Molecular Characterization of Neurotransmitter Transporters Saad Shafqat, Maria Velaz-Faircloth, Ana Guadaiio-Ferraz, and Robert T. Fremeau, Jr. Downloaded from https://academic.oup.com/mend/article/7/12/1517/2714704 by guest on 06 October 2020 Departments of Pharmacology and Neurobiology Duke University Medical Center Durham, North Carolina 27710 INTRODUCTION ogical processes. For example, blockade and/or rever- sal of the high affinity plasma membrane L-glutamate transporter during ischemia or anoxia elevates the ex- Rapid chemical signaling between neurons and target tracellular concentration of L-glutamate to neurotoxic cells is dependent upon the precise control of the levels resulting in nerve cell damage (7). Conversely, concentration and duration of neurotransmitters in syn- specific GABA uptake inhibitors are being developed as aptic spaces. After transmitter release from activated potential anticonvulsant and antianxiety agents (8). The nerve terminals, the principal mechanism involved in the ability of synaptic transporters to accumulate certain rapid clearance from the synapse of the biogenic amine neurotransmitter-like toxins, including N-methyW and amino acid neurotransmitters is active transport of phenylpyridine (MPP+), 6-hydroxydopamine, and 5,6- the transmitter back into presynaptic nerve terminals or dihydroxytryptamine, suggests a role for these active glial surrounding cells by one of a large number of transport proteins in the selective vulnerability of neu- specific, pharmacologically distinguishable membrane rons to exogenous agents (9). Therefore, a detailed transport proteins (1). High affinity, Na+-dependent up- understanding of the molecular structure, function, and take activities, analogous to the noradrenergic carrier regulation of neurotransmitter transporters will: 1) en- first described at peripheral synapses by Axelrod and hance our understanding of synaptic signaling, 2) elu- colleagues (2) have been identified in mammalian cen- cidate the potential role of transporter defects in neu- tral nervous system (CNS) nerve terminals for the bio- rologic and psychiatric disorders; and 3) aid in the genie amine neurotransmitters including norepinephrine development of novel therapeutic agents. Finally, the (NE), dopamine (DA), and serotonin (5HT) for the ex- discovery of novel synaptic transporters may contribute citatory amino acid neurotransmitters L-glutamate and to the identification of new synaptic regulatory sub- L-aspartate, and for the inhibitory amino acid neuro- stances. transmitters r-amino butyric acid (GABA) and glycine (3). The primary function of plasma membrane neuro- transmitter transporters is to clear the synapse of the TWO DISTINCT GENE FAMILIES ENCODE neurotransmitter between nerve impulses and to re- PLASMA MEMBRANE NEUROTRANSMllTER plenish neurotransmitter levels in presynaptic nerve TRANSPORTERS terminals. In certain cases, Na+-dependent transport processes may also mediate the presynaptic accumu- lation of neurotransmitter precursors as well. For ex- Neurotransmitter transporters are representative mem- ample, the rate-limiting step in the biosynthesis of ace- bers of a family of proteins that transduce free energy tylcholine appears to be Na+dependent choline uptake stored in an electrochemical gradient into work in the into cholinergic nerve terminals by a hemicholinium- form of a concentration gradient. Active transport of sensitive high affinity choline transporter (5). neurotransmitters across the plasma membrane is Neurotransmitter transporters represent critical tar- driven by the transmembrane Na+ gradient generated gets for therapeutic and pathological alterations of syn- by the plasma membrane Na+/K+ ATPase (reviewed in aptic function. This is particularly apparent with the ref. 4). Additional ions are also required for transport of tricyclic antidepressants, amphetamines, and cocaine, many neurotransmitters, including extracellular Cl- and agents that block biogenic amine transport and produce intracellular K’. These ion sensitivities reflect cotrans- dramatic behavioral changes (6). Abnormalities of neu- port of the ions with the neurotransmitter during each rotransmitter transport can contribute to neuropathol- translocation cycle. In recent years, molecular cloning studies have re- 0933-9909/93/1517-15!23903.00/0 vealed that two distinct gene families code for plasma Molecular Endocrin&qy Copyright 0 1993 by The Endocrine society membrane neurotransmitter transporter proteins. 