Physicochemical Properties of Serotonin 5-HT3Binding Sites Solubilized from Membranes of NG 108-15 Neuroblastoma-Glioma Cells

Abstract

Specific binding sites with pharmacological properties typical of serotonin 5‐HT₃ receptors were identified in membranes of the murine hybridoma cell line NG 108‐15, using [³H]zacopride as a ligand. Optimal solubilization of these sites (yield. 50%) could be achieved using the detergent 3–[3–(cholamidopropyl)dimethylammonio]‐1‐propane sul‐fonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris‐HCl, pH 7.4. Specific [³H]zacopride binding to soluble sites in the 100,000‐g CHAPS extract was saturable and showed characteristics (B_max= 425 ± 81 fmol/mg of protein; K_D= 0.19 ± 0.02 nM) closely related to those of membrane‐bound sites (B_max= 932 ± 183 fmol/mg of protein; K_D= 0.60 ± 0.03 nM). Determination of association (K₊₁=0.17 nM min⁻¹) and dissociation (k_‐1= 0.02 min⁻¹) rate constants for the soluble sites gave a K_D value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC₅₀) of 10 different drugs, the pharmacological profile of [³H]zacopride specific binding sites was essentially the same (r= 0.99) in the CHAPS‐soluble extract and in cell membranes, although some increase in the affinity for 5‐HT₃ antagonists (zacopride, ICS 205–930, and MDL 72222) and decrease in the affinity for 5‐HT₃ agonists (2‐methyl‐5‐hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS‐soluble extract gave a Svedberg coefficient of 12S for the material with [³H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S‐400 and wheat germ agglutinin‐agarose columns indicated marked enrichment (by 2.5– and 10‐fold, respectively) in [³H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5‐HT₃ receptors. Two soluble materials with apparent molecular masses of ∼600 and ∼36 kDa were found to bind [³H]zacopride specifically in the Sephacryl S‐400 eluate. Interestingly, molecular mass determination by radiation inactivation of [³H]zacopride binding sites in frozen NG 108–15 cells gave a value of ∼35 kDa.