tailieunhanh - Báo cáo khoa học: 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 cen-tral nervous system of the insectManduca sextaenabled us to define domains that affect antagonist action, particularly cocaine. | Eur. J. Biochem. 269 3934-3944 2002 FEBS 2002 doi A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction Sumandeep K. Sandhu1 2 Linda S. Ross2 and Sarjeet S. Gill1 2 1 Environmental Toxicology Graduate Program and 2Department of Cell Biology and Neuroscience University of California Riverside USA 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 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 M and cocaine IC50 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 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 M compared to the human IC50 M and Manduca .