Endogenous Regulator of G-Protein Signaling Proteins Regulate the Kinetics of G q/11-Mediated Modulation of Ion Channels in Central Nervous System Neurons

Slow synaptic potentials are generated when metabotropic G-protein-coupled receptors activate heterotrimeric G-proteins, which in turn modulate ion channels. Many neurons generate excitatory postsynaptic potentials mediated by G-proteins of the G q/11 family, which in turn activate phospholipase C. Accessory GTPase-activating proteins (GAPs) are thought to be required to accelerate GTP hydrolysis and rapidly turn off G-proteins, but the involvement of GAPs in neuronal G q/11 signaling has not been examined. Here, we show that regulator of G-protein signaling (RGS) proteins provide necessary GAP activity at neuronal G q/11 subunits. We reconstituted inhibition of native 2-pore domain potassium channels in cerebellar granule neurons by expressing chimeric G subunits that are activated by G i/o-coupled receptors, thus bypassing endogenous G q/11 subunits. RGS-insensitive variants of these chimeras mediated inhibition of potassium channels that developed and recovered more slowly than inhibition mediated by RGS-sensitive (wild-type) chimeras or native G q/11 subunits. These changes were not accompanied by a change in agonist sensitivity, as might be expected if RGS proteins acted primarily as effector antagonists. The slowed recovery from potassium channel inhibition was largely reversed by an additional mutation that mimics the RGS-bound state. These results suggest that endogenous RGS proteins regulate the kinetics of rapid G q/11-mediated signals in central nervous system neurons by providing GAP activity. Heterotrimeric G-protein signaling is initiated when an activated receptor stimulates the exchange of GDP for GTP by the G subunit of a G heterotrimer. This exchange promotes the dissociation of G -GTP and G subunits, both of which can directly or indirectly modify the function of ion channels. Many receptors couple exclusively to one family of G subunits and thereby activate a specific set of effector molecules. For example, G i/o proteins often activate inwardly rectifying potassium channels, whereas G q/11 proteins often inhibit potassium channels or activate nonselective cation channels (Hille, 1994). In neurons, these transduction pathways are responsible for metabotropic inhibitory and excitatory postsynaptic potentials (EPSPs), respectively. G-protein signaling terminates when G -GTP is hydrolyzed to G -GDP and the heterotrimer reassociates. Although G subunits possess intrinsic GTPase activity, this activity is too slow to account for the rapid termination of many G-protein-mediated signals. The timely termination of brief physiological signals thus depends on the activity of GTPase-activating proteins (GAPs), which bind to GTPbound G subunits and greatly accelerate the rate of GTP hydrolysis (Ross and Wilkie, 2000). Regulator of G-protein signaling (RGS) proteins are ubiquitous proteins that have GAP activity at G i/o, G t, and G q/11 proteins (Hepler, 1999; Neubig and Siderovski, 2002). As such, these proteins can regulate the strength and kinetics of a variety of G-protein signals. RGS proteins are the only known GAPs at G i/o and G t proteins, and rapid termination of events mediated by these subunits clearly depends on RGS proteins (Doupnik et al., 1997; Saitoh et al., 1997; Chen et al., 2000). In contrast, the GTPase activity of G q/11 proteins can be accelerated not only by RGS proteins but also by the effector molecule phospholipase C(PLC ) (Berstein et al., 1992; Hepler et al., 1997; Chidiac and Ross, 1999). Thus, it is not clear to what This work was supported by National Institutes of Health grant NS36455. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.019059. ABBREVIATIONS: EPSP, excitatory postsynaptic potential; GAP, GTPase-activating protein; RGS, regulator of G-protein signaling; PLC , phospholipase C; CNS, central nervous system; RGSi, regulator of G-protein signaling-insensitive; 2AR, 2 adrenoreceptor; EGFP, enhanced green fluorescent protein; CGN, cerebellar granule neuron; IKSO, standing outward K current; HEK, human embryonic kidney; ANOVA, analysis of variance; NE, norepinephrine; GS, G qi9 G188S; SD, G qi9 S211D; GS:SD, double mutant G qi9 G188S:S211D; WT, wild-type; RGSm, regulator of G-protein signaling-mimicking. 