Matthias A. Hediger

Cloning and characterization of a mammalian proton-coupled metal-ion transporter

Metal ions are essential cofactors for a wealth of biological processes, including oxidative phosphorylation, gene regulation and free-radical homeostasis. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, and certain neurodegenerative diseases. Despite their pivotal physiological roles, however, there is no molecular information on how metal ions are actively absorbed by mammalian cells. We have now identified a new metal-ion transporter in the rat, DCT1, which has an unusually broad substrate range that includes Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+ and Pb2+. DCT1 mediates active transport that is proton-coupled and depends on the cell membrane potential. It is a 561-amino-acid protein with 12 putative membrane-spanning domains and is ubiquitously expressed, most notably in the proximal duodenum. DCT1 is upregulated by dietary iron deficiency, and may represent a key mediator of intestinal iron absorption. DCT1 is a member of the ‘natural-resistance-associated macrophage protein’ (Nramp) family and thus its properties provide insight into how these proteins confer resistance to pathogens.

Knockout of Glutamate Transporters Reveals a Major Role for Astroglial Transport in Excitotoxicity and Clearance of Glutamate

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins

The Human Genome Organisation (HUGO) Nomenclature Committee Database provides a list of transporter families of the solute carrier (SLC) gene series (see http://www.gene.ucl.ac.uk/nomenclature/). Currently, it includes 43 families and 298 transporter genes. This special issue features mini-reviews on each of these SLC families written by the experts in each field. A WEB site has been established (http://www.pharmaconference.org/slctable.asp) that gives the latest updates for the SLC families and their members as well as relevant links to gene databases and reviews in the literature. A list of all currently known SLC families, a discussion of additional SLC families and family members as well as a brief summary of non-SLC transporter genes is included in this introduction.

A family of mammalian Na+-dependent L-ascorbic acid transporters.

Vitamin C (L-ascorbic acid) is essential for many enzymatic reactions, in which it serves to maintain prosthetic metal ions in their reduced forms (for example, Fe2+, Cu+),, and for scavenging free radicals in order to protect tissues from oxidative damage. The facilitative sugar transporters of the GLUT type can transport the oxidized form of the vitamin, dehydroascorbic acid, but these transporters are unlikely to allow significant physiological amounts of vitamin C to be taken up in the presence of normal glucose concentrations, because the vitamin is present in plasma essentially only in its reduced form. Here we describe the isolation of two L-ascorbic acid transporters, SVCT1 and SVCT2, from rat complementary DNA libraries, as the first step in investigating the importance of L-ascorbic acid transport in regulating the supply and metabolism of vitamin C. We find that SVCT1 and SVCT2 each mediate concentrative, high-affinity L-ascorbic acid transport that is stereospecific and is driven by the Na+ electrochemical gradient. Despite their close sequence homology and similar functions, the two isoforms of the transporter are discretely distributed: SVCT1 is mainly confined to epithelial systems (intestine, kidney, liver), whereas SVCT2 serves a host of metabolically active cells and specialized tissues in the brain, eye and other organs.

Molecular Cloning and Characterization of a Channel-like Transporter Mediating Intestinal Calcium Absorption

Calcium is a major component of the mineral phase of bone and serves as a key intracellular second messenger. Postnatally, all bodily calcium must be absorbed from the diet through the intestine. Here we report the properties of a calcium transport protein (CaT1) cloned from rat duodenum using an expression cloning strategy in Xenopus laevis oocytes, which likely plays a key role in the intestinal uptake of calcium. CaT1 shows homology (75% amino acid sequence identity) to the apical calcium channel ECaC recently cloned from vitamin D-responsive cells of rabbit kidney and is structurally related to the capsaicin receptor and the TRP family of ion channels. Based on Northern analysis of rat tissues, a 3-kilobase CaT1 transcript is present in rat duodenum, proximal jejunum, cecum, and colon, and a 6.5-kilobase transcript is present in brain, thymus, and adrenal gland. In situ hybridization revealed strong CaT1 mRNA expression in enterocytes of duodenum, proximal jejunum, and cecum. No signals were detected in kidney, heart, liver, lung, spleen, and skeletal muscle. When expressed inXenopus oocytes, CaT1 mediates saturable Ca2+uptake with a Michaelis constant of 0.44 mm. Transport of Ca2+ by CaT1 is electrogenic, voltage-dependent, and exhibits a charge/Ca2+uptake ratio close to 2:1, indicating that CaT1-mediated Ca2+ influx is not coupled to other ions. CaT1 activity is pH-sensitive, exhibiting significant inhibition by low pH. CaT1 is also permeant to Sr2+ and Ba2+ (but not Mg2+), although the currents evoked by Sr2+ and Ba2+ are much smaller than those evoked by Ca2+. The trivalent cations Gd3+ and La3+ and the divalent cations Cu2+, Pb2+, Cd2+, Co2+, and Ni2+ (each at 100 μm) do not evoke currents themselves, but inhibit CaT1-mediated Ca2+ transport. Fe3+, Fe2+, Mn2+, and Zn2+ have no significant effects at 100 μm on CaT1-mediated Ca2+ transport. CaT1 mRNA levels are not responsive to 1,25-dihydroxyvitamin D3 administration or to calcium deficiency. Our studies strongly suggest that CaT1 provides the principal mechanism for Ca2+ entry into enterocytes as part of the transcellular pathway of calcium absorption in the intestine.

The human kidney low affinity Na+/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose.

The major reabsorptive mechanism for D-glucose in the kidney is known to involve a low affinity high capacity Na+/glucose cotransporter, which is located in the early proximal convoluted tubule segment S1, and which has a Na+ to glucose coupling ratio of 1:1. Here we provide the first molecular evidence for this renal D-glucose reabsorptive mechanism. We report the characterization of a previously cloned human kidney cDNA that codes for a protein with 59% identity to the high affinity Na+/glucose cotransporter (SGLT1). Using expression studies with Xenopus laevis oocytes we demonstrate that this protein (termed SGLT2) mediates saturable Na(+)-dependent and phlorizin-sensitive transport of D-glucose and alpha-methyl-D-glucopyranoside (alpha MeGlc) with Km values of 1.6 mM for alpha MeGlc and approximately 250 to 300 mM for Na+, consistent with low affinity Na+/glucose cotransport. In contrast to SGLT1, SGLT2 does not transport D-galactose. By comparing the initial rate of [14C]-alpha MeGlc uptake with the Na(+)-influx calculated from alpha MeGlc-evoked inward currents, we show that the Na+ to glucose coupling ratio of SGLT2 is 1:1. Using combined in situ hybridization and immunocytochemistry with tubule segment specific marker antibodies, we demonstrate an extremely high level of SGLT2 message in proximal tubule S1 segments. This level of expression was also evident on Northern blots and likely confers the high capacity of this glucose transport system. We conclude that SGLT2 has properties characteristic of the renal low affinity high capacity Na+/glucose cotransporter as previously reported for perfused tubule preparations and brush border membrane vesicles. Knowledge of the structural and functional properties of this major renal Na+/glucose reabsorptive mechanism will advance our understanding of the pathophysiology of renal diseases such as familial renal glycosuria and diabetic renal disorders.

Molecular Characterization of a Broad Selectivity Neutral Solute Channel

In all living cells, coordination of solute and water movement across cell membranes is of critical importance for osmotic balance. The current concept is that these processes are of distinct biophysical nature. Here we report the expression cloning of a liver cDNA encoding a unique promiscuous solute channel (AQP9) that confers high permeability for both solutes and water. AQP9 mediates passage of a wide variety of non-charged solutes including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercury-sensitive manner, whereas amino acids, cyclic sugars, Na+, K+, Cl−, and deprotonated monocarboxylates are excluded. The properties of AQP9 define a new evolutionary branch of the major intrinsic protein family of aquaporin proteins and describe a previously unknown mechanism by which a large variety of solutes and water can pass through a single pore, enabling rapid cellular uptake or exit of metabolites with minimal osmotic perturbation.

SOD1 mutants linked to amyotrophic lateral sclerosis selectively inactivatea glial glutamate transporter

The mechanism by which Cu2+/Zn2+ superoxide dismutase (SOD1) mutants lead to motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS) is unknown. We show that oxidative reactions triggered by hydrogen peroxide and catalyzed by A4V and I113T mutant but not wild-type SOD1 inactivated the glutamate transporter human GLT1. Chelation of the copper ion of the prosthetic group of A4V prevented GLT1 inhibition. GLT1 was a selective target of oxidation mediated by SOD1 mutants, and its reactivity was confined to the intracellular carboxyl-terminal domain. The antioxidant Mn(III)TBAP rescued GLT1 from inhibition. Because inactivation of GLT1 results in neuronal degeneration, we propose that toxic properties of SOD1 mutants lead to neuronal death via an excitotoxic mechanism in SOD1-linked FALS.

CaT1 manifests the pore properties of the calcium-release-activated calcium channel

The calcium-release-activated Ca2+channel, ICRAC, is a highly Ca2+-selective ion channel that is activated on depletion of either intracellular Ca2+ levels or intracellular Ca2+ stores. The unique gating of ICRAC has made it a favourite target of investigation for new signal transduction mechanisms; however, without molecular identification of the channel protein, such studies have been inconclusive. Here we show that the protein CaT1 (ref. 4), which has six membrane-spanning domains, exhibits the unique biophysical properties of ICRAC when expressed in mammalian cells. Like ICRAC, expressed CaT1 protein is Ca2+ selective, activated by a reduction in intracellular Ca2+ concentration, and inactivated by higher intracellular concentrations of Ca2+. The channel is indistinguishable from ICRAC in the following features: sequence of selectivity to divalent cations; an anomalous mole fraction effect; whole-cell current kinetics; block by lanthanum; loss of selectivity in the absence of divalent cations; and single-channel conductance to Na+ in divalent-ion-free conditions. CaT1 is activated by both passive and active depletion of calcium stores. We propose that CaT1 comprises all or part of the ICRAC pore.

The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects

The solute carrier family 1 (SLC1) includes five high-affinity glutamate transporters, EAAC1, GLT-1, GLAST, EAAT4 and EAAT5 (SLC1A1, SLC1A2, SLC1A3, SLC1A6, and SLC1A7, respectively) as well as the two neutral amino acid transporters, ASCT1 and ASCT2 (SLC1A4 and ALC1A5, respectively). Although each of these transporters have similar predicted structures, they exhibit distinct functional properties which are variations of a common transport mechanism. The high-affinity glutamate transporters mediate transport of l-Glu, l-Asp and d-Asp, accompanied by the cotransport of 3 Na+ and 1 H+, and the countertransport of 1 K+, whereas ASC transporters mediate Na+-dependent exchange of small neutral amino acids such as Ala, Ser, Cys and Thr. The unique coupling of the glutamate transporters allows uphill transport of glutamate into cells against a concentration gradient. This feature plays a crucial role in protecting neurons against glutamate excitotoxicity in the central nervous system. During pathological conditions, such as brain ischemia (e.g. after a stroke), however, glutamate exit can occur due to “reversed glutamate transport”, which is caused by a reversal of the electrochemical gradients of the coupling ions. Selective inhibition of the neuronal glutamate transporter EAAC1 (SLC1A1) may be of therapeutic interest to block glutamate release from neurons during ischemia. On the other hand, upregulation of the glial glutamate transporter GLT1 (SLC1A2) may help protect motor neurons in patients with amyotrophic lateral sclerosis (ALS), since loss of function of GLT1 has been associated with the pathogenesis of certain forms of ALS.