Detection of nicotinic receptor ligands with a light addressable potentiometric sensor.
The nicotinic acetylcholine receptor, purified from Torpedo electric organ, was coupled to a light addressable potentiometric sensor (LAPS) to form a LAPS-receptor biosensor. Receptor-ligand complexes containing biotin and urease were captured on a biotinylated nitrocellulose membrane via a streptavidin bridge and detected with a silicon-based sensor. Competition between biotinylated alpha-bungarotoxin and nonbiotinylated ligands formed the basis of this assay. This biosensor detected both agonists (acetylcholine, carbamylcholine, succinylcholine, suberyldicholine, and nicotine) and competitive antagonists (d-tubocurarine, alpha-bungarotoxin, and alpha-Naja toxin) of the receptor with affinities comparable to those obtained using radioactive ligand binding assays. Consistent with agonist-induced desensitization of the receptor, the LAPS-receptor biosensor reported a time-dependent increase in affinity for the agonist carbamylcholine as expected, but not for the antagonists.
Rogers
K R
KR
Department of Pharmacology and Experimental Therapeutics, University of Maryland, School of Medicine, Baltimore 21201.
Fernando
J C
JC
Thompson
R G
RG
Valdes
J J
JJ
Eldefrawi
M E
ME
D000818:Animals; D015374:Biosensing Techniques; D001710:Biotin; D002038:Bungarotoxins / Q000032:analysis / Q000378:metabolism; D002794:Choline / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D003039:Cobra Neurotoxin Proteins / Q000032:analysis / Q000378:metabolism; D009538:Nicotine / Q000032:analysis / Q000378:metabolism; D018733:Nicotinic Antagonists; D010277:Parasympathomimetics / Q000032:analysis / Q000378:metabolism; D011199:Potentiometry; D011978:Receptors, Nicotinic / Q000378:metabolism*; D014101:Torpedo; D014403:Tubocurarine / Q000032:analysis / Q000378:metabolism; D014510:Urease
D002038:Bungarotoxins; D003039:Cobra Neurotoxin Proteins; D018733:Nicotinic Antagonists; D010277:Parasympathomimetics; D011978:Receptors, Nicotinic; D009538:Nicotine; D001710:Biotin; D014510:Urease; D002794:Choline; D014403:Tubocurarine