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Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100430

ISSN

1083-351X

Autores

Tatiana Sorkina, Mary Hongying Cheng, Tarique Rajasaheb Bagalkot, Callen T. Wallace, Simon C. Watkins, İvet Bahar, Alexander Sorkin,

Tópico(s)

Advanced Fluorescence Microscopy Techniques

Resumo

Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule–induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization. Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule–induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization. The plasma membrane dopamine transporter (DAT) regulates dopamine (DA) neurotransmission in the central nervous system by mediating the reuptake of DA following its release by dopaminergic neurons (1Giros B. el Mestikawy S. Bertrand L. Caron M.G. Cloning and functional characterization of a cocaine-sensitive dopamine transporter.FEBS Lett. 1991; 295: 149-154Crossref PubMed Scopus (276) Google Scholar, 2Jaber M. Jones S. Giros B. Caron M.G. The dopamine transporter: A crucial component regulating dopamine transmission.Movement Disord. 1997; 12: 629-633Crossref PubMed Scopus (192) Google Scholar). Thus, DAT plays a critical role in many functions of the brain DA system including locomotion control, motivation, and reward-seeking behaviors (3Mortensen O.V. Amara S.G. Dynamic regulation of the dopamine transporter.Eur. J. Pharmacol. 2003; 479: 159-170Crossref PubMed Scopus (128) Google Scholar, 4Iversen S.D. Iversen L.L. Dopamine: 50 years in perspective.Trends Neurosci. 2007; 30: 188-193Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). DAT is a primary target for psychostimulants, e.g., cocaine and amphetamines, and is involved in neurological disorders and neurodegenerative pathologies (5Gainetdinov R.R. Caron M.G. Monoamine transporters: From genes to behavior.Annu. Rev. Pharmacol. Toxicol. 2003; 43: 261-284Crossref PubMed Scopus (295) Google Scholar, 6Gowrishankar R. Hahn M.K. Blakely R.D. Good riddance to dopamine: Roles for the dopamine transporter in synaptic function and dopamine-associated brain disorders.Neurochem. Int. 2014; 73: 42-48Crossref PubMed Scopus (46) Google Scholar, 7Volkow N.D. Morales M. The brain on Drugs: From reward to Addiction.Cell. 2015; 162: 712-725Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 8Wise R.A. Dopamine and reward: The anhedonia hypothesis 30 years on.Neurotoxicity Res. 2008; 14: 169-183Crossref PubMed Scopus (367) Google Scholar). DAT and other monoamine transporters belong to the solute carrier 6 (SLC6) family of Na+-dependent neurotransmitter symporters (9Kristensen A.S. Andersen J. Jorgensen T.N. Sorensen L. Eriksen J. Loland C.J. Stromgaard K. Gether U. SLC6 neurotransmitter transporters: Structure, function, and regulation.Pharmacol. Rev. 2011; 63: 585-640Crossref PubMed Scopus (538) Google Scholar). They are composed of 12 transmembrane (TM) helices with both amino termini and carboxy termini projected intracellularly (10Amara S.G. Kuhar M.J. Neurotransmitter transporters: Recent progress.Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Scopus (990) Google Scholar, 11Chen N.H. Reith M.E. Quick M.W. Synaptic uptake and beyond: The sodium- and chloride-dependent neurotransmitter transporter family SLC6.Pflugers Arch. 2004; 447: 519-531Crossref PubMed Scopus (331) Google Scholar, 12Penmatsa A. Wang K.H. Gouaux E. X-ray structure of dopamine transporter elucidates antidepressant mechanism.Nature. 2013; 503: 85-90Crossref PubMed Scopus (425) Google Scholar). The molecular mechanism of substrate transport by neurotransmitter sodium symporter (NSS) family members has been extensively studied using biochemical, structural, and computational approaches. Our current understanding is an alternating access mechanism where binding of the substrate and Na+ ions to the extracellular (EC) vestibule of DAT in the outward-facing (OF) state triggers the transition to an occluded intermediate followed by an inward-facing (IF) state. The IF state, in turn, allows for the release of substrate and cations to the intracellular (IC) medium; and the transport cycle is completed by the return of DAT to its OF state (13Navratna V. Gouaux E. Insights into the mechanism and pharmacology of neurotransmitter sodium symporters.Curr. Opin. Struct. Biol. 2019; 54: 161-170Crossref PubMed Scopus (27) Google Scholar, 14Cheng M.H. Bahar I. Monoamine transporters: Structure, intrinsic dynamics and allosteric regulation.Nat. Struct. Mol. Biol. 2019; 26: 545-556Crossref PubMed Scopus (40) Google Scholar). Despite significant progress in characterizing DAT structure and function (13Navratna V. Gouaux E. Insights into the mechanism and pharmacology of neurotransmitter sodium symporters.Curr. Opin. Struct. Biol. 2019; 54: 161-170Crossref PubMed Scopus (27) Google Scholar, 14Cheng M.H. Bahar I. Monoamine transporters: Structure, intrinsic dynamics and allosteric regulation.Nat. Struct. Mol. Biol. 2019; 26: 545-556Crossref PubMed Scopus (40) Google Scholar), many aspects of DAT regulation remain to be understood. Perhaps one of the most significant limitations in our understanding is the process of DAT oligomerization and its role in DAT function and localization in the cell. For example while Drosophila DAT was crystallized as a monomer (12Penmatsa A. Wang K.H. Gouaux E. X-ray structure of dopamine transporter elucidates antidepressant mechanism.Nature. 2013; 503: 85-90Crossref PubMed Scopus (425) Google Scholar), biochemical and microscopy studies provide evidence for dimerization, high-order oligomerization, and cluster assembly of mammalian DAT expressed in nonneuronal cells and endogenous rodent DAT in dopaminergic neurons (15Hastrup H. Karlin A. Javitch J.A. Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306 at the extracellular end of the sixth transmembrane segment.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (171) Google Scholar, 16Hastrup H. Sen N. Javitch J.A. The human dopamine transporter forms a tetramer in the plasma membrane: Cross-linking of a cysteine in the fourth transmembrane segment is sensitive to cocaine analogs.J. Biol. Chem. 2003; 278: 45045-45048Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 17Torres G.E. Carneiro A. Seamans K. Fiorentini C. Sweeney A. Yao W.D. Caron M.G. Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical domains important for the functional expression of the transporter.J. Biol. Chem. 2003; 278: 2731-2739Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 18Sorkina T. Doolen S. Galperin E. Zahniser N.R. Sorkin A. Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy.J. Biol. Chem. 2003; 278: 28274-28283Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 19Chen N. Reith M.E. Substrates dissociate dopamine transporter oligomers.J. Neurochem. 2008; 105: 910-920Crossref PubMed Scopus (39) Google Scholar, 20Li Y. Cheng S.Y. Chen N. Reith M.E. Interrelation of dopamine transporter oligomerization and surface presence as studied with mutant transporter proteins and amphetamine.J. Neurochem. 2010; 114: 873-885Crossref PubMed Scopus (15) Google Scholar, 21Zhen J. Antonio T. Cheng S.Y. Ali S. Jones K.T. Reith M.E. Dopamine transporter oligomerization: Impact of combining protomers with differential cocaine analog binding affinities.J. Neurochem. 2015; 133: 167-173Crossref PubMed Scopus (30) Google Scholar, 22Zhen J. Reith M.E.A. Functional properties of dopamine transporter oligomers after copper linking.J. Neurochem. 2018; 144: 162-171Crossref PubMed Scopus (13) Google Scholar, 23Rahbek-Clemmensen T. Lycas M.D. Erlendsson S. Eriksen J. Apuschkin M. Vilhardt F. Jorgensen T.N. Hansen F.H. Gether U. Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains.Nat. Commun. 2017; 8: 740Crossref PubMed Scopus (46) Google Scholar, 24Das A.K. Kudlacek O. Baumgart F. Jaentsch K. Stockner T. Sitte H.H. Schutz G.J. Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.J. Biol. Chem. 2019; 294: 5632-5642Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 25Sorkina T. Ma S. Larsen M.B. Watkins S.C. Sorkin A. Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.Elife. 2018; 7: e32293Crossref PubMed Scopus (18) Google Scholar, 26Cheng M.H. Ponzoni L. Sorkina T. Lee J.Y. Zhang S. Sorkin A. Bahar I. Trimerization of dopamine transporter triggered by AIM-100 binding: Molecular mechanism and effect of mutations.Neuropharmacology. 