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• W2053181997 abstract "Na+/Cl−-dependent neurotransmitter transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat γ-aminobutyric acid transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of Förster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane. Na+/Cl−-dependent neurotransmitter transporters form constitutive oligomers, the significance of which is not known. In soluble proteins, leucine heptad repeats drive dimerization; the rat γ-aminobutyric acid transporter GAT-1 (rGAT) contains a motif reminiscent of a leucine heptad repeat in the second transmembrane helix (TM2). We substituted leucine residues in TM2 of rGAT by alanine and tested the ability of the resulting mutants to form oligomers by three methods of Förster resonance energy transfer (FRET) microscopy. Replacement of one leucine (L97A) resulted in considerable loss of energy transfer, replacing two or more ablated it completely. Furthermore, intracellular trapping increased with the number of leucine substitutions. Only rGAT-L97A reached the cell surface to a sufficient amount such that, in intact cells, it was indistinguishable from wild type rGAT with respect to substrate transport, binding of inhibitors, and regulation by protein kinase C. However, in membrane vesicles prepared from transfected cells, all mutants were still functional. In addition, FRET was readily detected during maturation of wild type rGAT, when the bulk of the protein resided in the endoplasmic reticulum. Hence, our findings strongly argue for a role of oligomer formation during biosynthesis and subsequent delivery of the multimer from the endoplasmic reticulum to the plasma membrane. Na+/Cl−-dependent neurotransmitter transporters (e.g. the transporters for dopamine, serotonin, or GABA) 1The abbreviations used are: GABA, γ-aminobutyric acid; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FRET, Förster resonance energy transfer; FRETM, FRET microscopy; DRAP, donor recovery after acceptor photobleaching; SKF 89976A, 1-(4,4-diphenyl-3-butenyl)-3-piperidine-carboxylic acid hydrochloride; rGAT, rat GABA-transporter GAT-1, CMV, cytomegalovirus; TM, transmembrane; AU, arbitrary units; βPMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; ER, endoplasmic reticulum; wt, wild type 1The abbreviations used are: GABA, γ-aminobutyric acid; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FRET, Förster resonance energy transfer; FRETM, FRET microscopy; DRAP, donor recovery after acceptor photobleaching; SKF 89976A, 1-(4,4-diphenyl-3-butenyl)-3-piperidine-carboxylic acid hydrochloride; rGAT, rat GABA-transporter GAT-1, CMV, cytomegalovirus; TM, transmembrane; AU, arbitrary units; βPMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; ER, endoplasmic reticulum; wt, wild typeretrieve neurotransmitters from the synaptic cleft into the presynaptic specialization (1Rudnick G. Clark J. Biochim. Biophys. Acta. 1993; 1144: 249-263Crossref PubMed Scopus (367) Google Scholar). The medical relevance of these proteins is obvious; for instance, it has long been known that antidepressant drugs block the transporter for norepinephrine and serotonin (2Axelrod J. Whitby L.G. Hertting G. Science. 1961; 133: 383-384Crossref PubMed Scopus (202) Google Scholar). Likewise, tiagabine, an inhibitor of GABA transport, is used as an anticonvulsant in the treatment of epileptic seizures (3Iversen L. Mol. Psychiatry. 2000; 5: 357-362Crossref PubMed Scopus (129) Google Scholar). Transporters support bidirectional flux of substrate, i.e. not only do they mediate influx of substrate but they also allow for non-exocytotic release of substrate (4Levi G. Raiteri M. Trends Neurosci. 1993; 16: 415-419Abstract Full Text PDF PubMed Scopus (343) Google Scholar). Compounds that induce reverse transport enjoy widespread popularity among illicit drug users; this is particularly true for amphetamine and its congeners, including ecstasy.Increasing evidence suggests that neurotransmitter transporters are oligomers (5Klingenberg M. Nature. 