Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor

Homomeric complexes of 5-HT(3A) receptor subunits form a ligand-gated ion channel. This assembly does not fully reproduce the biophysical and pharmacological properties of native 5-HT(3) receptors which might contain the recently cloned 5-HT(3B) receptor subunit. In the present study, heteromeric assemblies containing human 5-HT(3A) and 5-HT(3B) subunits were expressed in HEK 293 cells to detail the functional diversity of 5-HT(3) receptors. We designed patch-clamp experiments with homomeric (5-HT(3A)) and heteromeric (5-HT(3AB)) receptors to emphasize the kinetics of channel activation and desensitization. Co-expression of the 5-HT(3B) receptor subunit reduced the sensitivity for 5-HT (5-HT(3A) receptor: EC(50) 3 micro M, Hill coefficient 1.8; 5-HT(3AB) receptor: EC(50) 25 micro M, Hill coefficient 0.9) and markedly altered receptor desensitization. Kinetic modeling suggested that homomeric receptors, but not heteromeric receptors, desensitize via an agonist-induced open-channel block. Furthermore, heteromeric 5-HT(3AB) receptor assemblies recovered much faster from desensitization than homomeric 5-HT(3A) receptor assemblies. Unexpectedly, the specific 5-HT(3) receptor agonist mCPBG induced an open-channel block at both homomeric and heteromeric receptors. Because receptor desensitization and resensitization massively affect amplitude, duration, and frequency of synaptic signaling, these findings are evidence in favor of a pivotal role of subunit composition of 5-HT(3) receptors in serotonergic transmission.     

The extracellular subunit interface of the 5-HT3 receptors: a computational alanine scanning mutagenesis study

The functional serotonin 5-HT type-3 (5-HT(3)) receptor, the target of many neuroactive drugs, is known to be a pseudo-symmetric pentamer made either of five identical subunits A (homomeric 5-HT(3A)-R) or of subunits A and B (heteromeric 5-HT(3A/B)-R) in a still debated arrangement. The serotonin binding site is located in the extracellular region, at the interface between two monomers, called the principal and the complementary subunits. The results of molecular dynamics simulations and computational alanine scanning mutagenesis studies applied here to the homomeric human 5-HT(3A)-R disclose an aromatic "hot" cluster in the centre of the interface formed by residues W178 (principal subunit), Y68, Y83, W85 and Y148 (complementary subunit). Moreover, investigation of the coupling of agonist/antagonist binding to channel activation/inactivation points out the presence of two putative functional pathways at the subunit interface: W116-H180-L179-W178-E124-F125 (principal subunit) and Y136-Y138-Y148-W85-(P150) (complementary subunit), where W178 and Y148 appear to be critical residues for the binding/activation mechanism. Finally, direct comparison of the main features shown by the AA interface in the human 5-HT(3A)-R with those of the BB interface in the homopentameric human 5-HT(3B)-R provides interesting clues about the possible reasons that cause the 5-HT(3B)-R not to be functional.     

Agonists and Antagonists Bind to an A-A Interface in the Heteromeric 5-HT3AB Receptor

The 5-HT₃ receptor is a member of the Cys-loop family of transmitter receptors. It can function as a homopentamer (5-HT3A-only subunits) or as a heteropentamer. The 5-HT₃AB receptor is the best characterized heteropentamer. This receptor differs from a homopentamer in its kinetics, voltage dependence, and single-channel conductance, but its pharmacology is similar. To understand the contribution of the 5-HT3B subunit to the binding site, we created homology models of 5-HT₃AB receptors and docked 5-HT and granisetron into AB, BA, and BB interfaces. To test whether ligands bind in any or all of these interfaces, we mutated amino acids that are important for agonist and antagonist binding in the 5-HT3A subunit to their corresponding residues in the 5-HT3B subunit and vice versa. Changes in [³H]granisetron binding affinity (K_d) and 5-HT EC₅₀ were determined using receptors expressed in HEK-293 cells and Xenopus oocytes, respectively. For all A-to-B mutant receptors, except T181N, antagonist binding was altered or eliminated. Functional studies revealed that either the receptors were nonfunctional or the EC₅₀ values were increased. In B-to-A mutant receptors there were no changes in K_d, although EC₅₀ values and Hill slopes, except for N170T mutant receptors, were similar to those for 5-HT₃A receptors. Thus, the experimental data do not support a contribution of the 5-HT3B subunit to the binding pocket, and we conclude that both 5-HT and granisetron bind to an AA binding site in the heteromeric 5-HT₃AB receptor.     