1517 MOL ENDO.1993 Vol7No.12 1518 These include: 1) the Na+- (and Cl-)-dependent family and colleagues (10) on the purification and cloning of a that includes transporters for GABA, NE, DA, 5HT, high affinity Na+- (and Cl-)-dependent GABA trans- glycine, proline, taurine, betaine, and creatine; and 2) porter from rat brain (rGAT-1). Subsequently, Amara the Na+- (and K+)-dependent, but Cl--independent, fam- and colleagues (11) isolated a cDNA clone for a cocaine- ily of transporters that include transporters for the and antidepressant-sensitive human NE transporter excitatory amino acids L-glutamate and L-aspartate (hNET) by expression cloning (11). Despite dramatic (Fig. 1). These two families of plasma membrane trans- differences in pharmacological sensitivities to sub- porters exhibit significant differences in their ionic re- strates and antagonists, rGAT-1 and hNET possessed quirements and proposed structures which are likely to significant (46%) but dispersed amino acid sequence reflect underlying differences in transport mechanisms. identities and exhibited similar inferred membrane to- This review will discuss recent advances in our under- pographies. No significant amino acid sequence identity Downloaded from https://academic.oup.com/mend/article/7/12/1517/2714704 by guest on 06 October 2020 standing of the structure, expression, and extent of the was observed, however, between these two neuro- molecular diversity of these two neurotransmitter trans- transmitter transporters and other membrane transport porter gene families. proteins whose sequences had been determined, in- cluding the mammalian facilitated glucose transporters Na+- (and Cl-)-Dependent Neurotransmitter (12) the mammalian Na+/glucose cotransporter (13) Transporters the prokaryotic Na+dependent cotranspotters (14) or the ATP-binding cassette membrane transporters, in- The molecular characterization of neurotransmitter cluding the multidrug resistance P-glycoproteins (15) transporters began with the pioneering work of Kanner and the cystic fibrosis transmembrane conductance regulator (16). These results established the presence of a distinct gene family of neurotransmitter transport Na+- (and Cl-1 -Dependent Transporters proteins and paved the way for the rapid identification of additional members of this novel gene family. Polymerase chain reaction (PCR) and low stringency screening techniques have been used to isolate addi- tional members of the emerging family of Na+- (and Cl-)-dependent neurotransmitter transporters. Based upon amino acid sequence conservation (Fig. 2) these cDNAs can be divided into three subfamilies including: NH, 1 1) the GABA, betaine, taurine, and creatine transport- COOH 1 ers; 2) the biogenic amine (NE, DA, and 5HT) trans- Na+- (and K+) -Dependent Glutamate Transporters porters; and 3) the amino acid (L-proline and glycine) transporters. The observation that transporters for sub- Pl strates with no known neurotransmitter role, such as betaine and creatine, belong to this family of transport- ers indicates that this family may subserve a greater diversity of physiological functions, and should more correctly be referred to as the Na+- (and Cl-)-dependent plasma membrane transporter family. -2 COOH 2 As depicted in Fig. 1, a tentative structural model for “Orphan” clonea the Na+- (and Cl-)-dependent plasma membrane trans- Y porters predicts approximately 12 transmembrane (Y- helical domains, cytoplasmic NHP- and COOH-termini, and a large, glycosylated, extracellular loop separating putative transmembrane domains 3 and 4. This pro- posed topographical model remains to be experimen- tally verified. Although in each case a single cDNA is able to induce high affinity Na+- (and Cl-)-dependent NH, - transport in a nonneural cell line, the subunit stoichi- COOH - ometry of the native transporter complex has not been Fig. 1. Schematicmodels depicting the proposed transmem- determined for any member of this family of membrane brane topology of the two major familiesof plasma membrane transport proteins. neurotransmittertransporters. Potential N-linkedglycosylation sites on the presumed extracellulardomains are depicted. Due The Subfamily of GABA, Betaine, Taurine, and to the subjective nature of hydropathy analysis, the model Creatine Transporters proposed for the glutamate transporters is controversial(see text). Orphan clones refer to recentlydescribed putative trans- porters (56, 57) which bear sequence homology to the Na+- An exciting result to emerge from the molecular char- (and Cl-)-dependenttransporters but exhibit a distinct inferred acterization of neurotransmitter transporters was the membrane topology. discovery that multiple transporter subtypes exist for Minireview 1519 certain neurotransmitters. Thus four distinct GABA activity (20). Because the subfamily of biogenic amine transporter cDNAs have been described which exhibit transporters share this conserved arginine residue (see 50-69% amino acid sequence identity but differ in their Fig. 3), but the cognate biogenic amine substrates