; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044387:GTP-Binding Protein beta Subunits; D024661:Potassium Channels, Inwardly Rectifying; D019204:GTP-Binding Proteins