Heterogeneity amongst 5-HT₃ receptor subunits: is this significant?

The serotonin 3 (5-HT?) receptor is a ligand gated ion channel unlike the other 5-HT receptors which are G protein coupled receptors. The functional 5-HT? receptor forms a pentamer of five symmetrically arranged subunits surrounding a central pore. The 5-HT(3A) subunit was first identified at a molecular level and can form functional homomers or heteromers with the 5-HT(3B) subunit. Recently, three new 5-HT? subunits have been discovered and these can also form functional heteromers with the 5-HT(3A) subunit. In addition, splice variants of the 5-HT? subunits have also been reported. These findings have markedly increased the complexity of the 5-HT? receptor and may form part of the explanation of unresolved differences between studies investigating 5-HT? receptor function in cell lines compared with native tissues. In this review we discuss the properties of the different subunits and their distribution to determine if they contribute to functional changes in the 5-HT? receptor. Several recent pharmacogenomic studies have revealed single nucleotide polymorphisms (SNPs) and other variations in the different 5-HT? receptor subunits that are associated with various clinical conditions. We discuss the implications of these findings with respect to drug design and tailored pharmacogenomic therapies.     

Apparent homomeric NR1 currents observed in Xenopus oocytes are caused by an endogenous NR2 subunit

Functional N-methyl-d-aspartate receptors NMDARs are thought to be heteromeric receptor complexes consisting of NR1 and NR2 subunits. However, recombinant NR1 subunits expressed in Xenopus oocytes assemble functional ion channels even without exogenous NR2 subunits and with a different pharmacology, suggesting a homomeric subunit stoichiometry. To explain this phenomenon, we screened oocytes for Xenopus NR2 subunits and found all four subunit-encoding mRNAs (XenNR2A-XenNR2D) to be present endogenously, with those encoding the XenNR2B subunit being particularly abundant. We cloned the full-length XenNR2B cDNA and co-expressed it with NR1 in oocytes. A detailed electrophysiological characterization revealed that the pharmacology of NR1/XenNR2B was identical with that of the presumed homomeric NMDARs expressed from NR1 subunits. By contrast, heteromeric receptors containing the rat NR2B subunit showed significant pharmacological differences compared with NR1/XenNR2B receptors. These results demonstrate that recombinant NR1 subunits expressed in Xenopus oocytes interact with an endogenously expressed NR2B subunit and form hybrid heteromeric NMDARs. These findings confirm the current views that NMDARs are obligatory heteromeric complexes and that functional homomeric NMDARs do not exist.     

Aequorin luminescence-based assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization

The classical electrophysiological method to measure the function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT(3)) receptor, a cation-permeable ligand-gated ion channel, is time-consuming and not suitable for high-throughput screening. Therefore, we have optimized the conditions for a sensitive assay suitable to measure 5-HT(3) receptor responses in cell suspension based on aequorin bioluminescence caused by Ca(2+) influx. The assay, carried out in 96-well plates, was applied for the pharmacological characterization of 5-HT(3) receptors on human embryonic kidney (HEK) 293 cells transiently coexpressing apoaequorin and either the human homopentameric 5-HT(3A) receptor or the human heteromeric 5-HT(3A/B) receptor in the same subset of cells. Thus, the luminescence signal originates exclusively from transfected cells, leading to a high signal/noise ratio, a major advantage compared with fluorescence techniques using Ca(2+)-sensitive dyes. The potencies of two 5-HT(3A) receptor agonists and two antagonists as well as the potency and efficacy of serotonin at the heteromeric 5-HT(3A/B) receptor were comparable to those reported using other functional methods. In conclusion, the aequorin assay described here provides a convenient and highly sensitive method for functional characterization of 5-HT(3) receptors that is well suited for high-throughput screening.     