0026-895X/06/6904-1280–1287$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 4 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 19059/3092366 Mol Pharmacol 69:1280–1287, 2006 Printed in U.S.A. 1280 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from extent RGS proteins are essential for terminating transient G q/11-mediated signals such as slow EPSPs. In addition, RGS proteins could negatively regulate G q/11 signals by serving as “effector antagonists”, competing with the effector PLC for binding to active G-proteins (Hepler et al., 1997). We tested the hypothesis that endogenous RGS proteins regulate G q/11-mediated signals by providing GAP activity in CNS neurons. Several previous studies of endogenous RGS protein function in intact cells have relied on functional replacement of native G subunits with RGS-insensitive (RGSi) mutants (Chen and Lambert, 2000; Jeong and Ikeda, 2000, 2001). We adopted a similar strategy by expressing RGS-sensitive and RGSi variants of G subunit chimeras consisting of G q with nine amino acids at the carboxy terminus substituted for those found in G i (Conklin et al., 1993). These chimeric (G qi9) subunits are activated by receptors that normally activate G i/o subunits but couple to downstream effectors that normally interact with G q/11 subunits. Using this system, we reconstituted inhibition of “leak” two-pore domain potassium (K2P) channels with RGS-sensitive and RGSi G subunits. We found that signals mediated by RGSi subunits develop and decay more slowly than those mediated by endogenous G q/11 or RGS-sensitive G qi9 subunits. These results suggest that endogenous RGS proteins are essential for the rapid kinetics of G q/11-mediated synaptic potentials. Materials and Methods The coding sequence for the chimeric G-protein subunit G qi9 (provided by Dr. Bruce R. Conklin, University of California, San Francisco, San Francisco, CA) was subcloned into pcDNA 3.1 (Invitrogen, Carlsbad, CA). The plasmid coding for the 2 adrenoreceptor ( 2AR) was obtained from the Guthrie Research Institute cDNA resource (www.cdna.org). A plasmid coding for the enhanced green fluorescent protein (EGFP)-RGS2 fusion protein was provided by Dr. Peter Chidiac (University of Western Ontario, London, ON, Canada). QuikChange (Stratagene, La Jolla, CA) mutagenesis was carried out according to the manufacturer’s instructions, and mutagenesis was confirmed by automated oligonucleotide sequencing. Dissociated cultures of cerebellar granule neurons (CGNs) were prepared from 5to 8-day-old Sprague-Dawley rats as described previously (Chen et al., 2004). CGNs were maintained in minimal essential medium supplemented with 5% fetal bovine serum, 25 mM KCl, 2% B-27 (Invitrogen, Carlsbad, CA), 0.1% MITO serum extender (BD Collaborative, San Diego, CA), 0.6% glucose, 1 mM pyruvate, 50 IU/ml penicillin, and 50 g/ml streptomycin in 5% CO2 at 37°C. Neurons were transfected after 6 days in culture using polyethylenimine as described previously (Chen et al., 2004). Neurons were cotransfected with a plasmid coding for pEGFP-N1 (Clontech, Mountain View, CA) and were identified for recording by fluorescence microscopy. Control neurons were transfected with vector (pcDNA 3.1 ) and marker (pEGFP-N1) only. Transfection efficiency using this method was low ( 5%) but provided ample green fluorescent protein-positive cells for consistent recordings. Control experiments were consistent with the idea that the amount of protein expressed in individual cells was related to the amount of DNA added during transfection, although we made no attempt to quantify the levels of protein expressed in individual cells. Electrophysiological recordings were carried out 14 to 48 h after transfection. Recordings were carried out at room temperature (24°C) on the stage of an inverted fluorescence microscope. Neurons were constantly perfused with a solution containing 150 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM glucose, 0.5 mM CaCl2, and 0.5 mM MgCl2, pH 7.8; osmolality, 310 mOsm/kg H2O). Solution changes were made by switching between perfusion reservoirs using a series of pinch valves (Warner Instruments, Hamden, CT). All recordings were made using the whole-cell perforated patch configuration. Recording pipettes were filled with a solution containing 70 mM K gluconate, 70 mM KCl, 0.2 mM EGTA, 10 mM HEPES, and 0.03% amphotericin B, pH 7.3; 295 mOsm/kg H2O). Recordings began 5 min after seal formation, at which time series resistance reached a stable minimum because of patch perforation. Neurons were held in voltage-clamp mode at 20 mV and were stepped to 110 mV for 20 ms every 450 ms. Standing outward K current (IKSO) was measured as the absolute holding current at 20 mV. Currents were digitized and recorded using a multifunction I/O board (National Instruments, Austin, TX) and WinWCP software (provided by Dr. J. Dempster, University of Strathclyde, Glasgow, Scotland). For imaging EGFP-RGS2 translocation, HEK 293 cells were cotransfected with plasmids coding for EGFP-RGS2 and various G qi9 subunits using LipofectAMINE (Invitrogen) according to the manufacturer’s instructions. After at least 16 h, cells were imaged using a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc., Thornwood, NY). Single optical sections through the center of each cell were analyzed using ImageJ software (http://rsb.info.nih.gov/ij/) by drawing a profile normal to and centered on the plasma membrane and plotting the fluorescence intensity along this profile. Because of the limits of diffraction and imperfect centering of these profiles, some fluorescence seems to originate on the extracellular side of the plasma