TY - JOUR

T1 - Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants

AU - Kristensen, Anders S

AU - Larsen, Mads B

AU - Johnsen, Laust B

AU - Wiborg, Ove

PY - 2004/3

Y1 - 2004/3

N2 - The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

AB - The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

KW - Amino Acid Sequence

KW - Animals

KW - Binding Sites

KW - Biological Transport

KW - Blotting, Western

KW - COS Cells

KW - Carrier Proteins

KW - Cercopithecus aethiops

KW - Chimera

KW - Choline

KW - Cloning, Molecular

KW - Cocaine

KW - Dose-Response Relationship, Drug

KW - Humans

KW - Indicators and Reagents

KW - Inhibitory Concentration 50

KW - Membrane Glycoproteins

KW - Membrane Transport Proteins

KW - Mesylates

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Mutation

KW - Nerve Tissue Proteins

KW - Radiopharmaceuticals

KW - Serotonin

KW - Serotonin Plasma Membrane Transport Proteins

KW - Sodium

KW - Structure-Activity Relationship

KW - Transfection

U2 - 10.1111/j.1460-9568.2004.03202.x

DO - 10.1111/j.1460-9568.2004.03202.x

M3 - Journal article

C2 - 15066148

VL - 19

SP - 1513

EP - 1523

JO - European Journal of Neuroscience

JF - European Journal of Neuroscience

SN - 0953-816X

IS - 6

ER -