Cell surface expression of 5-hydroxytryptamine type 3 receptors is controlled by an endoplasmic reticulum retention signal

Two subunits of the 5-hydroxytryptamine type 3 (5-HT3) have been identified (5-HT3A and 5-HT3B) that assemble into homomeric (5-HT3A) and heteromeric (5-HT3A+5-HT3B) complexes. Unassembled 5-HT3B subunits are efficiently retained within the cell. In this study, we address the mechanism controlling the release of 5-HT3B from the endoplasmic reticulum (ER). An analysis of chimeric 5-HT3A receptor(R).5-HT3BR constructs suggests the presence of elements downstream of the first transmembrane domain of 5-HT3B subunits that inhibit cell surface expression. To investigate this possibility, truncated 5-HT3B subunits were constructed and assessed for their ability to access the cell surface in COS-7 and ts201 cells. Using this approach, we have identified the presence of an ER retention signal located within the first cytoplasmic loop between transmembrane domains I and II of 5-HT3B. Transplantation of this signal (CRAR) into the homologous region of 5-HT3A results in the ER retention of this subunit until rescued by co-assembly with wild-type 5-HT3A. The mutation of this ER retention signal in 5-HT3B (5-HT3BSGER) does not lead to cell surface expression, suggesting the presence of other signals or mechanisms to control the surface expression of 5-HT3BRs. The generation of truncated 5-HT3BSGER constructs confirmed that the CRAR signal does play an important role in the ER retention of 5-HT3B.     

Agonist binding to the GluK5 subunit is sufficient for functional surface expression of heteromeric GluK2/GluK5 kainate receptors

Trafficking of ionotropic glutamate receptors to the plasma membrane commonly requires occupation of the agonist binding sites. This quality control check does not typically involve receptor activation, as binding by competitive antagonists or to non-functional channels may also permit surface expression. The tetrameric kainate receptors can be assembled from five different subunits (GluK1–GluK5). While the “low-affinity” GluK1-3 subunits are able to produce functional homomeric receptors, the “high-affinity” GluK4 and GluK5 subunits require co-assembly with GluK1, 2, or 3 for surface expression. These two different types of subunits have distinct functional roles in the receptor. Therefore, we examined the relative importance of occupancy of the agonist site of the GluK2 or GluK5 subunit for surface expression of heteromeric receptors. We created subunits with a mutation within the S2 ligand-binding domain which decreased agonist affinity. Mutations at this site reduced functional surface expression of homomeric GluK2 receptors, but surface expression of these receptors could be increased with either a competitive antagonist or co-assembly with wild-type GluK5. In contrast, mutations in the GluK5 subunit reduced the production of functional heteromeric receptors at the membrane, and could not be rescued with either an antagonist or wild-type GluK2. These findings indicate that ligand binding to only the GluK5 subunit is both necessary and sufficient to allow trafficking of recombinant GluK2/K5 heteromers to the cell membrane, but that occupancy of the GluK2 site alone is not. Our results suggest a distinct role for the GluK5 subunit in regulating surface expression of heteromeric kainate receptors.     

Cloning and expression of the epsilon 4 subunit of the NMDA receptor channel.

The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon 4, has been revealed by cloning and sequencing the cDNA. The epsilon 4 subunit shares high amino acid sequence identity with the epsilon 1, epsilon 2 and epsilon 3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon 4 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon 4/zeta 1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon 4 subunit is thus distinct in functional properties from the epsilon 1, epsilon 2 and epsilon 3 subunits, and contributes further diversity of the NMDA receptor channel.     

