MOLECULAR CHARACTERISTICS OF MAMMALIAN AND INSECT AMINO-ACID TRANSPORTERS - IMPLICATIONS FOR AMINO-ACID HOMEOSTASIS

In mammalian cells, the uptake of amino acids is mediated by specializ ed, energy-dependent and passive transporters with overlapping substra te specificities, Most energy-dependent transporters are coupled eithe r to the cotransport of Na+ or Cl- or to the countertransport of K+. P assive transporters are either facilitated transporters or channels. A s a prelude to the molecular characterization of the different classes of transporters, we have isolated transporter cDNAs by expression-clo ning with Xenopus laevis oocytes and we have characterized the cloned transporters functionally by uptake studies into oocytes using radiola belled substrates and by electrophysiology to determine substrate-evok ed currents, Mammalian transporters investigated include the dibasic a nd neutral amino acid transport protein D2/NBAT (system b(0+)) and the Na+- and K+-dependent neuronal and epithelial high-affinity glutamate transporter EAAC1 (system X(AG)(-)). A detailed characterization of t hese proteins has provided new information on transport characteristic s and mechanisms for coupling to different inorganic ions, This work h as furthermore advanced our understanding of the roles these transport ers play in amino acid homeostasis and in various pathologies, For exa mple, in the central nervous system, glutamate transporters are critic ally important in maintaining the extracellular glutamate concentratio n below neurotoxic levels, and defects of the human D2 gene have been shown to account for the formation of kidney stones in patients with c ystinuria. Using similar approaches, we are investigating the molecula r characteristics of K+-coupled amino acid transporters in the larval lepidopteran insect midgut. In the larval midgut, K+ is actively secre ted into the lumen through the concerted action of an apical H+ V-ATPa se and an apical K+/2H(+) antiporter, thereby providing the driving fo rce for absorption of amino acids, In vivo, the uptake occurs at extre mely high pH (pH10) and is driven by a large potential difference (app roximately -200 mV). Studies with brush-border membrane vesicles have shown that there are several transport systems in the larval intestine with distinct amino acid and cation specificities, In addition to K+, Na+ can also be coupled to amino acid uptake at lower pH, but the Na/K+ ratio of the hemolymph is so low that K+ is probably the major cou pling ion in vivo. The neutral amino acid transport system of larval m idgut has been studied most extensively, Apart from its cation selecti vity, it appears to be related to the amino acid transport system B pr eviously characterized in vertebrate epithelial cells, Both systems ha ve a broad substrate range which excludes 2-(methylamino)-isobutyric a cid, an amino acid analog accepted by the mammalian Na+-coupled system A. In order to gain insights into the K+-coupling mechanism and into amino acid and K+ homeostasis in insects, current studies are designed to delineate the molecular characteristics of these insect transporte rs. Recent data showed that injection of mRNA prepared from the midgut of Manduca sexta into Xenopus laevis oocytes induced a 1.5- to 2.5-fo ld stimulation of the Na+-dependent uptake of both leucine and phenyla lanine (0.2 mmol l(-1), pH 8). The molecular cloning of these transpor ters is now in progress, Knowledge of their unique molecular propertie s could be exploited in the future to control disease vectors and inse ct pests.