Impact of RNA Editing on Functions of the Serotonin 2C Receptor in vivo

Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing 5-HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor

Glucose injections into the dorsal hippocampus or dorsolateral striatum of rats prior to T-maze training: Modulation of learning rates and strategy selection

The present experiments examined the effects of injecting glucose into the dorsal hippocampus or dorsolateral striatum on learning rates and on strategy selection in rats trained on a T-maze that can be solved by using either a hippocampus-sensitive place or striatum-sensitive response strategy. Percentage strategy selection on a probe trial (P(crit)) administered after rats achieved criterion (nine of 10 correct choices) varied by group. All groups predominately exhibited a response strategy on a probe trial administered after overtraining, i.e., after 90 trials. In experiment 1, rats that received intrahippocampal glucose injections showed enhanced acquisition of the T-maze and showed increased use of response solutions at P(crit) compared with that of unimplanted and artificial cerebral spinal fluid (aCSF)-treated groups. These findings suggest that glucose enhanced hippocampal functions to accelerate the rate of learning and the early adoption of a response strategy. In experiment 2, rats that received intrastriatal glucose injections exhibited place solutions early in training and reached criterion more slowly than did aCSF controls, with learning rates comparable to those of unoperated and operated-uninjected controls. Relative to unoperated, operated-uninjected and glucose-injected rats, rats that received intrastriatal aCSF injections showed enhanced acquisition of the T-maze and increased use of response solutions at P(crit). The unexpected enhanced acquisition seen after striatal aCSF injections suggests at least two possible interpretations: (1) aCSF impaired striatal function, thereby releasing competition with the hippocampus and ceding control over learning to the hippocampus during early training trials; and (2) aCSF enhanced striatal functioning to facilitate striatal-sensitive learning. With either interpretation, the results indicate that intrastriatal glucose injections compensated for the aCSF-induced effect. Finally, enhanced acquisition regardless of treatment was accompanied by rapid adoption of a response solution for the T-maze

Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5‑HT_2C Receptor Underlying the Pharmacology of Distinct Ligands

While exploring the structure–activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT_2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT_2C receptors. In HEK293 cells expressing human 5-HT_2C‑INI receptors, for example, (−)-<i>trans</i>-3′-Br-PAT and (−)-<i>trans</i>-3′-Cl-PAT are agonists regarding Gα_q-inositol phosphate signaling, whereas (−)-<i>trans</i>-3′-CF₃-PAT is an inverse agonist. To investigate the ligand–receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT_2C receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (<i>K</i>_i) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC₅₀ functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (−)-<i>trans</i>-3′-CF₃-PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (−)-<i>trans</i>-3′-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (<i>K</i>_D) of the antagonist radioligand [³H]­mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT_2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT_2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand