Solubilization and characterization of the serotonin 5-HT1c site from pig choroid plexus.
A novel type of serotonergic binding site, termed the 5-HT1c site, was recently identified on choroid plexus epithelial cells. In the present study, we describe the solubilization of pig choroid plexus 5-HT1c sites by the zwitterionic detergent 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS). High affinity labeling of both membrane-bound and solubilized 5-HT1c sites was obtained by use of the serotonergic radioligand N1-methyl-2-[125I]lysergic acid diethylamide (125I-MIL). In solubilized preparations, 125I-MIL exhibited a dissociation constant (Kd) of 2.0 nM and a 60-75% ratio of specific to total binding. Approximately 45% of the membrane binding sites were solubilized by CHAPS as measured by a postlabeling, polyethylene glycol precipitation method. The CHAPS-solubilized 5-HT1c site fulfilled the accepted criteria for receptor solubilization. The affinities of a series of serotonergic antagonists for the 5-HT1c site showed little or no change upon solubilization of the site. Serotonin, however, showed a 20-fold increase in affinity for the 5-HT1c site after solubilization, which may indicate the loss of a modulatory component during detergent treatment. Both gel filtration and equilibrium sedimentation experiments indicate that the CHAPS-solubilized site is a large molecular weight complex. These studies demonstrate that the pig choroid plexus 5-HT1c site can be solubilized with retention of its binding activity in a form suitable for further purification and characterization.
D000818:Animals; D001667:Binding, Competitive; D002793:Cholic Acids / Q000494:pharmacology; D002831:Choroid Plexus / Q000032:analysis*; D007457:Iodine Radioisotopes; D008238:Lysergic Acid Diethylamide / Q000031:analogs & derivatives / Q000378:metabolism; D008970:Molecular Weight; D011985:Receptors, Serotonin / Q000302:isolation & purification*; D012701:Serotonin / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000494:pharmacology; D012995:Solubility; D013552:Swine
D002793:Cholic Acids; D007457:Iodine Radioisotopes; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D012701:Serotonin; C048042:N(1)-methyl-2-lysergic acid diethylamide; D008238:Lysergic Acid Diethylamide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate