An automatic electrophysiological assay for the neuronal glutamate transporter mEAAC1.

A rapid and robust electrophysiological assay based on solid supported membranes (SSM) for the murine neuronal glutamate transporter mEAAC1 is presented. Measurements at different concentrations revealed the EAAC1 specific affinities for l-glutamate (K(m)=24microM), l-aspartate (K(m)=5microM) and Na(+) (K(m)=33mM) and an inhibition constant K(i) for dl-threo-beta-benzyloxyaspartic acid (TBOA) of 1microM. Inhibition by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) was not purely competitive with an IC(50) of 13microM. Experiments using SCN(-) concentration jumps yielded large transient currents in the presence of l-glutamate showing the characteristics of the glutamate-gated anion conductance of EAAC1. Thus, SSM-based electrophysiology allows the analysis of all relevant transport modes of the glutamate transporter on the same sample. K(+) and Na(+) gradients could be applied to the transporter. Experiments in the presence and absence of Na(+) and K(+) gradients demonstrated that the protein is still able to produce a charge translocation when no internal K(+) is present. In this case, the signal amplitude is smaller and a lower apparent affinity for l-glutamate of 144microM is found. Finally the assay was adapted to a commercial fully automatic system for SSM-based electrophysiology and was validated by determining the substrate affinities and inhibition constants as for the laboratory setup. The combination of automatic function and its ability to monitor all transport modes of EAAC1 make this system an universal tool for industrial drug discovery.

Flow injection amperometric detection of OP nerve agents based on an organophosphorus-hydrolase biosensor detector.

A flow-injection system with an organophosphorus-hydrolase (OPH)-biosensor detector has been developed and characterized for the rapid detection of organophosphorus (OP) nerve agents. The enzyme was immobilized onto a thin-film gold detector through a cystamine-glutaraldehyde coupling. Factors influencing the performance were optimized. The resulting flow system offered a fast, sensitive, selective, and stable response. The peak current increased linearly with the concentration of paraoxon and methyl parathion over the 1-10 microM range (sensitivity, 2.29 and 1.04 nA/microM, respectively). The OPH-biosensor flow injection systems offered low detection limits (e.g. 0.1 microM paraoxon), along with a good precision (R.S.D. of 3.6% for 20 successive injections of a 1.0 microM paraoxon solution). The OPH-biosensor flow detector offers great promise for rapid field screening of OP pesticides and nerve agents.