1981; 290: 449-454Crossref PubMed Scopus (214) Google Scholar, 6Veenhoff L.M. Heuberger E.H. Poolman B. Trends Biochem. Sci. 2002; 27: 242-249Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Constitutive oligomerization has been visualized in intact cells by FRET microscopy (7Schmid J.A. Scholze P. Kudlacek O. Freissmuth M. Singer E.A. Sitte H.H. J. Biol. Chem. 2001; 276: 3805-3810Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar); the oligomeric nature of neurotransmitter transporters has also been demonstrated by other, more disruptive, approaches, e.g. co-immunoprecipitation from detergent extracts and cross-linking (8Jess U. Betz H. Schloss P. FEBS Lett. 1996; 394: 44-46Crossref PubMed Scopus (54) Google Scholar, 9Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (188) Google Scholar, 10Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (171) Google Scholar) and by freeze-fracture electron microscopy (11Eskandari S. Kreman M. Kavanaugh M.P. Wright E.M. Zampighi G.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8641-8646Crossref PubMed Scopus (109) Google Scholar). However, the functional role of oligomer formation remains enigmatic. The hypothesis has been formulated that neurotransmitter transporters function in a manner analogous to ligand-gated ion channels, as binding of substrate induces an ion current (12Sonders M.S. Amara S.G. Curr. Opin. Neurobiol. 1996; 6: 294-302Crossref PubMed Scopus (205) Google Scholar, 13DeFelice L.J. Blakely R.D. Biophys. J. 1996; 70: 579-580Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 14Beckman M.L. Quick M.W. J. Membr. Biol. 1998; 164: 1-10Crossref PubMed Scopus (77) Google Scholar). There is still an ongoing debate whether the GAT operates in a channel-like mode (15Cammack J.N. Schwartz E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 723-727Crossref PubMed Scopus (84) Google Scholar, 16Risso S. DeFelice L.J. Blakely R.D. J. Physiol. (Lond.). 1996; 490: 691-702Crossref Scopus (65) Google Scholar) or according to the conservative “alternating access model” (17Lu C.C. Hilgemann D.W. J. Gen. Physiol. 1999; 114: 445-457Crossref PubMed Scopus (61) Google Scholar, 18Lu C.C. Hilgemann D.W. J. Gen. Physiol. 1999; 114: 429-444Crossref PubMed Scopus (101) Google Scholar, 19Hilgemann D.W. Lu C.C. J. Gen. Physiol. 1999; 114: 459-475Crossref PubMed Scopus (91) Google Scholar). In the former, binding of substrate induces an ion influx in excess, and in the latter, ions and substrates are translocated with a fixed stoichiometry. Regardless of which model is correct, it is attractive to speculate that the functional transporter unit contains more than one current membrane permeation pathway that allows for concomitant but segregated translocation of substrate and ions (12Sonders M.S. Amara S.G. Curr. Opin. Neurobiol. 1996; 6: 294-302Crossref PubMed Scopus (205) Google Scholar, 13DeFelice L.J. Blakely R.D. Biophys. J. 1996; 70: 579-580Abstract Full Text PDF PubMed Scopus (70) Google Scholar). A survey of channel structures indicates that oligomers are frequently encountered (20Jentsch T.J. Nature. 2002; 415: 276-277Crossref PubMed Scopus (43) Google Scholar), regardless of whether the channels contain a single pore (e.g. K+ channels and ligand-gated ion channels) or multiple pores (Cl− channels and aquaporins).We have therefore searched for mutations that abolish oligomer formation in the rat GABA transporter GAT-1 (rGAT). The rGAT contains a perfect leucine heptad repeat in its second transmembrane helix (TM2); 4 leucine residues (Leu83–Leu104) are separated by 6 intervening amino acids. This leucine heptad repeat conforms to the arrangement found in canonical leucine zippers. Thus, by analogy with transcription factors (21O'Shea E.K. Rutkowski R. Stafford III, W.F. Kim P.S. Science. 