Identification of residues involved in substrate recognition by a vesicular monoamine transporter.
To identify the residues involved in substrate recognition by recently cloned vesicular monoamine transporters (VMAT1 and VMAT2), we have mutagenized the conserved residues in a cytoplasmic loop between transmembrane domains two and three of VMAT2. Although studies of related bacterial antibiotic resistance proteins indicate an important functional role for this region, we found no effect of these mutations on VMAT2 activity. However, replacement of aspartate 33 in the first predicted transmembrane domain with an asparagine (D33N) eliminates transport. D33N shows normal levels of expression and normal binding at equilibrium to the potent inhibitor reserpine. However, in contrast to wild-type VMAT2, serotonin inhibits reserpine binding to D33N very poorly, indicating a specific defect in substrate recognition. Replacement of three serine residues in transmembrane domain three with alanine (Stmd3A) shows a similarly selective but even more profound defect in substrate recognition. The results suggest that by analogy to receptors and plasma membrane transporters for monoamines, the cationic amino group of the ligand interacts with an asparte in the first transmembrane domain of VMAT2 and hydroxyl groups on the catechol or indole ring interact with a group of serines in the third transmembrane domain. Importantly, D33N and Stmd3A retain coupling to the proton electrochemical gradient as measured by the delta microH(+)-induced acceleration of reserpine binding. This indicates that substrate recognition can be separated from coupling to the driving force.
Merickel
A
A
Interdepartmental Program in Neuroscience, UCLA School of Medicine 90024-1769, USA.
Rosandich
P
P
Peter
D
D
Edwards
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D015306:Biogenic Monoamines / Q000378:metabolism*; D001692:Biological Transport; D004252:DNA Mutational Analysis; D006023:Glycoproteins / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000378:metabolism*; D011487:Protein Conformation; D018892:Proton-Motive Force; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012110:Reserpine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012701:Serotonin / Q000378:metabolism; D013329:Structure-Activity Relationship; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D015306:Biogenic Monoamines; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; C493451:Slc18a1 protein, rat; C493454:Slc18a2 protein, rat; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D012701:Serotonin; D012110:Reserpine