Characterization of the Novel Human Serotonin Receptor Subunits 5-HT_3C,5-HT_3D, and 5-HT_3E

Abstract

Within the family of serotonin receptors, the 5-hydroxytryptamine-3 (5-HT 3 ) receptor is the only ligand-gated ion channel. It is composed of five subunits, of which the 5-HT 3A and 5-HT 3B subunits are best characterized. Several studies, however, have reported on the functional diversity of native 5-HT 3 receptors, which cannot solely be explained on the basis of the 5-HT 3A and 5-HT 3B subunits. After our discovery of further putative 5-HT 3 serotonin receptor-encoding genes, HTR3C, HTR3D, and HTR3E, we investigated whether these novel candidates and the isoform 5-HT 3Ea are able to form functional 5-HT 3 receptor complexes. Using immunofluorescence and immunoprecipitation studies of heterologously expressed proteins, we found that each of the respective candidates coassembles with 5-HT 3A . To investigate whether the novel subunits modulate 5-HT 3 receptor function, we performed radioligandbinding assays and calcium-influx studies in human embryonic kidney 293 cells. Our experiments revealed that the 5-HT 3C , 5-HT 3D , 5-HT 3E , and 5-HT 3Ea subunits alone cannot form functional receptors. Coexpression with 5-HT 3A , however, results in the formation of functional heteromeric complexes with different serotonin efficacies. Potencies of two agonists and antagonists were nearly identical with respect to homomeric 5-HT 3A and heteromeric complexes. However, 5-HT showed increased efficacy with respect to 5-HT 3A/D and 5-HT 3A/E receptors, which is consistent with the increased surface expression compared with 5-HT 3A receptors. In contrast, 5-HT 3A/C and 5-HT 3A/Ea receptors exhibited decreased 5-HT efficacy. These data show for the first time that the novel 5-HT 3 subunits are able to form heteromeric 5-HT 3 receptors, which exhibit quantitatively different functional properties compared with homomeric 5-HT 3A receptors.The 5-HT 3 receptor is the only ligand-gated ion channel (LGIC) within the family of serotonin (5-hydroxytryptamine, 5-HT) receptors (Hoyer et al., 2002). Based on structural and functional homologies, the nicotinic acetylcholine receptor and the 5-HT 3 receptor are most closely related; both are cation channels. The 5-HT 3 receptor is formed by a pentameric complex and is permeable to Na ϩ , K ϩ , and Ca 2ϩ . Binding of serotonin to the 5-HT 3 receptor leads to a fast excitatory response of the neuron. After cloning of the human HTR3A gene (Belelli et al., 1995;Miyake et al., 1995), findings concerning variable receptor compositions and properties led to the hypothesis that further 5-HT 3 receptor subunits and isoforms should exist (Hussy et al., 1994;Jackson and Yakel, 1995;Fletcher and Barnes, 1998). This hypothesis was confirmed by the cloning of the human HTR3B gene (Davies et al., 1999) and of two different human splice variants of the HTR3A gene (Brü ss et al., 2000). To date, HTR3A and HTR3B (Belelli et al., 1995;Miyake et al., 1995;Davies et al., 1999) are well characterized. 5-HT 3A subunits are able to form functional homooligomeric receptors after expression in Xenopus laevi...

A Coupled Array of Noncovalent Interactions Impacts the Function of the 5-HT3A Serotonin Receptor in an Agonist-Specific Way

“…It is interesting that although these hydrogen bonding interactions were first highlighted in structures of the acetylcholine binding protein (AChBP), comparable backbone mutations of the nAChR did not have strong effects on receptor function. 12 To establish that the backbone mutations are indeed influencing a hydrogen bond to D124, we performed double mutant cycle analyses 15,16 with the D124N mutation. We consider a ΔΔG°> 0.65 kcal/mol (i.e., a 3-fold deviation from additivity) to represent a meaningful interaction energy.…”

“…To establish that the backbone mutations are indeed influencing a hydrogen bond to D124, we performed double mutant cycle analyses 15,16 with the D124N mutation. We consider a ΔΔG°> 0.65 kcal/mol (i.e., a 3-fold deviation from additivity) to represent a meaningful interaction energy.…”

Successful treatment with risperidone increases 5‐HT 3A receptor gene expression in patients with paranoid schizophrenia – data from a prospective study

“…Among five identified 5‐HT3 receptor subtypes (A‐E), only subtypes A, B, and C are expressed in the brain (Niesler, Frank, Kapeller, & Rappold, 2003). While 5‐HT3A is the most important subtype of the receptor, 5‐HT3B subtype is functional only coexpressed with 5‐HT3A subtype (Davies et al., 1999) and the 5‐HT3C subtype may combine with 5‐HT3A to work (Niesler et al., 2007). …”

The structural basis of function in Cys-loop receptors

“…2). Only 5-HT3A subunits can form functional homomeric 5-HT 3 Rs, and appear to be obligatory in heteromeric receptors (Holbrook et al 2009 ;Niesler et al 2007). Of the heteromeric receptors, only 5-HT 3 AB receptors have been extensively characterized and, compared to homomeric 5-HT 3 A, 5-HT 3 AB receptors differ in their EC 50 , Hill slope, desensitization kinetics, shape of current-voltage relationship, and most noticeably, a much larger single-channel conductance (y16 pS in 5-HT 3 AB compared to <1 pS in 5-HT 3 A ; Davies et al 1999 ;Dubin et al 1999).…”