The neuronal and epithelial human high affinity glutamate transporter. Insights into structure and mechanism of transport.
High affinity transport of glutamate across plasma membranes of brain neurons and epithelial is mediated by a Na(+)- and K(+)-coupled electrogenic transporter. Here we report the primary structure and functional characterization of the human high affinity glutamate transporter (HEAAC1). A unique characteristic of HEAAC1-mediated transport is that the affinity for glutamate and the maximal transport rate are strongly dependent on membrane potential. Our data provide new insights into individual steps of high affinity glutamate transport and show that the transport mechanism is distinct from that of the gamma-aminobutyric acid transporter GAT-1 and the Na+/glucose transporter SGLT1. Under voltage clamp condition, HEAAC1 mediated large substrate-evoked inward currents (up to 1 microA). The substrate specificity, stereospecificity, the Km value (30 +/- 3 microM at -60 mV) of the L-glutamate-evoked current, and Northern analysis all agree with previously reported characteristics of high affinity glutamate transport in brain. In contrast to SGLT1 and GAT-1, voltage jump studies of HEAAC1 yielded only minor relaxation currents. Classic inhibitors of brain glutamate uptake such as DL-threo-beta-hydroxyaspartate, L-trans-pyrrolidine 2,4,-dicarboxylic acid (PDC), and dihydrokainate were found to be either transport substrates or to have no significant effect on glutamate transport. We also found that the maximal transport rate for PDC was markedly reduced compared to that for L-glutamate. We propose that PDC most likely reduces the turnover rate of the transporter. A search of the sequence data bases revealed weak homology of HEAAC1 to the H(+)-coupled vesicular monoamine transporter, suggesting an evolutionary link between plasma membrane and vesicular transporters.
Kanai
Y
Y
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Stelzner
M
M
Nussberger
S
S
Khawaja
S
S
Hebert
S C
SC
Smith
C P
CP
Hediger
M A
MA
D000328:Adult; D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001692:Biological Transport; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004848:Epithelium / Q000378:metabolism; D018691:Excitatory Amino Acid Antagonists; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D009435:Synaptic Transmission; D014981:Xenopus
D027322:Amino Acid Transport System X-AG; D018076:DNA, Complementary; D018691:Excitatory Amino Acid Antagonists; D005971:Glutamates; D006023:Glycoproteins; D018698:Glutamic Acid