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Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells.

ABSTRACT: The 5-HT3 hydroxytryptamine receptor from NCB 20 cells was solubilized and the molecular and hydrodynamic properties of the receptor were investigated. The receptor was identified by binding of the radioligand 3-NN'-[3H]dimethyl-8-azabicyclo[3.2.1]octanyl indol-3-yl carboxylate ester [( 3H]Q ICS 205-930) to NCB 20 membranes (Bmax = 1.19 +/- 0.31 pmol/mg of protein; Kd = 0.43 +/- 0.076 nM) and was optimally solubilized with 0.5% deoxycholate. [3H]Q ICS 205-930 labelled one population of sites in solution (Bmax = 1.11 +/- 0.4 pmol/mg of protein; Kd = 0.48 +/- 0.06 nM; n = 4). The characteristics of [3H]Q ICS 205-930 binding were essentially unchanged by solubilization, and competition for [3H]Q ICS 205-930 binding by a series of 5-HT3 agonists and antagonists was consistent with binding to a 5-HT3 receptor site and was similar to that observed for 5-HT3 receptors solubilized from rat brain [McKernan, Quirk, Jackson & Ragan (1990) J. Neurochem. 54, 924-930]. Some physical properties of the solubilized receptor were investigated. The molecular size (Stokes radius) of the [3H]Q ICS 205-930-binding site was measured by gel-exclusion chromatography in a buffer containing 0.2% Lubrol and 0.5 M-NaCl and was determined as 4.81 +/- 0.15 nm (mean +/- S.E.M.; n = 6). Sucrose-density-gradient centrifugation was also performed under the same detergent and salt conditions to determine the partial specific volume (v) of the detergent-receptor site complex. This was found to be 0.794 ml.g-1. Sucrose-density-gradient centrifugation was carried out in both 1H2O and 2H2O to allow correction for detergent binding to the receptor. The Mr of the 5-HT3 receptor under these conditions was calculated as 249,000 +/- 18,000 (n = 3). The size and physical properties of the 5-HT3 receptor are similar to those observed for members of the family of ligand-gated ion channels.

SUBMITTER: McKernan RM

PROVIDER: S-EPMC1131632 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

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Project description:5-HT3A receptors select among permeant ions based on size and charge. The membrane-associated (MA) helix lines the portals into the channel's cytoplasmic vestibule in the 4-Å resolution structure of the homologous acetylcholine receptor. 5-HT3A MA helix residues are important determinants of single-channel conductance. It is unknown whether the portals into the cytoplasmic vestibule also determine the size selectivity of permeant ions. We sought to determine whether the portals form the size selectivity filter. Recently, we showed that channels functioned when the entire 5-HT3A M3-M4 loop was replaced by the heptapeptide M3-M4 loop sequence from GLIC, a bacterial Cys-loop neurotransmitter gated ion channel homologue from Gloebacter violaceus. We used homomeric 5-HT3A receptors with either a wild-type (WT) M3-M4 loop or the chimeric heptapeptide (5-HT3A-glvM3M4) loop, i.e., with or without portals. In Na(+)-containing buffer, the WT receptor current-voltage relationship was inwardly rectifying. In contrast, the 5-HT3A-glvM3M4 construct had a negative slope conductance region at voltages less than -80 mV. Glutamine substitution for the heptapeptide M3-M4 loop arginine eliminated the negative slope conductance region. We measured the relative permeabilities and conductances of a series of inorganic and organic cations ranging from 0.9 to 4.5 Å in radius (Li(+), Na(+), ammonium, methylammonium, ethanolammonium, 2-methylethanolammonium, dimethylammonium, diethanolammonium, tetramethylammonium, choline, tris [hydroxymethyl] aminomethane, and N-methyl-d-glucamine). Both constructs had measurable conductances with Li(+), ammonium, and methylammonium (size range of 0.9-1.8-Å radius). Many of the organic cations >2.4 Å acted as competitive antagonists complicating measurement of conductance ratios. Analysis of the permeability ratios by excluded volume theory indicates that the minimal pore radius for 5-HT3A and 5-HT3-glvM3M4 receptors was similar, ? 5 Å. We infer that the 5-HT3A size selectivity filter is located in the transmembrane channel and not in the portals into the cytoplasmic vestibule. Thus, the determinants of size selectivity and conductance are located in physically distinct regions of the channel protein.

Project description:The somatostatin receptors on rat pancreatic acinar membranes were demonstrated by use of a radioiodinated (125I-) analogue of somatostatin (SMS 204-090 or [Tyr3]SMS). The tracer was found to bind to the receptor with a Kd of 58 pM. The number of sites detected by this tracer (4.7 pmol/mg of protein) was 5-10 times higher than the number of sites previously found with other tracers. Since the level of non-specific binding was also very low as compared with findings with other tracers, 125I-204-090 might be of interest in future attempts to characterize the somatostatin receptors in the pancreas. The prelabelled membranes were solubilized with 1% CHAPS, and the solubilized complexes were found to adsorb to wheat-germ-agglutinin-coupled agarose, from which they could be eluted with 4 mM-triacetylchitotriose. The complexes within this eluate were shown by gel filtration on Trisacryl GF-2000 to have an Mr of about 400,000. The dissociation of the complexes was augmented both within the membranes as well as in the solubilized state by incubation with the GTP analogue guanosine 5'-[gamma-thio]triphosphate, indicating that the complexes are probably functionally linked to a guanine-nucleotide-binding regulatory protein. After SDS/slab-gel electrophoresis and autoradiography of cross-linked complexes after treatment with the heterobifunctional reagent N-5-azido-2-nitrobenzoyloxysuccinimide, a broad band occurred at approximately Mr 90,000 both in the membranes and in the eluates of complexes after lectin-adsorption chromatography. We conclude that the augmentation of the number of detectable sites for binding of somatostatin, as well as the very low level of non-specific binding obtained by the use of 125I-[Tyr3]SMS as tracer, has made it possible for us to demonstrate the solubilization of the somatostatin receptor in conjunction with its ligand and a GTP-binding regulatory protein, and we have succeeded in cross-linking 125I-[Tyr3]SMS to a binding subunit of Mr 90,000 in the membranes and in demonstrating the presence of the same labelled binding subunit within complexes solubilized and chromatographed on a lectin column before cross-linking.

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Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells.

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