2019; 161: 107676Crossref PubMed Scopus (4) Google Scholar). Furthermore, the molecular mechanisms mediating and regulating DAT oligomerization and their relation to substrate transport properties and IC trafficking remain largely unknown. We recently showed that furopyrimidine AIM-100, an inhibitor of the cytoplasmic activated CDC42 tyrosine kinase (ACK1/TNK2), induced dramatic oligomerization and clustering in cellular membranes of human DAT stably expressed in various cultured cells and endogenous mouse DAT (25Sorkina T. Ma S. Larsen M.B. Watkins S.C. Sorkin A. Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.Elife. 2018; 7: e32293Crossref PubMed Scopus (18) Google Scholar). It was proposed that oligomerization and clustering promoted DAT clathrin-independent and dynamin-independent endocytosis, which did not require ACK1 activity. However, ACK1 has been proposed to regulate constitutive DAT endocytosis (27Wu S. Bellve K.D. Fogarty K.E. Melikian H.E. Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 15480-15485Crossref PubMed Scopus (36) Google Scholar, 28Wu S. Fagan R.R. Uttamapinant C. Lifshitz L.M. Fogarty K.E. Ting A.Y. Melikian H.E. The dopamine transporter Recycles via a Retromer-dependent Postendocytic mechanism: Tracking studies using a novel Fluorophore-coupling Approach.J. Neurosci. 2017; 37: 9438-9452Crossref PubMed Scopus (34) Google Scholar). ACK1 has also been implicated in the regulation of endosomal sorting of signaling receptors (29Grovdal L.M. Johannessen L.E. Rodland M.S. Madshus I.H. Stang E. Dysregulation of Ack1 inhibits down-regulation of the EGF receptor.Exp. Cell Res. 2008; 314: 1292-1300Crossref PubMed Scopus (34) Google Scholar). Therefore, the involvement of ACK1, if any, remained unclear as it might be conceivable that AIM-100–induced accumulation of DAT in endosomes resulted from a combination of ACK1-dependent and ACK1-independent effects. To eliminate the confounding effect of ACK1 and analyze ACK1-independent mechanisms that underlie DAT endocytosis, we searched for AIM-100–like (AL) compounds that induce DAT oligomerization and endocytosis but do not inhibit ACK1. Our analysis using these small molecules and DAT mutagenesis revealed the key role of the preferred conformational state and dynamics of DAT as well as selected amino acids in TM helices TM4 and TM9 in augmenting oligomerization and promoting endocytosis. The results expose a striking coupling between intramolecular conformational dynamics, intermolecular association, and endocytosis events and suggest novel strategies for modulating DAT activity. Treatment of cells with AIM-100 induces DAT oligomerization, detected by SDS-PAGE as a robust accumulation of high molecular mass SDS-resistant species and promotes DAT clustering in the plasma membrane and endocytosis (25Sorkina T. Ma S. Larsen M.B. Watkins S.C. Sorkin A. Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.Elife. 2018; 7: e32293Crossref PubMed Scopus (18) Google Scholar). To examine the ability of AL compounds, shortly designated as ALs, to induce DAT oligomerization and endocytosis, we performed screening assays with 16 small molecules in porcine aortic endothelial (PAE) cells stably expressing YFP- and HA-epitope tagged DAT (YFP-HA-DAT) (18Sorkina T. Doolen S. Galperin E. Zahniser N.R. Sorkin A. Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy.J. Biol. Chem. 2003; 278: 28274-28283Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 30Sorkina T. Miranda M. Dionne K.R. Hoover B.R. Zahniser N.R. Sorkin A. RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.J. Neurosci. 2006; 26: 8195-8205Crossref PubMed Scopus (152) Google Scholar). These compounds were structurally similar to AIM-100 with or without the two phenyl groups attached to a central scaffold, with a variable third side chain or with di-phenyl-modified central scaffold, including the DA reuptake inhibitor, GBR12935 (Figs. 1A and S1). Among ALs, AL3, 4 and 8 caused the largest redistribution of the YFP-HA-DAT immunoreactivity—from monomeric ∼90 to 100 kDa (M-DAT) to ∼270 to 300 kDa and additional larger species (Fig. 1B), even though the extent of oligomerization was typically smaller than that induced by AIM-100. We will refer to the 270 to 300 kDa SDS-resistant species as a trimer (T-DAT), notwithstanding that it could be an oligomer of a different stoichiometry in intact cells. A small amount of the same trimer species was detected in lysates of vehicle-treated cells (Fig. 1B). Importantly, chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS3) in untreated or vehicle-treated cells significantly increased the fraction of YFP-HA-DAT trimers (Fig. 1C). Likewise, BS3 amplified AL-induced increase in DAT trimerization and high-order oligomerization (example with AL3 is shown; Fig. 1C). Cross-linking did not result in the appearance of a band, corresponding to YFP-HA-DAT dimer, indicating that the 270 to 300 kDa species is a minimal BS3-cross-linkable constitutive oligomer that can be stabilized by ALs. AL3 and AL4 are highly similar to AIM-100 with furopyrimidine as the central scaffold, whereas AL8 has a different scaffold—triazine (Fig. 1A). Triazine-based AL9 that lacks the pyridyl sidechain (Fig. 1A) was ineffective and used as the negative control to AL8 (Fig. 1B). The effects on DAT oligomerization were studied at concentrations of AL3, AL4, and AL8 of 10 to 40 μM. For consistency with previous work (25Sorkina T. Ma S. Larsen M.B. Watkins S.C. Sorkin A. Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.Elife. 2018; 7: e32293Crossref PubMed Scopus (18) Google Scholar), we adopted concentrations of 20 to 40 μM for all ALs in subsequent experiments. However, AL3, AL4, and AL8 as well as AIM-100 are poorly soluble in aqueous liquids (see Fig. S1). As a result, they had effects of variable strengths on DAT and may be equally effective at lower concentrations. AL3 and AL4 significantly increased oligomerization of endogenous HA-DAT in mouse striatal synaptosomes isolated from HA-DAT knock-in mice (31Rao A. Richards T.L. Simmons D. Zahniser N.R. Sorkin A. Epitope-tagged dopamine transporter knock-in mice reveal rapid endocytic trafficking and filopodia targeting of the transporter in dopaminergic axons.FASEB J. 2012; 26: 1921-1933Crossref PubMed Scopus (31) Google Scholar) (Fig. 1D; left panel). The minimal SDS-resistant oligomer was ∼200 kDa, which precisely corresponds to a trimer of the 65 kDa monomeric HA-DAT. AL8 did not induce significant DAT oligomerization in synaptosomes that were routinely prepared and maintained in the glucose-supplemented Krebs-Ring buffer (KRHG) (Fig. 1D; left panel) but increased the fraction of HA-DAT oligomers by 35% (SD = 5%) in the presence of 1:1 mixture of KRHG and F12 medium (Fig. 1D, right panel). Given the metal-binding properties of AL8 (32Bellam R. Raju G.G. Anipindi N.R. Jaganyi D. Kinetics and mechanism of base hydrolysis of tris(3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine)iron(II) in aqueous and micellar media.Transit. Metal Chem. 2016; 41: 271-278Crossref Scopus (5) Google Scholar), it is possible that the presence of metals in F12 medium was necessary for its effect on DAT. Direct fluorescence microscopy imaging of YFP-HA-DAT showed that a 2-h treatment of cells with AL3 and AL4 strongly induced the endocytosis of YFP-HA-DAT (Fig. 2A). The accumulation of YFP-HA-DAT in vesicular compartments was accompanied by a reduction in its levels at the filopodium-like plasma membrane protrusions and ruffles (Fig. 2A). AL8 had a less pronounced effect on the appearance of YFP-containing vesicles, which were typically dimmer (apparent smaller size) as compared with those vesicles in cells treated with AIM-100 or AL3/4 (Fig. 2A). However, an accumulation of YFP-HA-DAT in vesicles induced by AL8 was evident when images were compared with those of cells treated with AL9 that had no visible effect on YFP-HA-DAT localization. Vesicular YFP-HA-DAT was significantly co-localized with early endosomal antigen EEA.1, a marker of early endosomes, in cells treated with AIM-100, AL3, AL4, or AL8 but not in cells treated with vehicle or AL9 (Figs. 2B and S2). Cell-surface biotinylation assays confirmed robust AIM-100/AL-induced DAT endocytosis manifested by downregulation of the plasma membrane YFP-HA-DAT (Fig. 2, C and D). Notably, monomeric ∼70-kD (iM-DAT) and putative trimeric ∼200-kD (iT-DAT) species of immature YFP-HA-DAT were detected only in lysates but not in the plasma membrane fraction ("NeuAv"; Fig. 2B), indicating that both these species were of IC (endoplasmic reticulum) origin. Prompted by observations of AL-induced oligomerization and endocytosis, we used fluorescence resonance energy transfer (FRET) microscopy to demonstrate the effect of ALs on DAT oligomerization in single living cells. Constitutive and AIM-100–induced oligomerization of DAT had been previously demonstrated by live-cell FRET microscopy (18Sorkina T. Doolen S. Galperin E. Zahniser N.R. Sorkin A. Oligomerization of dopamine transporters visualized in living cells by fluorescence resonance energy transfer microscopy.J. Biol. Chem. 2003; 278: 28274-28283Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 25Sorkina T. Ma S. Larsen M.B. Watkins S.C. Sorkin A. Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.Elife. 2018; 7: e32293Crossref PubMed Scopus (18) Google Scholar). We used the same method of sensitized FRET measurements in cells co-expressing CFP-DAT and YFP-DAT treated with ALs. The intensity of FRET from CFP to YFP was found to be significantly higher in the plasma membrane clusters and endosomes in AL3-, AL4-, and AL8-treated cells compared with that in vehicle-treated cells, where FRET measurements were taken in the regions of DAT accumulation, such as filopodia, ruffles, and cell–cell contacts. (Fig. 2, E and F). An increased FRET in AL-treated cells is indicative of the close proximity (<5 nm) of multiple DAT molecules, in support of AL-induced DAT oligomerization in living cells. AL3, AL4, and AL8 did not cause any detectable SDS-resistant oligomerization and endocytosis of norepinephrine (NET) and serotonin transporters (SERT) (Fig. S3). This type of selectivity against DAT and not against other NSS family members is a feature shared with AIM-100 (25Sorkina T. Ma S. Larsen M.B. Watkins S.C. Sorkin A. Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.Elife. 2018; 7: e32293Crossref PubMed Scopus (18) Google Scholar). Furthermore, to test whether ALs affect constitutive or stimuli-induced clathrin-mediated and clathrin-independent endocytosis of other cargoes, we used PAE cells stably expressing high level of GFP-tagged epidermal growth factor receptor (EGFR-GFP) (33Carter R.E. Sorkin A. Endocytosis of functional epidermal growth factor receptor- green fluorescent protein chimera.J. Biol. Chem. 1998; 273: 35000-35007Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). When stimulated with saturating ligand concentrations, EGFR has been shown to internalize via a clathrin-independent pathway (34Sigismund S. Woelk T. Puri C. Maspero E. Tacchetti C. Transidico P. Di Fiore P.P. Polo S. Clathrin-independent endocytosis of ubiquitinated cargos.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2760-2765Crossref PubMed Scopus (651) Google Scholar). Fig. S4 demonstrates that ALs did not increase endocytosis of EGFR-GFP over its basal constitutive level and did not alter the clathrin-independent endocytosis of EGFR-GFP stimulated by 100 ng/ml EGF. In the same cells, clathrin-mediated endocytosis, assessed by imaging of the uptake of fluorescent transferrin, was also not affected by ALs (Fig. S4). Importantly, unlike AIM-100, AL3, AL4, and AL8 did not inhibit the kinase activity of ACK1, as measured by probing the tyrosine autophosphorylation of ACK1 in HeLa cells in which ACK1 was activated upon cell stimulation with EGF (Fig. 2G). These data confirm that the effects of AIM-100 and ALs on DAT oligomerization and endocytic trafficking are independent of ACK1 activity. AIM-100–induced DAT oligomerization and endocytosis were shown to be inhibited by the DAT antagonist, cocaine (25Sorkina T. Ma S. Larsen M.B. Watkins S.C. Sorkin A. Small molecule induced oligomerization, clustering and clathrin-independent endocytosis of the dopamine transporter.Elife. 2018; 7: e32293Crossref PubMed Scopus (18) Google Scholar). Here, we show that all effects of AL3, AL4, and AL8 on DAT were also inhibited by cocaine (Fig. 3). The inhibition of DAT oligomerization by cocaine was partial but significant in all experiments (Fig. 3, A and B). Cocaine strongly blocked small molecule–induced endocytosis measured using the HA antibody uptake assay (Fig. 3, C and D). Notably, the accumulation of YFP-HA-DAT in endosomes induced by 20 μM AL8 was statistically significant but much smaller than that in cells treated with AL3 or AL4 (Fig. 3D), although further increase in DAT endocytosis was observed at high AL8 concentrations (40 μM). Because AL8 had a weaker effect on DAT endocytosis, we classify it as "partially effective" (Fig. S1). Altogether, the data in Figure 1, Figure 2, Figure 3 and their supplements demonstrate the DAT-specific and cocaine-sensitive effects of ALs on the oligomerization and