The catalytic domain of human neuropathy target esterase mediates an organophosphate-sensitive ionic conductance across liposome membranes.
In humans and other vertebrates, reaction of organophosphates with a neuronal membrane protein, neuropathy target esterase (NTE), initiates events which culminate in axonal degeneration. The initiation process appears to involve modification of a property of the protein distinct from its esterase activity, subsequent to formation of a negatively charged adduct with the active site serine residue. Here, we show that membrane patches from liposomes containing NEST, a recombinant hydrophobic polypeptide comprising the esterase domain of human NTE, display a transmembrane ionic conductance with both stable and high-frequency flickering components. An asymmetric current-voltage relationship suggested that ion flow was favoured in one direction relative to the membrane and its associated NEST molecules. Flow of anions was slightly favoured compared with cations. The flickering current formed a much larger proportion of the overall conductance in patches containing wild-type NEST compared with the catalytically inactive S966A mutant form of the protein. The conductance across patches containing NEST, but not those with the S966A mutant, was significantly reduced after adding neuropathic organophosphates to the bathing medium. By contrast, non-neuropathic covalent inhibitors of the catalytic activity of NEST did not reduce NEST-mediated conductance. Future work may establish whether NTE itself mediates an organophosphate-sensitive ion flux across intracellular membranes within intact cells.
Forshaw
P J
PJ
MRC Toxicology Unit, University of Leicester, Leicester, UK.
Atkins
J
J
Ray
D E
DE
Glynn
P
P
D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology*; D002384:Catalysis; D004553:Electric Conductivity; D006801:Humans; D007477:Ions; D007531:Isoflurophate / Q000494:pharmacology*; D008081:Liposomes; D009943:Organophosphorus Compounds / Q000494:pharmacology*; D017434:Protein Structure, Tertiary / Q000502:physiology; D013997:Time Factors
D007477:Ions; D008081:Liposomes; D009943:Organophosphorus Compounds; D007531:Isoflurophate; C046357:phenylsaligenin cyclic phosphate; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase