Substrate translocation kinetics of excitatory amino acid carrier 1 probedwith laser-pulse photolysis of a new photolabile precursor of D-aspartic acid

Here we report the synthesis and photochemical and biological characterizat ion of a new photolabile precursor of D-aspartic acid, alpha -carboxynitrob enzyl-caged D-aspartate (alpha -CNB-caged D-aspartate), and its application for studying the molecular mechanism of the neuronal excitatory amino acid carrier 1 (EAAC1). Investigation of the photochemical properties of alpha -CNB-caged D-aspartate by transient absorption spectroscopy of the aci-nitr o intermediate revealed that it photolyzes with a quantum yield of 0.19 at pH 7.0. The major component of the nci-nitro intermediate (77% of the total absorbance) decays with a time constant of 26 mus. This decay is slowed by only a factor of 2 when increasing the pH to 10, A minor component (21%) d ecays with a time constant of 410 mus and is pH insensitive, The compound w as tested with respect to its biological activity with the glutamate transp orter EAAC1 expressed in HEK293 cells. Whole-cell current recordings from t hese cells in the presence and absence of alpha -CNB-caged D-aspartate demo nstrated that the compound neither activates nor inhibits EAAC1. Upon photo lysis, D-aspartate-mediated whole-cell currents were generated. In contrast to laser-pulse photolysis experiments with alpha -CNB-caged L-glutamate, o nly a minor and much slower transient current component was observed, These results indicate that the substrate translocation step, which is not rate- limiting for the overall turnover of the transporter with L-glutamate, beco mes rate-limiting when D-aspartate is translocated, The results demonstrate that the new caged D-aspartate derivative is a useful tool for the investi gation of the molecular mechanism of glutamate transporters and probably ot her aspartate translocating systems using rapid chemical kinetic techniques .