Occurrence of the a subunits of G proteins in cerebral cortex synaptic membrane and postsynaptic density fractions: Modulation of ADP-ribosylation by Ca²⁺/calmodulin

We have examined the isolated postsynaptic density (PSD) fraction for the presence of a G protein. First, we found specific binding of guanosine 5′-[γ-[³⁵S]thio]triphosphate to the PSD. Second, pertussis toxin-activated ADP-ribosylation of the isolated PSD fraction resulted in the appearance of a G protein with an apparent molecular mass of 41 kDa, and two G proteins with apparent molecular masses of 41 kDa and 39 kDa in synaptic membrane (SM) fraction and total homogenate (H). The amount of the 41-kDa G protein per unit protein was in the order of SM > H > PSD. Anti-G_i antibodies recognized the 41-kDa G protein in both PSD and SM, whereas anti-G_o antibodies reacted with the 39-kDa G protein in the SM. The absence of G_o protein in the PSD suggested that there was no contamination with SM. Moreover, unlabeled PSD incubated with an extract of SM that contained the labeled G proteins resulted in no label in the subsequently reisolated PSD, suggesting that the G protein found in the PSD was not due to adsorption of the G protein onto the PSD during its isolation from the SM. PSD pretreated with EGTA gave an 11-fold increase in the ADP-ribosylation reaction of the G_i protein; similar effects on the G₁ and G_o proteins of SM were obtained. Restoration of Ca²⁺/calmodulin to the PSD, but not of either Ca²⁺ or calmodulin alone, removed the effect of EGTA, indicating a strong complex formation between G_i and Ca²⁺/calmodulin that decreased the ADP-ribosylation reaction. Preincubation with the Ca²⁺-channel blocker nifedipine decreased the ADP-ribosylation reaction in the PSD. We conclude that G_i is present in the PSD, that it may interact with calmodulin and that it is involved in the regulation of voltage-dependent Ca²⁺ channel. We present a theory of the involvement of the G protein and calmodulin in postsynaptic neurophysiological events.