1989; 245: 646-648Crossref PubMed Scopus (395) Google Scholar), soluble proteins (22Bartkiewicz M. Houghton A. Baron R. J. Biol. Chem. 1999; 274: 30887-30895Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 23Su H.P. Brugnera E. Van Criekinge W. Smits E. Hengartner M. Bogaert T. Ravichandran K.S. J. Biol. Chem. 2000; 275: 9542-9549Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and soluble domains of membrane proteins (24Bauer M.F. Sirrenberg C. Neupert W. Brunner M. Cell. 1996; 87: 33-41Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), TM2 in individual rGAT molecules may drive dimerization by adopting a leucine zipper-like conformation.Our experiments show that substituting leucine residues by alanine abrogates intermolecular FRET most likely by interruption of oligomer formation in rGAT1. One of those mutants, namely L97A, still binds and transports substrate in a manner indistinguishable from the wild type. Mutations that replace more than one leucine result in intracellular retention of transporters that are still capable of translocating substrate across the bilayer. These data indicate that assembly of rGAT into a stable oligomeric complex is required for export of rGAT1 from the endoplasmic reticulum.DISCUSSIONThe mechanisms that support oligomer formation of membrane proteins are still poorly understood, because the forces that stabilize protein-protein interfaces in a hydrophobic milieu differ from those operating in aqueous solution. In the present study, we have focused on the leucine heptad repeat TM2 of the rat GABA transporter-1 and employed three independent techniques of FRET microscopy to investigate its contribution in supporting oligomer formation. Our approach has the advantage that it allows the visualization of protein-protein interactions in the native environment, i.e.in the membrane of a living cell. Based on all data sets (ratio imaging, donor photobleaching, and donor recovery after acceptor photobleaching), we conclude that the propensity to form oligomeric complexes is considerably impaired upon substitution of a single leucine residue in TM2 (Leu97 by alanine) and completely abrogated by removal of additional leucine side chains. We cannot rule out an overall change in the conformation that would move the amino-terminally attached fluorescent tags in an otherwise intact oligomer beyond the Förster distance (∼50 Å). However, the fact that bidirectional transport of substrate, binding of inhibitors, and regulation by protein kinase C was not affected in the mutants (cf. Figs. 3, 4, and 8) argues against a global conformational change of this magnitude. The observations are more likely accounted for by a loss in the physical interaction of neighboring amino termini of YFP- and CFP-tagged rGAT-L97A, because their ability to form oligomers is impaired. However, our results obviously do not rule out that the oligomer may be stabilized by interaction of helices other than TM2; in fact, it has been shown that cysteines on top of TM6 can support intermolecular cross-links in the human dopamine transporter (10Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (171) Google Scholar). Finally, the possibility has to be considered that the effect of the leucine residues is indirect; they may be required for packing of the helices in the hydrophobic core of an individual transporter and thereby indirectly stabilize the interface that supports oligomer formation.Apart from the current observations, the following three lines of evidence support the assumption that leucine heptad repeats may mediate homophilic interactions of proteins within the membrane: (i) studies of the small protein phospholamban which contains a single transmembrane span (36Arkin I.T. Adams P.D. MacKenzie K.R. Lemmon M.A. Brunger A.T. Engelman D.M. EMBO J. 1994; 13: 4757-4764Crossref PubMed Scopus (174) Google Scholar, 37Cornea R.L. Autry J.M. Chen Z. Jones L.R. J. Biol. Chem. 2000; 275: 41487-41494Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar); (ii) experiments studying the homophilic interactions of single artificial transmembrane segments (38Gurezka R. Laage R. Brosig B. Langosch D. J. Biol. Chem. 1999; 274: 9265-9270Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 39Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (345) Google Scholar, 40Zhou F.X. Merianos H.J. Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (316) Google Scholar); and (iii) a related approach in which the amphipathic segment comprising the leucine zipper of the transcription factor GCN4 was converted into a hydrophobic helix (41Zhou F.X. Cocco M.J. Russ W.P. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 2000; 7: 154-160Crossref PubMed Scopus (356) Google Scholar). There are, however, several arguments that cast doubt on a role of simplistic extension of the leucine zipper model to membrane proteins, in particular those containing multiple transmembrane domains. First, voltage-sensitive Shaker K+ channels, for instance, are assembled from identical or related subunits that contain a conserved leucine heptad repeat within their S4–S5 region; these leucine residues, however, are not involved in subunit assembly (28McCormack K. Tanouye M.A. Iverson L.E. Lin J.W. Ramaswami M. McCormack T. Campanelli J.T. Mathew M.K. Rudy B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2931-2935Crossref PubMed Scopus (172) Google Scholar). Similar results have been found for L-type calcium channels, where the heptad motif is thought to play a fundamental role in channel activation rather than helix packing (29Garcia J. Nakai J. Imoto K. Beam K.G. Biophys. J. 1997; 72: 2515-2523Abstract Full Text PDF PubMed Scopus (54) Google Scholar). Second, it is difficult to envisage the driving force that would stabilize a leucine zipper in a membrane protein; in aqueous medium helices that contain leucine heptad repeats form a “zipper” because this conformation shields the bulky hydrophobic residue from the aqueous medium. This need to minimize the solvent-exposed surface obviously does not arise in the lipid environment of the membrane. In fact, polyleucine stretches haveper se little propensity to support helix-helix interactions in a hydrophobic environment (41Zhou F.X. Cocco M.J. Russ W.P. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 2000; 7: 154-160Crossref PubMed Scopus (356) Google Scholar). The association (as dimers and trimers) is greatly enhanced by hydrogen-bonding donors such as asparagine (39Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (345) Google Scholar, 41Zhou F.X. Cocco M.J. Russ W.P. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 2000; 7: 154-160Crossref PubMed Scopus (356) Google Scholar) or aspartate and glutamate (40Zhou F.X. Merianos H.J. Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (316) Google Scholar) in the position “a” of a heptad repeat “dnefgabcdn+1,” where the positions “dn” and “dn+1” are held by leucines (or isoleucines), i.e. in the helix the hydrophilic residue is sandwiched between two hydrophobic side chains. In this context, it is worth pointing out that a glutamate residue is found in TM2 of all members of Na+-dependent (monoamine) neurotransmitter transporters. This invariant glutamate (Glu101 in rGAT) is found at the a position between two leucine residues (Leu97 and Leu104 in rGAT, see Fig. 1). It is also worth noting that the constitutive activity of the neu oncogene results from a point mutation, which replaces Val664 of the Her/erbB2 receptor by glutamate. This substitution results in constitutive (i.e.ligand-independent) oligomerization and hence persistent signaling (42Weiner D.B. Liu J. Cohen J.A. Williams W.V. Greene M.I. Nature. 1989; 339: 230-231Crossref PubMed Scopus (357) Google Scholar, 43Bargmann C.I. Weinberg R.A. EMBO J. 1988; 7: 2043-2052Crossref PubMed Scopus (271) Google Scholar).Based on these arguments, we propose that the leucine heptad repeat and the invariant glutamate in TM2 are important components of the oligomeric interface of rGAT1. The role of Glu101 in rat Gat1 is currently being explored. Importantly, we stress, however, that our data do not allow us to draw any conclusions about the structure of the leucine heptad repeat nor about the stoichiometry of transporters in the oligomeric complex. For appropriate synthetic artificial leucine heptad repeats can exist as dimers and trimers (39Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (345) Google