endocytosis of recombinant human YFP-HA-DAT and endogenous mouse HA-DAT and confirm that these effects are not associated with the inhibition ACK1. To determine which DAT residues play a dominant role in the AIM-100/AL-induced oligomerization and endocytosis, we conducted site-directed mutagenesis experiments. Our recent study pointed to the involvement of TM4 and TM9, the latter containing a putative leucine heptad or "zipper" motif, in the AIM-100–induced stabilization of DAT trimer (26Cheng M.H. Ponzoni L. Sorkina T. Lee J.Y. Zhang S. Sorkin A. Bahar I. Trimerization of dopamine transporter triggered by AIM-100 binding: Molecular mechanism and effect of mutations.Neuropharmacology. 2019; 161: 107676Crossref PubMed Scopus (4) Google Scholar). Alanine-substitution of multiple TM9 leucine residues revealed that the L459 A mutation impaired AIM-100/AL-induced endocytosis when the mutant was transiently expressed. However, this effect was not statistically significant in cells stably expressing this mutant. Recent studies by Navratna et al. revealed that the mutation of I248 (TM4) to an aromatic residue resulted in a π-stacking interaction between F/Y248 and F457 of TM9 (35Navratna V. Tosh D.K. Jacobson K.A. Gouaux E. Thermostabilization and purification of the human dopamine transporter (hDAT) in an inhibitor and allosteric ligand bound conformation.PLoS One. 2018; 13: e0200085Crossref PubMed Scopus (13) Google Scholar). The TM4–TM9 association prompted us to test the effects of the I248Y substitution. The I248Y mutant of YFP-HA-DAT displayed a partially reduced response to AIM-100 and ALs, although this reduction was again not statistically significant. Encouraged by the trends observed with I248Y and L459A mutants and because the AIM-100/AL-effects are unique to DAT, we generated a quadruple mutant where we replaced four TM4 and TM9 residues by their SERT counterparts: I248F, V249T, L458F, and L459G. This mutant will be shortly referred to as the "TM4-9" mutant (Fig. 4A). We hypothesized that the introduction of SERT-like residues, including two phenylalanines, into TM4 and TM9 of DAT could rewire the interactions between these helices, such that AIM-100/AL binding and/or AIM-100/AL-induced DAT oligomerization might be impeded. In support of this hypothesis, we observed a substantial decrease in the fraction of oligomers (T-DAT) induced by AIM-100 and ALs in the TM4-9 mutant compared with that in the WT YFP-HA-DAT (Fig. 4, B and C). Moreover, total internal reflection fluorescence microscopy demonstrated only a minimal, if any, AIM-100 induced clustering of the TM4-9 mutant at the cell surface (Fig. S5). Interestingly, the constitutive oligomerization of the TM4-9 mutant (∼270–300 kDa species) was slightly increased compared with WT YFP-HA-DAT (Fig. 4, B and C). In parallel with the decrease in AIM-100/AL-induced oligomerization, AIM-100 and ALs did not induce significant endocytosis of the TM4-9 mutant as observed on the images of direct YFP fluorescence and using the antibody uptake internalization assay (Figs. 4, D, E and S6). By contrast, endocytosis triggered by phorbol ester (phorbol 12-myristate 13-acetate) was similar in WT YFP-HA-DAT and TM4-9 mutant expressing cells (Fig. 4, D and E), demonstrating that this mutant was otherwise capable of clathrin-mediated endocytosis (36Sorkina T. Hoover B.R. Zahniser N.R. Sorkin A. Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism.Traffic. 2005; 6: 157-170Crossref PubMed Scopus (135) Google Scholar). Overall, the data in Figure 4 and its supplements showed that residues I248/V249 in TM4 and L458/L459 in TM9 are involved in the oligomerization and clathrin-independent endocytosis of DAT induced by AIM-100 and ALs. The above experiments showed that cocaine binding and quadruple mutations had similar effects, both suppressing the oligomerization and internalization of DAT, which would be otherwise induced by AIM-100 or ALs. This observation suggests that both had a comparable effect on the molecular structure and mechanics, resulting in similar observables. To understand the mechanistic basis of the observed phenomena at the molecular level, we examined the change in the conformational dynamics of the quadruple mutant compared with that of WT DAT by carrying

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