Locking the dimeric GABA(B) G-protein-coupled receptor in its active state.

G-protein-coupled receptors (GPCRs) play a major role in cell-cell communication in the CNS. These proteins oscillate between various inactive and active conformations, the latter being stabilized by agonists. Although mutations can lead to constitutive activity, most of these destabilize inactive conformations, and none lock the receptor in an active state. Moreover, GPCRs are known to form dimers, but the role of each protomer in the activation process remains unclear. Here, we show that the heterodimeric GPCR for the main inhibitory neurotransmitter, the GABA(B) receptor, can be locked in its active state by introducing two cysteines expected to form a disulphide bridge to maintain the binding domain of the GABA(B1) subunit in a closed form. This constitutively active receptor cannot be inhibited by antagonists, but its normal functioning, activation by agonists, and inhibition by antagonists can be restored after reduction with dithiothreitol. These data show that the closed state of the binding domain of GABA(B1) is sufficient to turn ON this heterodimeric receptor and illustrate for the first time that a GPCR can be locked in an active conformation.

Ca2+ and phosphatidylinositol 4,5-bisphosphate stabilize a Gbeta gamma-sensitive state of Ca V2 Ca 2+ channels.

Direct interactions between G-protein betagamma subunits and N- or P/Q-type Ca(2+) channels mediate the inhibitory action of several neurotransmitters in the brain. Membrane potential, channel phosphorylation, or auxiliary subunit association tightly regulate these interactions and the consequent inhibition of Ca(2+) current. We now provide evidence that intracellular Ca(2+) concentration and phosphoinositides play a stabilizing role in this direct voltage-dependent inhibition. Lowering resting cytosolic Ca(2+) concentration in Xenopus oocytes expressing Ca(V)2Ca(2+) channels strongly decreased basal as well as phasic, agonist-dependent inhibition of Ca(2+) channels by G-proteins. Decreasing phosphoinositide levels also suppressed G-protein inhibition and completely occluded the effects of a subsequent injection of Ca(2+) chelator. Similar regulations are observed in mouse dorsal root ganglia neurons. Alteration of G-protein block by these agents is independent of protein phosphorylation, cytoskeleton dynamics, and GTPase or GDP/GTP exchange activity, suggesting a direct action at the level of the Ca(2+) channel/Gbetagamma-protein interaction. Moreover, affinity binding experiments of intracellular loops of the Ca(V)2.1 Ca(2+) channels to different phospholipids revealed specific interactions between the C-terminal tail of the channel and phosphoinositides. Taken together these data indicate that a Ca(2+)-sensitive interaction of the C-terminal tail of P/Q channels with the plasma membrane is important for G-protein regulation.