No abstract

‘Regulators of G protein Signalling’ (RGSs) accelerate the activation and deactivation kinetics of G protein‐gated inwardly rectifying K+ (GIRK) channels. In an apparent paradox, RGSs do not reduce steady‐state GIRK current amplitudes as expected from the accelerated rate of deactivation when reconstituted in Xenopus oocytes. We present evidence here that this kinetic anomaly is dependent on the degree of G protein‐coupled receptor (GPCR) precoupling, which varies with different Gαi/o‐RGS complexes. The gating properties of GIRK channels (Kir3.1/Kir3.2a) activated by muscarinic m2 receptors at varying levels of G protein expression were examined with or without the co‐expression of either RGS4 or RGS7 in Xenopus oocytes. Different levels of specific m2 receptor‐Gα coupling were established by uncoupling endogenous pertussis toxin (PTX)‐sensitive Gαi/o subunits with PTX, while expressing varying amounts of a single PTX‐insensitive subunit (Gαi1(C351G), Gαi2(C352G), Gαi3(C351G), GαoA(C351G), or GαoB(C351G)). Co‐expression of each of the PTX‐insensitive Gαi/o subunits rescued acetylcholine (ACh)‐elicited GIRK currents (IK,ACh) in a concentration‐dependent manner, with Gαo isoforms being more effective than Gαi isoforms. Receptor‐independent ‘basal’ GIRK currents (IK,basal) were reduced with increasing expression of PTX‐insensitive Gα subunits and were accompanied by a parallel rise in IK,ACh. These effects together are indicative of increased Gβγ scavenging by the expressed Gα subunit and the subsequent formation of functionally coupled m2 receptor‐G protein heterotrimers (Gα(GDP)βγ). Co‐expression of RGS4 accelerated all the PTX‐insensitive Gαi/o‐coupled GIRK currents to a similar extent, yet reduced IK,ACh amplitudes 60‐90 % under conditions of low Gαi/o coupling. Kinetic analysis indicated the RGS4‐dependent reduction in steady‐state GIRK current was fully explained by the accelerated deactivation rate. Thus kinetic inconsistencies associated with RGS4‐accelerated GIRK currents occur at a critical threshold of G protein coupling. In contrast to RGS4, RGS7 selectively accelerated Gαo‐coupled GIRK currents. Co‐expression of Gβ5, in addition to enhancing the kinetic effects of RGS7, caused a significant reduction (70‐85 %) in steady‐state GIRK currents indicating RGS7‐Gβ5 complexes disrupt Gαo coupling. Altogether these results provide further evidence for a GPCR‐Gαβγ‐GIRK signalling complex that is revealed by the modulatory affects of RGS proteins on GIRK channel gating. Our functional experiments demonstrate that the formation of this signalling complex is markedly dependent on the concentration and composition of G protein‐RGS complexes.

No abstract

The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [3H]phosphatidylinositol ([3H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca2+ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration‐dependently activated by guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and by NaF. GTPγS‐stimulated [3H]PI hydrolysis was markedly lower (50%) at all tested concentrations (0.3–10 µM GTPγS) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTPγS in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTPγS‐stimulated [3H]PI hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans‐1‐aminocyclopentyl‐1,3‐dicarboxylic acid, serotonin, and ATP each activated [3H]PI hydrolysis above that obtained with GTPγS alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTPγS alone. Thus, among the three cortical regions examined there was a selective impairment in G protein‐stimulated [3H]PI hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder.

The D2 subtype of dopamine receptor is thought to be an important site of action for antipsychotic drugs (Creese et al. 1976;Seeman et al. 1976). Recently, two new D2-like receptor subtypes, termed D3 and D4

Both opioids and cannabinoids bind to G-protein-coupled receptors to inhibit adenylyl cyclase in neurons. These reactions were assayed in brain membranes, where maximal inhibitory activity occurred in the following regions: mu-opioid inhibition in rat thalamus, delta-opioid inhibition in rat striatum, kappa-opioid inhibition in guinea pig cerebellum, and cannabinoid inhibition in cerebellum. The inhibition of adenylyl cyclase by both cannabinoid and opioid agonists was typical of G-protein-linked receptors: they required GTP, they were not supported by non-hydrolyzable GTP analogs, and they were abolished (in primary neuronal cell culture) by pertussis toxin treatment. The immediate targets of this system were determined by assaying protein phosphorylation in the presence of receptor agonists and App(NH)p, a substrate for adenylyl cyclase. In striatal membranes, opioid agonists inhibited the phosphorylation of at least two bands of MW 85 and 63 kDa, which may be synapsins I and II, respectively. Other experiments determined the long-term effects of this second messenger system. In primary neuronal cultures, opioid-inhibited adenylyl cyclase attenuated forskolin-stimulated pro-enkephalin mRNA levels, thus providing a feedback regulation of opioid synthesis. Finally, in cerebellar granule cells, both cannabinoid and opioid receptors may exist on the same cells. In these cells, agonists which bind to different receptor types may produce similar biological responses.

No abstract

Assessing the function of the phosphoinositide signal transduction system in membranes prepared from postmortem human brain by measuring the hydrolysis of exogenous labeled phosphoinositides has been applied to studies of a variety of CNS disorders in recent years. Two issues concerning such studies were addressed in the current investigation: how do [3H]phosphatidylinositol and [3H]phosphatidylinositol 4,5‐bisphosphate compare as substrates, and how do dopamine D1 receptors influence phosphoinositide signaling? Comparisons of [3H]phosphatidylinositol and [3H]phosphatidylinositol 4,5‐bisphosphate hydrolysis stimulated by guanosine‐5′‐O‐(3‐thiotriphosphate)‐activated G proteins and by several receptor agonists demonstrated that in most cases each substrate gave similar relative results in membranes prepared from prefrontal cortices of six individuals. However, using optimal assay conditions, [3H]phosphatidylinositol produced a greater signal‐to‐noise ratio compared with [3H]phosphatidylinositol 4,5‐bisphosphate. Dopamine D1 receptors were demonstrated to be directly coupled to phosphoinositide hydrolysis in human brain membranes, and this response was shown to be mediated by the Gq/11 G protein subtype and by the β‐subtype of phospholipase C. Therefore, these results demonstrate that [3H]phosphatidylinositol is a suitable substrate to measure phosphoinositide hydrolysis in human brain membranes and that dopamine D1 receptors directly stimulate this signaling system.