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 (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.
McKernan
R M
RM
Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, U.K.
Biggs
C S
CS
Gillard
N
N
Quirk
K
K
Ragan
C I
CI
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D007211:Indoles / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011985:Receptors, Serotonin / Q000145:classification* / Q000378:metabolism; D012702:Serotonin Antagonists / Q000378:metabolism; D014316:Tritium; D000077526:Tropisetron; D014407:Tumor Cells, Cultured / Q000648:ultrastructure
D007211:Indoles; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D014316:Tritium; D000077526:Tropisetron