Scholar). In phospholamban, the leucine heptad repeat in the single transmembrane span supports pentamer formation. Obviously, in rGAT (and related transporters) the 12 transmembrane helices impose a space constraint; hence, the number of monomers that can be recruited into a complex via homophilic association of TM2 is limited. Nevertheless, trimeric complexes can, for instance, readily be envisaged. In addition, as mentioned earlier, there may be additional contact sites that stabilize the oligomer (10Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (171) Google Scholar).Our data point to a role of the leucine heptad repeat in supporting the delivery of the transporter from the endoplasmic to the plasma membrane. A role for oligomerization in this process can be inferred from earlier domain swapping experiments in the glucose transporters 1 and 4 (44Asano T. Takata K. Katagiri H. Tsukuda K. Lin J.L. Ishihara H. Inukai K. Hirano H. Yazaki Y. Oka Y. J. Biol. Chem. 1992; 267: 19636-19641Abstract Full Text PDF PubMed Google Scholar), and this interpretation is supported by the following observations. Oligomer formation can be observed in the very early stage of rGAT synthesis and maturation. Replacing leucine residues in TM2 interferes with oligomer formation and results in the intracellular retention of (otherwise transport competent) rGAT mutants. These findings suggest that the assembly into a higher ordered complex is indeed important for the export from the ER. However, whereas oligomer formation is necessary, it is clearly not sufficient; resonance energy transfer was similar in magnitude in the carboxyl-terminal deletion mutant rGATΔC and in wild type rGAT. Thus, it is safe to conclude that this mutant existed in oligomeric form. Nevertheless, rGATΔC was not inserted into the plasma membrane. This indicates that an intact carboxyl terminus is also required for ER export. Presumably, the carboxyl terminus represents a docking site for a component of the machinery that recruits rGAT into the budding vesicles and/or targets it to the subsequent compartment. The amino terminus of rGAT is known to bind syntaxin-1A (45Quick M.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5686-5691Crossref PubMed Scopus (39) Google Scholar), a member of the t-SNARE family; proteins that bind to the carboxyl terminus of rGAT have not yet been identified. In our search for transporters, which are retained in intracellular compartments but have presumably normal transport characteristics, we focused on the carboxyl terminus, because its proteolytic removal does not affect the ability of purified rGAT to transport substrate (46Mabjeesh N.J. Kanner B.I. J. Biol. Chem. 1992; 267: 2563-2568Abstract Full Text PDF PubMed Google Scholar). Truncation of the carboxyl terminus by 38 amino acids reducedV max by 75% relative to cells expressing the full-length transporter (47Bendahan A. Kanner B.I. FEBS Lett. 1993; 318: 41-44Crossref PubMed Scopus (34) Google Scholar); the reason for the reduction was not explored, but it may presumably reflect intracellular retention of the truncated transporter. rGATΔC, the mutant investigated in the present work, lacks 45 amino acids; this mutant was not present at the cell surface to any appreciable extent. Conversely, a mutant of rGAT, in which the carboxyl terminus is truncated by 36 amino acids, was delivered to the apical membrane of Madin-Darby canine kidney cells in a manner similar to wild type rGAT (48Perego C. Bulbarelli A. Longhi R. Caimi M. Villa A. Caplan M.J. Pietrini G. J. Biol. Chem. 1997; 272: 6584-6592Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The interpretation of our results and those obtained in the earlier two studies is limited by the fact that three different cell types were used. With this obvious caveat in mind, it is possible to postulate that the first 7–9 amino acids immediately adjacent to the last trans-membrane span play an important role in supporting export of rGAT from the ER.Although the phenomenon of FRET has been known for some 5 decades (49Förster T. Ann. d. Physik (Leipzig). 