Charged residues in transmembrane domains II and XI of a vesicular monoamine transporter form a charge pair that promotes high affinity substrate recognition.
Vesicular monoamine transporters package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. Both vesicular monoamine transporter 1 and 2 contain several charged residues predicted to reside within transmembrane domains (TMDs), and conservation of these residues in multiple species and in other members of the gene family suggest important roles in transporter structure and function. To determine the role of these residues, we have used site-directed mutagenesis. Replacement of Asp-263 in TMD6 with Asn (D263N) had no effect on transport activity. However, replacement of Lys-139 in TMD2 with Ala (K139A), Asp-400 in TMD10 with Asn (D400N), or Asp-427 in TMD11 with Asn (D427N) eliminated transport activity despite normal levels of protein expression. Remarkably, the double mutant K139A/D427N showed substantial transport activity, suggesting that Lys-139 and Asp-427 interact to form an ion pair in the native protein and hence that TMD2 occurs next to TMD11. Nonetheless, the double mutant showed reduced apparent affinity for serotonin and reduced ability of serotonin to inhibit reserpine binding, suggesting that although not required for activity, the ion pair promotes high affinity interaction with the substrate. In addition, a double mutant in which the polarity of the charged residues was reversed (K139D/D427K) showed no active transport. Remarkably, however, this mutant displayed normal reserpine binding that remained coupled to DeltaH+, but serotonin failed to inhibit reserpine binding, suggesting that the charge reversal specifically disrupts substrate recognition.
Merickel
A
A
Interdepartmental Program in Neuroscience, Molecular Biology Institute, Howard Hughes Medical Institute, School of Medicine, UCLA, Los Angeles, California 90095, USA.
Kaback
H R
HR
Edwards
R H
RH
D000818:Animals; D001216:Asparagine; D001224:Aspartic Acid; D001692:Biological Transport; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012110:Reserpine / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D001224:Aspartic Acid; D012701:Serotonin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D001216:Asparagine; D012110:Reserpine; D008239:Lysine