Differential Expression of the 5-HT3A and 5-HT3B Receptor in Differentiated NG108-15 Cells

Previous work from this laboratory has shown that the serotonin (5-HT) induced response is significantly augmented in differentiated NG108-15 (NG) cells treated with dibutyryl cAMP (Bt(2)cAMP) due to qualitative and quantitative changes in the expression of the 5-HT(3) receptor as demonstrated by specific [(3)H] LY-278584 (a selective 5HT(3) receptor antagonist) binding. In this study, we investigated whether there is any change in the relative expression of the 5-HT(3A) and 5-HT(3B) subunits in NG cells differentiated following Bt(2)cAMP treatment cells. The major findings of this study were that the relative amount of 5-HT(3B) subunit mRNA in Bt(2)cAMP-treated NG cells 5 days following Bt(2)cAMP-treatment was greater than that in the untreated cells. In contrast, the relative expression of the 5-HT(3B) subunit protein in the Bt(2)cAMP-treated NG cells was much less than in the untreated cells, but the relative expression of the 5-HT(3A) subunit in the Bt(2)cAMP-treated NG cells was similar to the untreated cells. Therefore, no relationship between mRNA and protein expression for 5-HT(3A) and 5-HT(3B) subunits in Bt(2)cAMP treated and untreated NG cells were observed. It was also found that fluorescent intensity for the 5-HT(3B) subunit in the cell body of the Bt(2)cAMP treated and untreated NG cells gradually decreased from the day 1-5 after Bt(2)cAMP treatment. However, in specific areas such as the varicosity and nerve endings of the Bt(2)cAMP treated cells, staining intensity for the 5-HT(3B) subunits was stronger than in the untreated cells at the all time points, peaking at day 5 post-treatment. These results suggest that the augmented response induced by 5-HT acting via 5-HT(3) receptors in differentiated NG cells may be due to changes in the relative amount of the 5-HT(3B) subunit, particularly the ratio and distribution of the 5-HT(3A) to (3B) subunits.     

Complete structure of the mouse mast cell receptor for IgE (Fc epsilon RI) and surface expression of chimeric receptors (rat-mouse-human) on transfected cells

The high affinity receptor for IgE (Fc epsilon RI) found on mast cells and basophils is a tetrameric complex of a single alpha subunit, a single beta subunit, and two identical gamma subunits. The genes for the three subunits of mouse Fc epsilon RI have now been cloned from the mast cell line, PT18. When compared at the DNA level, the rat and mouse subunits are similarly conserved. However, at the protein level the homology between mouse and rat alpha is surprisingly low (71% identities) especially in the cytoplasmic regions (57% identities) which are of different length (25 and 20 residues, respectively). By contrast the beta and gamma are homogeneously conserved between mouse and rat (83 and 93% identities, respectively). The consensus amino acid sequence of the alpha subunit derived from three species (rat, mouse, and human) shows that the cytoplasmic tail diverges to the same extent as the leader peptide. Conversely, the transmembrane domain of the alpha is highly conserved and contains 10 consecutive residues that are identical. Comparisons between mouse Fc epsilon RI and other mouse proteins reveal regions of high homology between the alpha subunit and Fc gamma RIIa and between the gamma subunit and the zeta chain of the T cell receptor. Cells transfected with the alpha gene express the alpha subunit on their surface very inefficiently. Efficient expression is only achieved after co-transfection of the three rodent genes or of the human alpha gene together with the rodent gamma without apparent need for beta. The subunits are completely interchangeable upon transfection so that various chimeric mouse-rat-human receptors can be expressed.     

The identification of N-glycosylated residues of the human 5-HT3B receptor subunit: importance for cell membrane expression

The 5-hydroxytryptamine 3 (5-HT(3)) receptor is a pentameric ligand-gated ion channel with potential molecular isoforms arising from different subunit combinations and/or different post-translational modifications of the individual subunits. Since N-glycosylation of the 5-HT3A subunit impacts cell surface trafficking, the presence of N-glycosylation of the human (h) 5-HT3B subunit and the influence upon cell membrane expression was investigated. Following transient expression of the h5-HT3B subunit by human embryonic kidney cells (HEK293 cells) stably expressing the h5-HT3A subunit, the N-glycosylation inhibitor tunicamycin reduced the size of the predominant h5-HT3B-immunoreactive protein (～ 55 kDa reduced to ～ 40 kDa). Disruption of each consensus N-glycosylation sequences in the h5-HT3B subunit (N31S, N75S, N117S, N147S and N182S) resulted in a reduced molecular weight (by ～ 2-4 kDa) of each mutant when expressed by HEK293 cells stably expressing the h5-HT3A subunit. Immunocytochemical studies demonstrated that disruption of each of the N-glycosylation sequences (individually or combined) reduced the expression of the mutant h5-HT3B subunit protein in the cell membrane when co-expressed with the h5-HT3A subunit. The present study has identified utilised N-glycosylation sites of the h5-HT3B subunit and demonstrated that they promote subunit expression in the cell membrane; a prerequisite for 5-HT(3) receptor function.     

