MOLECULAR-SIZE OF THE 5-HT3 RECEPTOR SOLUBILIZED FROM NCB 20 CELLS

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 (B(max.) = 1.19 ± 0.31 pmol/mg of protein; K(d) = 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 (B(max.) = 1.11 ± 0.4 pmol/mg of protein; K(d) = 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 and 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 (v̄) 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 M(r) of the 5-HT3 receptor under these conditions was calculated as 249,00 ± 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.