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Regulators of G Protein Signaling (RGS Proteins) Regulate Presynaptic Inhibition at Rat Hippocampal SynapsesPresynaptic inhibition o f elicited neurotransmitter release mediated by G protein-coupled receptors (GPCRs) can develop and decay in a few seconds. This time course is too rapid to be accounted for by the intrinsic GTPase activity o f Ga subunits alone. Here we test the hypothesis that endogenous regulators o f G protein signaling (RGS proteins), which are GTPase activating proteins (GAPs) for Ga, are required for rapid, brief presynaptic inhibition. Endogenous G protein a subunits were uncoupled from GPCRs by treating hippocampal microisland cultures with pertussis toxin (PTX). Adenovirusmediated expression o f mutant PTX-insensitive (PTX-i) Gau.3 or Ga0 subunits rescued adenosine-induced presynaptic inhibition in neurons. Expression o f double mutant Gan or Ga0 subunits that were both PTX-insensitive and unable to bind RGS proteins (PTX/RGS-i) also rescued presynaptic inhibition. Presynaptic inhibition mediated by PTX/RGS-i subunits decayed much more slowly after agonist removal than that mediated by PTX-i subunits or native G proteins. The onset o f presynaptic inhibition mediated by PTX/RGS-i Ga0 was also slower than that mediated by PTX-i Ga0. In contrast, the onset o f presynaptic inhibition mediated by PTX/RGS-i Gan was similar to that mediated by PTX-i Gan. These results suggest that endogenous RGS proteins are present in presynaptic terminals and essential for fast recovery o f presynaptic inhibition. The effect o f endogenous RGS proteins on the onset o f presynaptic inhibition appears dependent on the particular Ga subunits involved. We also performed experiments to test whether the functions o f RGS proteins are sensitive to upregulation. Over-expression o f RGS8 in neurons without pretreatment o f PTX not only accelerated the time course o f the onset but also increased the steady state level o f presynaptic inhibition. Overexpression o f RGS4 also enhanced the steady state. These results suggest that RGS8 and probably RGS4 as well can be transported to presynaptic terminals and upregulate the activation o f Gy0 protein signaling. Interestingly, overexpression o f these RGS proteins failed to accelerate the recovery o f presynaptic inhibition, although it is well established that both RGS8 and RGS4 are strong GAPs for GcCj/0. This result suggests GAP activity for Gai/0 in presynaptic terminals is physiologically “ saturated” by endogenous RGS proteins.