Cavβ-subunit displacement is a key step to induce the reluctant state of P/Q calcium channels by direct G protein regulation

摘要

P/Q Ca²⁺ channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gβγ binding onto the pore-forming subunit, Cav2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary β3-subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of β3. Along with the observation that Gβγ has no effect on the I-V curve of Cav2.1 alone, we propose that the reluctant mode promoted by Gβγ corresponds to a state in which the β3-subunit has been displaced from its channel-binding site. We validate this hypothesis with a β3-I-II2.1 loop chimera construct. Gβγ binding onto the I-II2.1 loop portion of the chimera releases the β3-binding domain and makes it available for binding onto the I-II loop of Cav1.2, a G protein-insensitive channel. This finding is extended to the full-length Cav2.1 channel by using fluorescence resonance energy transfer. Gβγ injection into Xenopus oocytes displaces a Cy3-labeled β3-subunit from a GFP-tagged Cav2.1 channel. We conclude that β-subunit dissociation from the channel complex constitutes a key step in P/Q calcium channel regulation by G proteins that underlies the reluctant state and is an important process for modulating neurotransmission through G protein-coupled receptors.