ACCELERATED COMMUNICATION RNA Editing of the Human Serotonin 5-HT2C Receptor Delays Agonist-Stimulated Calcium Release

RNA encoding the human 5-HT2C receptor undergoes adenosine-to-inosine RNA editing events at five positions in the putative second intracellular loop, with a corresponding reduction in receptor/G-protein coupling. Agonist-stimulated calcium release was examined in NIH-3T3 fibroblasts stably expressing the nonedited human INI (hINI) or the edited hVSV or hVGV variants. We hypothesized that different receptor isoforms would show altered dynamics of agonist-induced calcium release. The three isoforms showed a rightward shift in agonist concentration-response curves for eliciting calcium release (EC50 values: hINI, 2.2 nM; hVSV, 15 nM; hVGV, 49 nM). Additionally, the hVGV receptor showed a blunted and delayed [Ca]i peak compared with the hINI or hVSV receptor isoforms. These distinctions in agonist-induced [Ca]i release imply that edited 5-HT2C receptors may produce distinct physiological responses within the central nervous system. The serotonin 5-HT2C receptor (5-HT2CR) signals through the heterotrimeric G-protein, Gq, to activate phospholipase C (Chang et al., 2000), leading to the intracellular accumulation of inositol trisphosphate and subsequent calcium release. RNA editing within the putative second intracellular loop of the 5-HT2CR produces at least 14 different receptor isoforms (Burns et al., 1997; Niswender et al., 1998). In the human 5-HT2CR, editing occurs at five potential sites named A, B, C, D, and E (Fig. 1; Fitzgerald et al., 1999; Niswender et al., 1999). Editing at A, B, C, and D sites changes amino acids 156, 158, and 160 from INI (hINI) to VSV (hVSV); editing at all five sites produces VGV (hVGV). Since hVSV is the predominant isoform in human brain and hVGV has the most prominent phenotypic differences (Niswender et al., 1999), the present study focuses on calcium responses produced by these two edited receptors. Previous work has shown that 5-HT and (6)-1-(4-iodo-2,5dimethoxyphenyl)-2-aminopropane (DOI), a 5-HT2A/2C receptor agonist, exhibit decreased potency when interacting with the hVSV and hVGV isoforms of the 5-HT2CR, reflected in a rightward shift in the dose-response curve for [H]inositol monophosphate generation (Fitzgerald et al., 1999; Niswender et al., 1999) and decreased efficiency of receptor/ G-protein coupling (Herrick-Davis et al., 1999; Niswender et al., 1999). In the current study, we examine the timing of agonist-induced increases in levels of [Ca]i, hypothesizing that edited isoforms will exhibit altered calcium release dynamics. Such alterations in the dynamics of calcium signaling have the potential to dramatically alter neuronal function (for review, see Berridge, 1998). Experimental Procedures Materials. Ionomycin was purchased from Sigma Chemical Co. (St. Louis, MO). Fura-2/acetoxymethyl ester (fura-2/AM) and pluronic acid were purchased from Molecular Probes (Eugene, OR). DOI was purchased from Research Biochemicals, Inc. (Natick, MA). Cell Culture. NIH-3T3 cells stably expressing hINI, hVSV, and hVGV 5-HT2CR s were generated as described previously (Niswender et al., 1999); receptor densities were 2047, 1292, and 5375 fmol/mg of protein, respectively. Cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 units of penicillin/ml and 100 mg of streptomycin/ml under 5% CO2 at 37°C. Calcium Imaging. Cells were plated on 35-mm tissue culture plates and incubated in serum-free Dulbecco’s modified Eagle’s medium overnight. They were then loaded with fura-2 by incubation in This work was supported by National Institutes of Health Grants NS35891, GM07623-22, and MH34007. ABBREVIATIONS: 5-HT2CR, serotonin 5-HT2C receptor; HBSS, Hanks’ balanced salt solution; DOI, (6)-1-(4-iodo-2,5-dimethoxyphenyl)-2aminopropane; fura-2/AM, fura-2 acetoxymethyl ester. 0026-895X/00/040859-04$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 4 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 273/855921 Mol Pharmacol 58:859–862, 2000 Printed in U.S.A. 859 at A PE T Jornals on N ovem er 7, 2016 m oharm .aspeurnals.org D ow nladed from 0.5 mM fura-2/AM in Hanks’ balanced salt solution (HBSS) for 60 min at room temperature, followed by two washes with HBSS. Cells were superfused with either HBSS (with or without calcium), DOI, or ionomycin. [Ca]i was measured in individual cells by dual-wavelength spectrofluorometry using a Nikon inverted microscope attached to a Compix Calcium Imaging System consisting of a charge coupled device camera (Dage-MTI CCD-72, Michigan City, IN) attached to an IBM compatible computer executing SIMCA C-Imaging software (Compix, Cranberry Township, PA). Cells were exposed to excitation wavelengths of 340 and 380 nm every 2 s and the emitted fluorescence was measured in real time at 510 nm. The ratio of emission at 340 and 380 nm excitation was used as an index of [Ca]i. Data Analysis. Data were analyzed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA) and Microsoft Excel 97. Statistical analyses were performed by two-way ANOVA with Tamhane post-tests. Statistics were only calculated for these doses of DOI: 28, 27.5, 27, 26.5, 26, 25.5 (log M). For t1/2 decay data, doses were grouped by low (log M 28, 27.5), medium (27, 26.5), and high (26, 25.5) doses. Level of significance was set