Castagna M, Shayakul C, Trotti D, Sacchi V F, Harvey W R, Hediger M A
Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA.
J Exp Biol. 1997 Jan;200(Pt 2):269-86. doi: 10.1242/jeb.200.2.269.
In mammalian cells, the uptake of amino acids is mediated by specialized, energy-dependent and passive transporters with overlapping substrate specificities. Most energy-dependent transporters are coupled either to the cotransport of Na+ or Cl- or to the countertransport of K+. Passive transporters are either facilitated transporters or channels. As a prelude to the molecular characterization of the different classes of transporters, we have isolated transporter cDNAs by expression-cloning with Xenopus laevis oocytes and we have characterized the cloned transporters functionally by uptake studies into oocytes using radiolabelled substrates and by electrophysiology to determine substrate-evoked currents. Mammalian transporters investigated include the dibasic and neutral amino acid transport protein D2/NBAT (system b0+) and the Na(+)- and K(+)-dependent neuronal and epithelial high-affinity glutamate transporter EAAC1 (system XAG-). A detailed characterization of these proteins has provided new information on transport characteristics and mechanisms for coupling to different inorganic ions. This work has furthermore advanced our understanding of the roles these transporters play in amino acid homeostasis and in various pathologies. For example, in the central nervous system, glutamate transporters are critically important in maintaining the extracellular glutamate concentration below neurotoxic levels, and defects of the human D2 gene have been shown to account for the formation of kidney stones in patients with cystinuria. Using similar approaches, we are investigating the molecular characteristics of K(+)-coupled amino acid transporters in the larval lepidopteran insect midgut. In the larval midgut, K+ is actively secreted into the lumen through the concerted action of an apical H+ V-ATPase and an apical K+/2H+ antiporter, thereby providing the driving force for absorption of amino acids. In vivo, the uptake occurs at extremely high pH (pH 10) and is driven by a large potential difference (approximately -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 coupling ion in vivo. The neutral amino acid transport system of larval midgut has been studied most extensively. Apart from its cation selectivity, it appears to be related to the amino acid transport system B previously characterized in vertebrate epithelial cells. Both systems have a broad substrate range which excludes 2-(methylamino)-isobutyric acid, 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 transporters. 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-fold stimulation of the Na(+)-dependent uptake of both leucine and phenylalanine (0.2 mmoll-1, pH 8). The molecular cloning of these transporters is now in progress. Knowledge of their unique molecular properties could be exploited in the future to control disease vectors and insect pests.
在哺乳动物细胞中，氨基酸的摄取由具有重叠底物特异性的特殊、能量依赖性和被动转运蛋白介导。大多数能量依赖性转运蛋白与Na⁺或Cl⁻的共转运或与K⁺的反向转运偶联。被动转运蛋白要么是易化转运蛋白，要么是通道蛋白。作为对不同类型转运蛋白进行分子表征的前奏，我们通过用非洲爪蟾卵母细胞进行表达克隆分离出了转运蛋白cDNA，并通过使用放射性标记底物对卵母细胞进行摄取研究以及通过电生理学来确定底物诱发电流，对克隆的转运蛋白进行了功能表征。所研究的哺乳动物转运蛋白包括二价和中性氨基酸转运蛋白D2/NBAT（b0⁺系统）以及Na⁺和K⁺依赖性神经元和上皮高亲和力谷氨酸转运蛋白EAAC1（XAG⁻系统）。对这些蛋白质的详细表征提供了关于转运特征以及与不同无机离子偶联机制的新信息。这项工作进一步增进了我们对这些转运蛋白在氨基酸稳态和各种病理过程中所起作用的理解。例如，在中枢神经系统中，谷氨酸转运蛋白对于将细胞外谷氨酸浓度维持在神经毒性水平以下至关重要，并且已证明人类D2基因的缺陷是胱氨酸尿症患者肾结石形成的原因。使用类似的方法，我们正在研究鳞翅目幼虫中肠中K⁺偶联氨基酸转运蛋白的分子特征。在幼虫中肠中，K⁺通过顶端H⁺ V-ATP酶和顶端K⁺/2H⁺反向转运体的协同作用被主动分泌到肠腔中，从而为氨基酸的吸收提供驱动力。在体内，摄取发生在极高的pH值（pH 10）下，并由较大的电位差（约 -200 mV）驱动。对刷状缘膜囊泡的研究表明，幼虫肠道中有几种具有不同氨基酸和阳离子特异性的转运系统。除了K⁺之外，Na⁺在较低pH值下也可以与氨基酸摄取偶联，但血淋巴中的Na⁺/K⁺比值非常低，以至于K⁺可能是体内主要的偶联离子。幼虫中肠的中性氨基酸转运系统研究最为广泛。除了其阳离子选择性外，它似乎与先前在脊椎动物上皮细胞中表征的氨基酸转运系统B有关。这两个系统都有广泛的底物范围，不包括2-（甲氨基）异丁酸，这是一种被哺乳动物Na⁺偶联系统A接受的氨基酸类似物。为了深入了解昆虫中的K⁺偶联机制以及氨基酸和K⁺稳态，目前的研究旨在描绘这些昆虫转运蛋白的分子特征。最近的数据表明，将从烟草天蛾中肠制备的mRNA注射到非洲爪蟾卵母细胞中会导致亮氨酸和苯丙氨酸（0.2 mmol·L⁻¹，pH 8）的Na⁺依赖性摄取增加1.5至2.5倍。这些转运蛋白的分子克隆正在进行中。对它们独特分子特性的了解未来可用于控制病媒和害虫。
