Modification of the pH profile and tetrabenazine sensitivity of rat VMAT1 by replacement of aspartate 404 with glutamate.
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+.
Steiner-Mordoch
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Alexander Silberman Institute of Life Sciences, Hebrew University, Jerusalem, 91904 Israel.
Shirvan
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Schuldiner
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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
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