Solubilization and characterization of [3H] 5HT high affinity binding sites (5HT1 and 5HT3).
The solubilization of the serotonergic 5HT1 and 5HT3 sites was performed with digitonin and sodium cholate at 1% (final concentration). Two binding sites for [3H]5HT were observed on rat or horse brain synaptosomal membranes solubilized with these detergents. The corresponding dissociation constants (KD) were 1-3 nM and 13-30 nM respectively. These values were closely similar to those corresponding to 5HT1 and 5HT3 sites located in intact membranes. The solubilized sites specifically bound 5HT. The effect of GTP decreasing the binding to 5HT1 sites was lost on solubilized 5HT1 sites; it was recovered, however, by addition of phospholipids (asolectin 0,2%). The apparent molecular weights of these sites were determined using the gel filtration method (438 and 235 K daltons). The photoactivation of [3H]5HT by U.V. light was used to label 5HT1 and 5HT3 sites irreversively in membranes. The binding of [3H]5HT following U.V. irradiation was not dissociated after dilution; it was saturable and prevented by serotonergic drugs and not by adrenergic or dopaminergic antagonists. Moreover, GTP added prior to the irradiation reduced it markedly thus showing that 5HT1 sites were labelled. Electrophoretic and fluorographic analyses of the labelled material evidenced a 60 K dalton-band specifically labelled with [3H]5HT (5 or 20 nM). These results tend to indicate that the 60 K dalton-proteic band might represent a proteic subunit constituting part of 5HT1 and 5HT3 sites.
D000818:Animals; D001923:Brain Chemistry; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D008297:Male; D010743:Phospholipids / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D011985:Receptors, Serotonin / Q000302:isolation & purification*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013574:Synaptosomes / Q000032:analysis
D010743:Phospholipids; D011985:Receptors, Serotonin