title: Modification of the pH profile and tetrabenazine sensitivity of rat VMAT1 by replacement of aspartate 404 with glutamate.
abstract: Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine, and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient (Delta micro H+) across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. Mutagenic analysis of the role of two conserved Asp residues located in transmembrane segments X and XI of rat VMAT type I reveals an important role of these two residues in catalysis. Replacement of Asp 431 with either Glu or Ser inhibits VMAT-mediated [3H]serotonin transport. The mutated proteins are unimpaired in ligand recognition as measured with the high affinity ligand [3H]reserpine or coupling to the proton electrochemical gradient as judged by its ability to accelerate [3H]reserpine binding. Therefore, the Asp residue is needed as such in this position and even a conservative replacement with Glu generates a protein that can catalyze only partial reactions but cannot complete the transport cycle. Replacement of Asp 404 with either Ser or Cys inhibits all VMAT-mediated reactions measured. However, replacement with Glu generated a protein that catalyzed [3H]serotonin transport with modified properties. Whereas the mutated protein binds [3H]reserpine to normal levels and the pH optimum of this reaction is only slightly affected, the optimum pH for transport activity shifted to the acid side and became very sharp; in addition the sensitivity to the inhibitor tetrabenazine increased significantly in this mutated protein. The results point to the need of a carboxyl moiety in position 404. A slight change in its relative location or in the environment around it has a significant effect on the pK of group(s) involved in steps after ligand recognition and coupling to the first H+.
authors.affiliation: Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, 91904 Israel.
authors.forename: S
authors.initials: S
authors.lastname: Steiner-Mordoch
authors.forename: A
authors.initials: A
authors.lastname: Shirvan
authors.forename: S
authors.initials: S
authors.lastname: Schuldiner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D002384:Catalysis; D002460:Cell Line; D018698:Glutamic Acid / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides; D051381:Rats; D012701:Serotonin / Q000378:metabolism; D013747:Tetrabenazine / Q000494:pharmacology*; D014316:Tritium; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
chemical_list: D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; C493451:Slc18a1 protein, rat; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D014316:Tritium; D001224:Aspartic Acid; D012701:Serotonin; D018698:Glutamic Acid; D013747:Tetrabenazine