1948; 2: 57-75Google Scholar), FRET microscopy has only been applied recently to the study of membrane proteins in living cells. A standard argument raised against this approach is the conjecture that membrane proteins, in particular when heterologously overexpressed, are tightly packed in the bilayer (e.g. Ref. 50Wilson M.C. Meredith D. Halestrap A.P. J. Biol. Chem. 2002; 277: 3666-3672Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar); this accumulation and the restricted mobility of membrane proteins (often referred to as “membrane crowding”) are invoked as a nonspecific source of FRET (thought to support FRET in a nonspecific manner). Our experiments refute this argument in a definitive manner; wild type YFP-rGAT and YFP-rGAT-L97A accumulated to levels in the plasma membrane that allowed for equivalent V max of inward (and outward) transport. Nevertheless, the lack of 9 atoms in YFP-rGAT-L97A sufficed to abolish or greatly blunt resonance energy transfer. This establishes the specificity of FRET microscopy beyond reasonable doubt. Na+/Cl−-dependent neurotransmitter transporters (e.g. the transporters for dopamine, serotonin, or GABA) 1The abbreviations used are: GABA, γ-aminobutyric acid; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FRET, Förster resonance energy transfer; FRETM, FRET microscopy; DRAP, donor recovery after acceptor photobleaching; SKF 89976A, 1-(4,4-diphenyl-3-butenyl)-3-piperidine-carboxylic acid hydrochloride; rGAT, rat GABA-transporter GAT-1, CMV, cytomegalovirus; TM, transmembrane; AU, arbitrary units; βPMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; ER, endoplasmic reticulum; wt, wild type 1The abbreviations used are: GABA, γ-aminobutyric acid; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FRET, Förster resonance energy transfer; FRETM, FRET microscopy; DRAP, donor recovery after acceptor photobleaching; SKF 89976A, 1-(4,4-diphenyl-3-butenyl)-3-piperidine-carboxylic acid hydrochloride; rGAT, rat GABA-transporter GAT-1, CMV, cytomegalovirus; TM, transmembrane; AU, arbitrary units; βPMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; ER, endoplasmic reticulum; wt, wild typeretrieve neurotransmitters from the synaptic cleft into the presynaptic specialization (1Rudnick G. Clark J. Biochim. Biophys. Acta. 1993; 1144: 249-263Crossref PubMed Scopus (367) Google Scholar). The medical relevance of these proteins is obvious; for instance, it has long been known that antidepressant drugs block the transporter for norepinephrine and serotonin (2Axelrod J. Whitby L.G. Hertting G. Science. 1961; 133: 383-384Crossref PubMed Scopus (202) Google Scholar). Likewise, tiagabine, an inhibitor of GABA transport, is used as an anticonvulsant in the treatment of epileptic seizures (3Iversen L. Mol. Psychiatry. 2000; 5: 357-362Crossref PubMed Scopus (129) Google Scholar). Transporters support bidirectional flux of substrate, i.e. not only do they mediate influx of substrate but they also allow for non-exocytotic release of substrate (4Levi G. Raiteri M. Trends Neurosci. 1993; 16: 415-419Abstract Full Text PDF PubMed Scopus (343) Google Scholar). Compounds that induce reverse transport enjoy widespread popularity among illicit drug users; this is particularly true for amphetamine and its congeners, including ecstasy. Increasing evidence suggests that neurotransmitter transporters are oligomers (5Klingenberg M. Nature. 1981; 290: 449-454Crossref PubMed Scopus (214) Google Scholar, 6Veenhoff L.M. Heuberger E.H. Poolman B. Trends Biochem. Sci. 2002; 27: 242-249Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Constitutive oligomerization has been visualized in intact cells by FRET microscopy (7Schmid J.A. Scholze P. Kudlacek O. Freissmuth M. Singer E.A. Sitte H.H. J. Biol. Chem. 2001; 276: 3805-3810Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar); the oligomeric nature of neurotransmitter transporters has also been demonstrated by other, more disruptive, approaches, e.g. co-immunoprecipitation from detergent extracts and cross-linking (8Jess U. Betz H. Schloss P. FEBS Lett. 1996; 394: 44-46Crossref PubMed Scopus (54) Google Scholar, 9Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (188) Google Scholar, 10Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (171) Google Scholar) and by freeze-fracture electron microscopy (11Eskandari S. Kreman M. Kavanaugh M.P. Wright E.M. Zampighi G.