PHYSICOCHEMICAL PROPERTIES OF SEROTONIN 5-HT3 BINDING-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 (Bmax= 425 ± 81 fmol/mg of protein; KD= 0.19 ± 0.02 nM) closely related to those of membrane‐bound sites (Bmax= 932 ± 183 fmol/mg of protein; KD= 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 KD 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. Copyright © 1990, Wiley Blackwell. All rights reserved