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  • Title: Modification of the pH profile and tetrabenazine sensitivity of rat VMAT1 by replacement of aspartate 404 with glutamate.
    Author: Steiner-Mordoch S, Shirvan A, Schuldiner S.
    Journal: J Biol Chem; 1996 May 31; 271(22):13048-54. PubMed ID: 8662678.
    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+.
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