A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction.
Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the central nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV-1 monkey kidney cells exhibits saturable, high affinity Na+ and Cl- dependent serotonin uptake, with estimated Km and Vmax values of 436 +/- 19 nm and 3.8 +/- 0.6 x 10-18 mol.cell.min-1, respectively. The Manduca high affinity Na+/Cl- dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC50 = 1.23 micro m) and cocaine (IC50 = 12.89 micro m). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148-152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC50 = 1.54 micro m). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1-146/MasSERT106-587, which involved N-terminal swaps including transmembrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC50 = 180 micro m) compared to the human (IC50 = 0.431 micro m) and Manduca serotonin transporters. The chimera MasSERT1-67/hSERT109-630, which involved only the TMD1 swap, showed greater sensitivity to cocaine (IC50 = 0.225 micro m) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca serotonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sensitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine.
Sandhu
Sumandeep K
SK
Environmental Toxicology Graduate Program and Department of Cell Biology and Neuroscience, University of California, Riverside 92521, USA.
Ross
Linda S
LS
Gill
Sarjeet S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003042:Cocaine / Q000494:pharmacology*; D004351:Drug Resistance; D005473:Fluoxetine / Q000494:pharmacology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018402:Manduca / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D017366:Serotonin Receptor Agonists / Q000378:metabolism; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D013045:Species Specificity; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D002712:Chlorides; D003042:Cocaine; D005473:Fluoxetine; D019476:Insect Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D012701:Serotonin; D012702:Serotonin Antagonists; D050486:Serotonin Plasma Membrane Transport Proteins; D017366:Serotonin Receptor Agonists; D017367:Selective Serotonin Reuptake Inhibitors; D012964:Sodium; C493230:SLC6A4 protein, human