Cavbeta-subunit displacement is a key step to induce the reluctant state of P/Q calcium channels by direct G protein regulation.
P/Q Ca(2+) channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gbetagamma binding onto the pore-forming subunit, Ca(v)2.1. 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 beta(3)-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 beta(3). Along with the observation that Gbetagamma has no effect on the I-V curve of Ca(v)2.1 alone, we propose that the reluctant mode promoted by Gbetagamma corresponds to a state in which the beta(3)-subunit has been displaced from its channel-binding site. We validate this hypothesis with a beta(3)-I-II(2.1) loop chimera construct. Gbetagamma binding onto the I-II(2.1) loop portion of the chimera releases the beta(3)-binding domain and makes it available for binding onto the I-II loop of Ca(v)1.2, a G protein-insensitive channel. This finding is extended to the full-length Ca(v)2.1 channel by using fluorescence resonance energy transfer. Gbetagamma injection into Xenopus oocytes displaces a Cy3-labeled beta(3)-subunit from a GFP-tagged Ca(v)2.1 channel. We conclude that beta-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.
Sandoz
Guillaume
G
Institut National de la Santé et de la Recherche Médicale, Unité 607, Canaux Calciques, Fonctions et Pathologies, Commissariat á l'Energie Atomique, Université Joseph Fourier, Département Recherche et Dynamique Cellulaire, Grenoble Cedex 09, France.
Lopez-Gonzalez
Ignacio
I
Grunwald
Didier
D
Bichet
Delphine
D
Altafaj
Xavier
X
Weiss
Norbert
N
Ronjat
Michel
M
Dupuis
Alain
A
De Waard
Michel
M
D000818:Animals; D015220:Calcium Channels / Q000502:physiology*; D002417:Cattle; D004735:Energy Transfer; D019204:GTP-Binding Proteins / Q000502:physiology*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D015220:Calcium Channels; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins