Competitive and Synergistic Interactions of G Protein β2 and Ca2+ Channel β1b Subunits with Cav2.1 Channels, Revealed by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Measurements

• Published in: The Journal of biological chemistry 2003-12, Vol.278 (49), p.49386-49400
• Main Authors: Hümmer, Alexander, Delzeith, Oliver, Gomez, Shannon R., Moreno, Rosa L., Mark, Melanie D., Herlitze, Stefan
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M306645200

Presynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein βγ subunits modulate the binding of the Ca2+ channel β subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel β and G protein β subunits on the two separate binding sites, i.e. the loopI–II and the C terminus, and on the full-length P/Q-type α12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel β1b subunit induces a strong fluorescent signal when interacting with the loopI–II but not with the C terminus. In contrast, the G protein β subunit induces FRET signals on both the C terminus and loopI–II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel β1b and G protein β subunits bind to the α1 subunit at the same time. Coexpression of the G protein increases the FRET signal between α1/β1b FRET pairs but not for α1/β1b FRET pairs where the C terminus was deleted from the α1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel β and α12.1 subunits, which involves the C terminus of the α1 subunit and may corresponds to a new conformational state of the channel.