Substrate translocation kinetics of excitatory amino acid carrier 1 probed with laser-pulse photolysis of a new photolabile precursor of D-aspartic acid.
Here we report the synthesis and photochemical and biological characterization of a new photolabile precursor of D-aspartic acid, alpha-carboxynitrobenzyl-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-nitro intermediate revealed that it photolyzes with a quantum yield of 0. 19 at pH 7.0. The major component of the aci-nitro intermediate (77% of the total absorbance) decays with a time constant of 26 s. This decay is slowed by only a factor of 2 when increasing the pH to 10. A minor component (21%) decays with a time constant of 410 s and is pH insensitive. The compound was tested with respect to its biological activity with the glutamate transporter EAAC1 expressed in HEK293 cells. Whole-cell current recordings from these cells in the presence and absence of alpha-CNB-caged D-aspartate demonstrated that the compound neither activates nor inhibits EAAC1. Upon photolysis, D-aspartate-mediated whole-cell currents were generated. In contrast to laser-pulse photolysis experiments with alpha-CNB-caged L-glutamate, only 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, becomes rate-limiting when D-aspartate is translocated. The results demonstrate that the new caged D-aspartate derivative is a useful tool for the investigation of the molecular mechanism of glutamate transporters and probably other aspartate translocating systems using rapid chemical kinetic techniques.
Grewer
C
C
Max-Planck-Institut für Biophysik, Kennedyallee 70, D-60596 Frankfurt, Germany. grewer@mpibp-frankfurt.mpg.de
Madani Mobarekeh
S A
SA
Watzke
N
N
Rauen
T
T
Schaper
K
K
D027322:Amino Acid Transport System X-AG; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism* / Q000502:physiology; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D005971:Glutamates / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007834:Lasers; D008954:Models, Biological; D008956:Models, Chemical; D018408:Patch-Clamp Techniques; D010648:Phenylacetates / Q000138:chemical synthesis / Q000378:metabolism*; D010782:Photolysis; D011789:Quantum Theory; D027981:Symporters
C102508:(carboxy-2-nitrobenzyl)glutamic acid; C419878:4-O-(alpha-carboxy-2-nitrobenzyl)aspartic acid; D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D005971:Glutamates; D010648:Phenylacetates; C493400:SLC1A1 protein, human; D027981:Symporters; D001224:Aspartic Acid