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8641-8646Crossref PubMed Scopus (109) Google Scholar). However, the functional role of oligomer formation remains enigmatic. The hypothesis has been formulated that neurotransmitter transporters function in a manner analogous to ligand-gated ion channels, as binding of substrate induces an ion current (12Sonders M.S. Amara S.G. Curr. Opin. Neurobiol. 1996; 6: 294-302Crossref PubMed Scopus (205) Google Scholar, 13DeFelice L.J. Blakely R.D. Biophys. J. 1996; 70: 579-580Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 14Beckman M.L. Quick M.W. J. Membr. Biol. 1998; 164: 1-10Crossref PubMed Scopus (77) Google Scholar). There is still an ongoing debate whether the GAT operates in a channel-like mode (15Cammack J.N. Schwartz E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 723-727Crossref PubMed Scopus (84) Google Scholar, 16Risso S. DeFelice L.J. Blakely R.D. J. Physiol. (Lond.). 1996; 490: 691-702Crossref Scopus (65) Google Scholar) or according to the conservative “alternating access model” (17Lu C.C. Hilgemann D.W. J. Gen. Physiol. 1999; 114: 445-457Crossref PubMed Scopus (61) Google Scholar, 18Lu C.C. Hilgemann D.W. J. Gen. Physiol. 1999; 114: 429-444Crossref PubMed Scopus (101) Google Scholar, 19Hilgemann D.W. Lu C.C. J. Gen. Physiol. 1999; 114: 459-475Crossref PubMed Scopus (91) Google Scholar). In the former, binding of substrate induces an ion influx in excess, and in the latter, ions and substrates are translocated with a fixed stoichiometry. Regardless of which model is correct, it is attractive to speculate that the functional transporter unit contains more than one current membrane permeation pathway that allows for concomitant but segregated translocation of substrate and ions (12Sonders M.S. Amara S.G. Curr. Opin. Neurobiol. 1996; 6: 294-302Crossref PubMed Scopus (205) Google Scholar, 13DeFelice L.J. Blakely R.D. Biophys. J. 1996; 70: 579-580Abstract Full Text PDF PubMed Scopus (70) Google Scholar). A survey of channel structures indicates that oligomers are frequently encountered (20Jentsch T.J. Nature. 2002; 415: 276-277Crossref PubMed Scopus (43) Google Scholar), regardless of whether the channels contain a single pore (e.g. K+ channels and ligand-gated ion channels) or multiple pores (Cl− channels and aquaporins). We have therefore searched for mutations that abolish oligomer formation in the rat GABA transporter GAT-1 (rGAT). The rGAT contains a perfect leucine heptad repeat in its second transmembrane helix (TM2); 4 leucine residues (Leu83–Leu104) are separated by 6 intervening amino acids. This leucine heptad repeat conforms to the arrangement found in canonical leucine zippers. Thus, by analogy with transcription factors (21O'Shea E.K. Rutkowski R. Stafford III, W.F. Kim P.S. Science. 1989; 245: 646-648Crossref PubMed Scopus (395) Google Scholar), soluble proteins (22Bartkiewicz M. Houghton A. Baron R. J. Biol. Chem. 1999; 274: 30887-30895Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 23Su H.P. Brugnera E. Van Criekinge W. Smits E. Hengartner M. Bogaert T. Ravichandran K.S. J. Biol. Chem. 2000; 275: 9542-9549Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and soluble domains of membrane proteins (24Bauer M.F. Sir" @default.
• W2053181997 created "2016-06-24" @default.
• W2053181997 creator A5060360807 @default.
• W2053181997 creator A5077296742 @default.
• W2053181997 creator A5079264101 @default.
• W2053181997 date "2002-11-01" @default.
• W2053181997 modified "2023-10-13" @default.
• W2053181997 title "Mutations within an Intramembrane Leucine Heptad Repeat Disrupt Oligomer Formation of the Rat GABA Transporter 1" @default.
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