A detergent-solubilized nicotinic acetylcholine receptor of Caenorhabditis elegans.
We have used a spin column assay to study the detergent-solubilized levamisole receptor, a nicotinic acetylcholine receptor of the nematode Caenorhabditis elegans. The receptor can be successfully solubilized in detergent solutions of Triton X-100, Lubrol PX, or sodium cholate. Centrifugal gel filtration assay using the tritiated ligand [3H]meta-aminolevamisole ([3H]MAL) provides a greater signal and a better signal-to-noise ratio for soluble levamisole receptor binding than either polyethylene glycol precipitation or DEAE filter assay with the same ligand. As for membrane-bound receptor, the detergent-solubilized levamisole receptor consists of more than one affinity state. Detergent solubilization appears to increase the affinity of all states for [3H]MAL (Kd for the highest affinity solubilized [3H]MAL binding state, 41 +/- 5 pM). Data is presented on the equilibrium binding and the association and dissociation reaction rates of the receptor. The similar relative efficacy with which various compounds inhibit specific [3H]MAL binding and deficiencies in solubilizable high affinity specific [3H]MAL binding in two receptor mutants show that the solubilized receptor is the same nicotinic acetylcholine receptor that is detected by assaying membrane-bound specific [3H]MAL binding. The detergent-solubilized levamisole receptor is stable at 0 degree to 4 degrees C, making receptor purification feasible.
Lewis
J A
JA
Division of Life Sciences, University of Texas, San Antonio 78249.
Berberich
S
S
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology; D007978:Levamisole / Q000494:pharmacology; D009154:Mutation; D011506:Proteins / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics / Q000302:isolation & purification*
D003902:Detergents; D011506:Proteins; D011978:Receptors, Nicotinic; D007978:Levamisole