Gustavo J Rodríguez

Publications (2)7.05 Total impact

• Article: Conformational flexibility of transmembrane helix VII of the human serotonin transporter impacts ion dependence and transport.

  Cody J Wenthur, Gustavo J Rodríguez, Charles P Kuntz, Eric L Barker
  [show abstract] [hide abstract]
  ABSTRACT: The serotonin transporter (SERT) regulates the serotonin concentration in the synapse and is a target of several antidepressant and psychostimulant drugs. Previous work suggested that the middle transmembrane helices (TMHs) of the biogenic amine transporters (TMHs) play a role in substrate and ion recognition. We focused our present studies on exploring the role of TMH VII in transporter function and ion recognition. Residues divergent between human SERT and Drosophila SERT (hSERT and dSERT, respectively) were identified and mutated in hSERT to the corresponding identity in dSERT. hSERT mutants V366S, M370L, S375A, and T381S exhibited a decrease in transport capacity. To further explore the role of these residues in the transport process, we generated cysteine mutants at multiple positions. Pretreatment with [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) caused a decrease in transport of [(3)H]5-HT in the V366C and M370C mutants. The hSERT V366S, M370L, and M370C mutations also altered the sodium and chloride dependence for substrate transport. Interpretation of our results in the context of a homology model of SERT based on the crystal structure of the Aquifex aeolicus leucine transporter suggests flexibility in the conformation of TMH VII that impacts ion dependence and substrate transport.
  Biochemical pharmacology 11/2010; 80(9):1418-26. · 4.25 Impact Factor
• Article: Helix XI contributes to the entrance of the serotonin transporter permeation pathway.

  Melissa I Torres-Altoro, Kellie J White, Gustavo J Rodríguez, David E Nichols, Eric L Barker
  [show abstract] [hide abstract]
  ABSTRACT: The sodium-dependent transporters for dopamine, norepinephrine, and serotonin that regulate neurotransmission, also translocate the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Previous studies implicated residues in transmembrane helix (TMH) XI of DAT as important sites for MPP(+) transport. We examined the importance of TMH XI residues F551 and F556 for MPP(+) translocation by human SERT. Mutations at hSERT F556, but not F551, reduced both 5-HT and MPP(+) transport compared to wild type. However, F556S/hSERT showed a reduction in surface expression explaining the decrease of transport activity for 5-HT, but did not account for the decrease in MPP(+) transport observed. Cysteine mutants at those positions confirmed the accessibility of hSERT/F556 to different methanethiosulfonate (MTS) reagents, suggesting its presence in a hydrophilic environment of the protein. In the presence of MTSET, current induced by 5-HT and MPP(+) was inhibited at the F556C mutant. In agreement with our homology model of SERT, based on the leucine transporter (LeuT(Aa)) from Aquifex aeolicus structure, these results are consistent with the hypothesis that a portion of TMH XI lines the entrance into the substrate permeation pathway.
  Protein Science 10/2008; 17(10):1761-70. · 2.80 Impact Factor