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon

Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine samples tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.     

Molecular cloning and functional characterization of a human 5-HT1B serotonin receptor: a homologue of the rat 5-HT1B receptor with 5-HT1D-like pharmacological specificity.

We describe a genomic clone encoding the human 5-HT1B receptor. This apparently intronless gene encodes a 390 amino acid polypeptide homologous to the rat 5-HT1B serotonin receptor, with which it shares 93% amino acid sequence identity. Remarkably, [3H]5-hydroxytryptamine binding studies with transfected HeLa cells show that the human 5-HT1B receptor has a pharmacological profile that is markedly different from that of the corresponding rat receptor. Instead, human 5-HT1B drug specificity is highly similar to that of the human 5-HT1D receptor, with which it shares 59% amino acid sequence identity. The human 5-HT1B receptor, like the 5-HT1D receptor, can couple to Gi proteins. The presence of the threonine355 in the human receptor rather than an asparagine, as found in the corresponding rat gene product, may explain much of the marked pharmacological difference between the human and rat 5-HT1B receptors.     

Nucleotide Sequence, Genomic Organization, and Chromosomal Localization of Genes Encoding the Human NMDA Receptor Subunits NR3A and NR3B

The N-methyl-D-aspartate (NMDA) receptors are glutamate-regulated ion channels that are critically involved in important physiological and pathological functions of the mammalian central nervous system. We have identified and characterized the gene encoding the human NMDA receptor subunit NR3A (GRIN3A), as well as the gene (GRIN3B) encoding an entirely novel subunit that we named NR3B, as it is most closely related to NR3A (57.4% identity). GRIN3A localizes to chromosome 9q34, in the region 13-34, and consists of nine coding exons. The deduced protein contains 1115 amino acids and shows 92.7% identity to rat NR3A. GRIN3B localizes to chromosome 19p13.3 and contains, as does the mouse NR3B gene (Grin3b), eight coding exons. The deduced proteins of human and mouse NR3B contain 901 and 900 amino acid residues, respectively (81.6% identity). In situ hybridization shows a widespread distribution of Grin3b mRNA in the brain of the adult rat.     

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.     

Subunit-specific redox modulation of nmda receptors expressed in xenopus oocytes

Abstract

We have examined the effects of oxidizing and reducing agents on a number of subtypes of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes. Oocytes were injected with cRNA for the ε1 subunit from mouse to express homomeric receptors or with ε1 in combination with either ε1, ε2, ε3 or ε4 subunits to express heteromeric receptors. All heteromeric combinations resulted in receptors that were affected by the redox reagents, dithiothreitol (DTT) and 5-5-dithio-bis-2-nitrobenzoic acid (DTNB). However, the effects on the small currents from homomeric receptors were quite variable. The ε1/ε3 combination showed a greater enhancement by DTT than any of the other combinations. All four receptors expressed showed both a component of persistent potentiation and a slowly reversible component. The reversible component was largest for ε1/ε3. Additional experiments were done with S-nitrosocysteine (SNOC), a nitric oxide donor that may affect NMDA receptors by oxidation. SNOC had transient effects on the four heteromeric subunit combinations. The different sensitivities of particular subunit combinations may have pharmacological and clinical significance.     

Subunit assembly of N-methyl-d-aspartate receptors analyzed by fluorescence resonance energy transfer

N-methyl-d-aspartate (NMDA) receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. Here we used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, our data indicated that the conformation of NR1 subunit homodimers was affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function.