Endogenous regulators of G protein signaling proteins regulate presynaptic inhibition at rat hippocampal synapses

Abstract

Presynaptic inhibition 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 of G␣ subunits alone. Here, we test the hypothesis that endogenous regulators of G protein signaling (RGS proteins) are required for rapid, brief presynaptic inhibition. Endogenous G protein ␣ subunits were uncoupled from GPCRs by treating cultures with pertussis toxin (PTX). Adenoviral expression of mutant PTX-insensitive (PTX-i) G␣i1-3 or G␣o subunits rescued adenosine-induced presynaptic inhibition in cultured hippocampal neurons. Expression of double mutant G␣ i1 or G␣o subunits that were both PTX-insensitive and unable to bind RGS proteins (PTX͞RGS-i) also rescued presynaptic inhibition. Presynaptic inhibition mediated by PTX͞RGS-i subunits decayed much more slowly after agonist removal than that mediated by PTX-i subunits or native G proteins. The onset of presynaptic inhibition mediated by PTX͞RGS-i G␣o was also slower than that mediated by PTX-i G␣ o. In contrast, the onset of presynaptic inhibition mediated by PTX͞RGS-i G␣i1 was similar to that mediated by PTX-i G␣ i1. These results suggest that endogenous RGS proteins regulate the time course of G protein signaling in mammalian central nervous system presynaptic terminals. The release of neurotransmitter from most presynaptic terminals is regulated by activation of G protein-coupled receptors (GPCRs) (1, 2). These receptors may be autoreceptors for ligands released at the same synapse, or heteroreceptors for a wide variety of small molecules [e.g., ␥-aminobutyric acid (GABA), glutamate, or adenosine] or peptides. The most common type of regulation is presynaptic inhibition mediated by heterotrimeric G proteins composed of pertussis toxin (PTX)-sensitive ␣ subunits (G␣ i and͞or G␣ o ) and ␤␥ dimers. Receptor activation promotes nucleotide exchange, which is followed by dissociation of ␣-GTP and ␤␥. Free ␤␥ dimers directly alter the gating of the calcium channels that mediate neurotransmitter release in a manner that makes these channels reluctant to open (3). The resulting inhibition of presynaptic calcium influx can largely account for presynaptic inhibition mediated by GPCRs (4, 5), although mechanisms downstream from calcium influx also may be important in some cases (5). Presynaptic inhibition is terminated by heterotrimer reassociation after hydrolysis of ␣-GTP to ␣-GDP (6).Presynaptic inhibition decays in a few seconds after agonist removal (7,8). This agrees well with the time course of inhibition of somatic calcium channels (7, 9-11) and inhibition of presynaptic calcium influx (8). Activity-dependent release of endogenous agonists produces presynaptic inhibition with a similarly rapid time course (8,(12)(13)(14). The decay of presynaptic inhibition is far too rapid to be accounted for by the intrinsic GTPase activity of PTX-sensitive ␣ subunits alone (15). In other systems regulators of G protein signaling (RGS proteins) act as GTPaseaccelerating protein...

Expression of Rem2, an RGK Family Small GTPase, Reduces N-Type Calcium Current without Affecting Channel Surface Density

“…Single DRG neurons were isolated from 1-to 2-week-old Sprague Dawley rats using essentially the same procedure except that the trypsin concentration was reduced to 0.1 mg/ml. Hippocampal neurons isolated from newborn Sprague Dawley rats were cultured essentially as described previously (Chen and Lambert, 2000) except that neurons were grown on glass bottom dishes coated with poly-D-lysine (0.1 mg/ml). HEK293 cells and COS-7 cells were cultured in growth media as recommended by the provider (American Type Culture Collection, Manassas, VA).…”

Modulation of Ion Channels and Synaptic Transmission by a Human Sensory Neuron-Specific G-Protein-Coupled Receptor, SNSR4/mrgX1, Heterologously Expressed in Cultured Rat Neurons

“…Single DRG neurons were isolated from 1-to 2-weekold Sprague Dawley rats using essentially the same procedure, except the collagenase concentration was reduced to 0.1 mg/ml. Hippocampal neurons were grown on collagen/poly-D-lysine microislands as reported previously (Bekkers and Stevens, 1991;Chen and Lambert, 2000). Briefly, hippocampi were dissected from newborn Sprague Dawley rats and digested with papain (ϳ25 U/ml) at 37°C for 1 hr.…”

Pleiotropic Phenotype of a Genomic Knock-In of an RGS-Insensitive G184S Gnai2 Allele

“…Thus, the only known effect of the G184S mutation in G␣ o and G␣ i is to prevent RGS action on G␣. Several publications using overexpression of these RGS-insensitive G␣ subunits revealed profound slow-ing of channel response kinetics and/or increased potency of agonist responses in neurons (8,(11)(12)(13)37).…”